Chimie Farma - Structure and Function of GPCRs (2019) PDF

Topics in Medicinal Chemistry 30
Guillaume Lebon Editor
Structure
and
Function of
GPCRs
30
Topics in Medicinal Chemistry
Series Editors
P.R. Bernstein, Philadelphia, USA
A.L. Garner, Ann Arbor, USA
G.I. Georg, Minneapolis, USA
T. Kobayashi, Tokyo, Japan
J.A. Lowe, Stonington, USA
N.A. Meanwell, Princeton, USA
A.K. Saxena, Lucknow, India
U. Stilz, Boston, USA
C.T. Supuran, Sesto Fiorentino, Italy
A. Zhang, Pudong, China
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Guillaume Lebon
Editor
Structure and Function

of GPCRs
With contributions by
F. Acher J.-L. Banères S.S. Bhunia J. Carlsson
M. Casiraghi L.J. Catoire T. Durroux O. Faklaris
A. Falco C. Goudet E. Goyet J. Heuninck
F.G. Jean-Alphonse G. Lebon A. Llebaria C. Mendre
B. Mouillac C. Nasrallah J. Perroy J.-P. Pin
A. Ranganathan D. Rodríguez P. Rondard X. Rovira
A.K. Saxena M. Saxena I. Sutkeviciute M. Tian
A.J. Venkatakrishnan J.-P. Vilardaga Q. Wang S. Ye
C. Yuan J.M. Zwier
Editor
Guillaume Lebon
Institut de Génomique Fonctionnelle
Centre National de la Recherche
Scientifique (CNRS), Institut National de
la Santé et de la Recherche Médicale
(INSERM)
University of Montpellier
Montpellier, France
ISSN 1862-2461 ISSN 1862-247X (electronic)

Topics in Medicinal Chemistry
ISBN 978-3-030-24589-4 ISBN 978-3-030-24591-7 (eBook)
https://doi.org/10.1007/978-3-030-24591-7
© Springer Nature Switzerland AG 2019

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A Decade of GPCR Structure and Signalling
in Three Dimensions
G protein-coupled receptors (GPCRs) are localised at the plasma membrane of

eukaryotic cells, and by sensing external stimuli they are key receptors for cellular
communication [1]. They are involved in all physiological function and many
pathological disorders. They are tractable drug targets as illustrated by the large
numbers of molecules approved by the Food and drug Administration, and they still
offer a large amount of opportunities for the conception of therapeutic molecules [2].
However, designing drugs will be for sure facilitated by a deep and thorough
understanding of their biological function, 3D structure and conformation as well
as the structure–function relation that is now supported by a large number of ligand-
bound GPCR high-resolution structures. Nasrallah and Lebon present several strat-
egies that were developed and successfully used to facilitate the structural determi-
nation of GPCR ligand-bound conformation [3–6]. This chapter also discusses the
future challenges to be undertaken for solving structure of GPCR signalling com-
plexes. Indeed, the first view of a GPCR signalling complex X-ray structure – the
β2-adrenergic receptor bound to heterotrimeric G protein – was reported in 2012 by
the laboratory of Brian Kobilka [7]. Since then, we now have access to the high-
resolution structures of GPCR signalling complexes for several GPCR types. Indeed,
recent technological development in cryogenic electron microscopy (cryoEM),
direct electron detector and images processing have strongly facilitated the structure
determination of flexible GPCR signalling complexes [8–11]. We can only expect
even deeper understanding of the signalling, ligand-binding kinetics and ligand
selectivity of this large family of receptors during the next decade and hopefully
the discovery of first in class molecules.
GPCRs are not only a seven transmembrane receptor that can be modulated by a
ligand that binds to the orthosteric site localised within the seven transmembrane
domain (7TM), but should also be seen as highly dynamic proteins for which several
strategies can be envisioned in order to modulate their biological activity [1, 12, 13].
For most receptor classes, ligands bind inside the 7TM helix bundle, at various
depths from the extracellular side. Several examples reported the binding of ligands
in unusual pockets, sometimes buried within the lipid bilayers or within an extra-
cellular vestibule as for the PAR2 receptor, or even down to intracellular side for the
v
vi A Decade of GPCR Structure and Signalling in Three Dimensions
CCR9 and CCR2 receptor [14–16]. Such observations illustrate the dynamic aspect
of GPCR 7TM domains, pushing the concept of modulating the GPCR activity to
almost no limitation. AJ Venkatashriman describes here a general view of structural
feature of GPCR ligand-binding sites and how drugs can stabilise receptor confor-
mations. Class A GPCR activation mechanism is also discussed in detail based on
recent structural information and their unbiased computational analysis [17]. It
clearly appears that despite a large number of X-ray structures available, from
different classes of receptors, including class A, B, C and F, we still have to explore
the conformational landscape of GPCRs and how ligands and intracellular signalling
partners are modulating together the type and efficacy of signal transduction.
Dynamic properties of GPCRs require special care and attention in order to
perform structural characterisation. Nuclear magnetic resonance (NMR) is a power-
ful technique for investing receptor dynamics and conformational changes that are
required for GPCR activation upon agonist binding [18–20]. Despite the challenge
of labelling the protein, NMR offers unique possibilities to identify and characterise
discrete receptor states at atomic resolution. Casiraghi and colleagues present a
detailed update on NMR spectroscopy for the characterization of GPCR energy
landscape and associated kinetic barriers [21].
Structural studies of GPCRs have delivered new insights into their ligand-binding
mode and activation mechanism at an atomic level, offering unprecedented infor-
mation for designing and discovering new drugs with therapeutic potential [22–24].
One can ask the real impact of GPCR structures on drug discovery process. Two
chapters in this book contributed by Saxena and colleagues and Ranganathan and
colleagues highlight the importance and also the limitations of GPCR high-
resolution structures for ligand docking and screening to identify new compounds.
Top hits are specific of the receptor conformation trapped during the crystallisation
process. They also describe and highlight the importance of the starting model and
the choice of ligand for generating an accurate homology model. Structure-based
drug discovery (SBDD) provides an additional strategy to develop and optimise
therapeutic molecules. As a consequence, pharmaceutical industry has now engaged
in the determination of GPCR high-resolution structures, reinforcing their drug
discovery pipeline and their chance of success in identifying new molecules.
GPCRs are signalling receptors for which understanding of their major function
has also undertaken major evolution during the last decade [13, 25]. Internalisation
and desensitization of receptor signalling were considered for a long time as a
secondary effect of agonist stimulation interfering with G protein signalling. How-
ever, by interacting with β-arrestin and G protein-coupled receptor kinases (GRKs),
GPCRs have additional G protein-independent signallings to their signalling reper-
toire. The variety of the signalling repertoire led to the major concepts of biased
signalling and consequently of ligand selectivity. These broaden further the possi-
bility of developing new molecules, selective of a given receptor but also with a
specific signalling signature. Indeed, the scientific community aims at designing
molecules biased toward a single signalling pathway and that likely represents the
solution for developing drugs devoid of side effect [26]. The concept of biased
A Decade of GPCR Structure and Signalling in Three Dimensions vii
agonists is presented by Bernard Mouillac and Christiane Mendre. In addition, they

also discuss a peculiar example at the Vasopressin 2 receptor subtype for which
mutations found in patients induce the production of misfolded and inactive recep-
tors. In this specific case biased agonist also acts as chaperone by rescuing misfolded
receptors to the plasma membrane and by specifically activating the Gs signalling
pathway, balancing undesired biological effect observed in the pathology.
GPCRs are activated at the plasma membrane triggering G protein activation, and
later internalised by interacting with the β-arrestin protein or desensitised, but in both
cases G protein coupling is alleviated. New paradigm in GPCRs is about intracellular
and compartmentalized signalling. It was recently reported that the internalised
β-adrenergic receptor can maintain a certain level of signal transduction whilst
being redirected and trafficked in endosomes within the cellular cytoplasm [27].
Sutkeviciute and colleagues describe this new phenomenon for the parathyroid
hormone (PTH) type 1 receptor (PTHR) and discuss the whole process of GPCR
signalling in endosomes and its biological consequences.
GPCRs display an important signalling plasticity, in time, and must be looked at
as unusual proteins, highly flexible for which dynamics is the basis of their biolog-
ical functions. New concepts about their biological function rise from the increasing
interest and arousing number of investigations aiming at deciphering their biology.
Several methodologies play important role in understanding the GPCR pharmacol-
ogy and function. In addition to the important breakthrough due to the improvement
of the structural biology techniques, additional technologies are available and in
continuous development to favour the understanding of GPCR molecular
mechanisms.
Among these technologies, we can cite the genetic code expansion and the
development of unnatural amino acids (Uaas). Over the last decade, it has gained
interest for monitoring GPCR dynamics, ligands–receptor interaction and protein–
protein interactions [28–30]. Tian and colleagues detail the methodology available
and the use of photo-cross-linking Uaas and site-specific fluorescent labelling to
select and analyse GPCR conformational changes. This chapter is illustrated with
several examples for which Uaa technology allowed to track either helical confor-
mational changes using infrared Uaas, ligand-induced conformational dynamics or
mapping protein–protein interaction between via Uaa cross linking [28, 31, 32].
Other technologies use fluorescent-based strategies and contributed to the under-
standing of GPCR signalling and oligomerisation. Flakaris and colleagues present
the principle of resonance energy transfer strategies that include BRET, FRET and
time-resolved FRET as well as fluorescent strategy such as fluorescence correlation
microscopy that is used to analyse GPCR dynamics and oligomerisation. They are
indispensable for investigating the molecular pharmacology of GPCRs [33–35]. The
devolvement of fluorescent–based technology allows to follow all steps of a GPCR
life cycle, from ligand binding to G protein activation, second messenger production,
protein kinase-dependent phosphorylation, internalisation and the even more chal-
lenging phenomenon of GPCR oligomerisation.
As illustrated in this book, GPCRs are very diverse family of protein. Amongst
the GPCR classes from, A, B, C, Adhesion and F, Class C is a peculiar small class,
viii A Decade of GPCR Structure and Signalling in Three Dimensions
with only 22 members that include metabotropic receptors for glutamate and GABA
that are respectively the main excitatory and inhibitory neurotransmitters of the
central nervous system [36]. Goudet and colleagues give a large overview of class
C metabotropic glutamate receptor family. Metabotropic glutamate receptors are
composed of a large extracellular domain (ECD) where glutamate binds and of a
conserved 7TM domain. They present this unique and obligatory dimeric molecular
organisation, in addition to having the binding site for glutamate in the ECD and for
allosteric modulators in the 7TM. They discuss the several possibilities recently
developed for modulating their activity. Nanobodies targeting the extracellular
domain were discovered recently opening new avenue for allosteric modulation
mGlu receptors [37]. Finally photo-switchable ligands targeting the 7TM also
offer the possibility to turn on and off the receptor activity on demands by using
laser light sources [38–40]. Indeed, photopharmacology makes possible to target
endogenous receptors and to have a spatial and time control of compound activity,
allowing the tuning of the receptor activity on request and in a tissue-specific
manner. This methodology has great promise for therapeutic application and also
complements the large and diverse toolbox available for scientists wishing to
investigate this fascinating family of GPCRs.
This book neither pretends to cover all aspects of GPCRs nor to present all
techniques available to date for investigating GPCR biological functions. The
chapters presented here cover some of the most recent advances in the field and
provide accessible understanding of recent achievements and also of the major
challenges that remain to be tackled in the GPCR field. A decade ago, GPCRs
were considered as 7TM domain receptors, composed of an orthosteric ligand-
binding site that accommodates ligands and activates intracellular G protein and of
potential distinct allosteric binding site. Indeed, allosteric modulation was already of
high interest as well as dimerization and oligomerisation. But today, the knowledge
about GPCRs has built up, and I hope that reading the book will provide a totally
different view on GPCRs and foster ideas for an even better understanding and also
for new concepts that hopefully will lead to discovering new therapeutic molecules.
IGF, CNRS UMR, INSERM, University Guillaume Lebon

of Montpellier, Montpellier, France
References
1. Venkatakrishnan AJ et al (2013) Molecular signatures of G-protein-coupled

receptors. Nature 494:185–194
2. Hauser AS et al (2018) Pharmacogenomics of GPCR drug targets. Cell 172:41–
54.e19
A Decade of GPCR Structure and Signalling in Three Dimensions ix
3. Tate CG, Schertler GFX (2009) Engineering G protein-coupled receptors to

facilitate their structure determination. Curr Opin Struct Biol 19:386–395
4. Warne T et al (2008) Structure of a beta1-adrenergic G-protein-coupled recep-
tor. Nature 454:486–491
5. Manglik A, Kobilka BK, Steyaert J (2017) Nanobodies to study G protein-
coupled receptor structure and function. Annu Rev Pharmacol Toxicol 57:19–37
6. Rosenbaum DM et al (2007) GPCR engineering yields high-resolution struc-
tural insights into beta2-adrenergic receptor function. Science 318:1266–1273
7. Rasmussen SGF et al (2011) Crystal structure of the β2 adrenergic receptor-Gs
protein complex. Nature 477:549–555
8. García-Nafría J, Nehmé R, Edwards PC, Tate CG (2018) Cryo-EM structure of
the serotonin 5-HT1B receptor coupled to heterotrimeric Go. Nature 558:620–
623
9. Kang Y et al (2015) Crystal structure of rhodopsin bound to arrestin by
femtosecond X-ray laser. Nature 523:561–567
10. Draper-Joyce CJ et al (2018) Structure of the adenosine-bound human adeno-
sine A1 receptor-Gi complex. Nature 558:559–563
11. Liang Y-L et al (2017) Phase-plate cryo-EM structure of a class B GPCR-G-
protein complex. Nature 546:118–123
12. Thal DM, Glukhova A, Sexton PM, Christopoulos A (2018) Structural insights
into G-protein-coupled receptor allostery. Nature 559:45–53
13. Wootten D, Christopoulos A, Marti-Solano M, Babu MM, Sexton PM (2018)
Mechanisms of signalling and biased agonism in G protein-coupled receptors.
Nat Rev Mol Cell Biol. https://doi.org/10.1038/s41580-018-0049-3
14. Oswald C et al (2016) Intracellular allosteric antagonism of the CCR9 receptor.
Nature 540:462–465
15. Zheng Y et al (2016) Structure of CC chemokine receptor 2 with orthosteric and
allosteric antagonists. Nature 540:458–461
16. Cheng RKY et al (2017) Structural insight into allosteric modulation of
protease-activated receptor 2. Nature 545:112–115
17. Venkatakrishnan AJ et al (2016) Diverse activation pathways in class A GPCRs
converge near the G-protein-coupling region. Nature 536:484–487
18. Sounier R et al (2015) Propagation of conformational changes during μ-opioid
receptor activation. Nature 524:375–378
19. Casiraghi M et al (2016) Functional modulation of a G protein-coupled receptor
conformational landscape in a lipid bilayer. J Am Chem Soc 138:11170–11175
20. Isogai S et al (2016) Backbone NMR reveals allosteric signal transduction
networks in the β1-adrenergic receptor. Nature 530:237–241
21. Casiraghi M, Damian M, Lescop E, Banères J-L, Catoire LJ (2018) Illuminating
the energy landscape of GPCRs: the key contribution of solution-state NMR
associated with Escherichia coli as an expression host. Biochemistry 57:2297–
2307
22. Congreve M, Dias JM, Marshall FH (2014) Structure-based drug design for G
protein-coupled receptors. Prog Med Chem 53:1–63
x A Decade of GPCR Structure and Signalling in Three Dimensions
23. Congreve M et al (2012) Discovery of 1,2,4-triazine derivatives as adenosine A

(2A) antagonists using structure based drug design. J Med Chem 55:1898–1903
24. Christopher JA et al (2018) Structure-based optimization strategies for G
protein-coupled receptor (GPCR) allosteric modulators: a case study from
analyses of new metabotropic glutamate receptor 5 (mGlu5) X-ray structures.
J Med Chem. https://doi.org/10.1021/acs.jmedchem.7b01722
25. Violin JD, Lefkowitz RJ (2007) Beta-arrestin-biased ligands at seven-
transmembrane receptors. Trends Pharmacol Sci 28:416–422
26. Manglik A et al (2016) Structure-based discovery of opioid analgesics with
reduced side effects. Nature 537:185–190
27. Irannejad R et al (2013) Conformational biosensors reveal GPCR signalling
from endosomes. Nature 495:534–538
28. Damian M et al (2015) Ghrelin receptor conformational dynamics regulate the
transition from a preassembled to an active receptor:Gq complex. Proc Natl
Acad Sci U S A 112:1601–1606
29. Grunbeck A, Huber T, Sachdev P, Sakmar TP (2011) Mapping the ligand-
binding site on a G protein-coupled receptor (GPCR) using genetically encoded
photocrosslinkers. Biochemistry 50:3411–3413
30. Naganathan S, Grunbeck A, Tian H, Huber T, Sakmar TP (2013) Genetically-
encoded molecular probes to study G protein-coupled receptors. J Vis Exp.
https://doi.org/10.3791/50588
31. Tian H et al (2014) Bioorthogonal fluorescent labeling of functional G-protein-
coupled receptors. Chembiochem 15:1820–1829
32. Ray-Saha S, Huber T, Sakmar TP (2014) Antibody epitopes on g protein-
coupled receptors mapped with genetically encoded photoactivatable cross-
linkers. Biochemistry 53:1302–1310
33. Scholler P et al (2013) Time-resolved Förster resonance energy transfer-based
technologies to investigate G protein-coupled receptor machinery: high-
throughput screening assays and future development. Prog Mol Biol Transl
Sci 113:275–312
34. Briddon SJ, Kilpatrick LE, Hill SJ (2018) Studying GPCR pharmacology in
membrane microdomains: fluorescence correlation spectroscopy comes of age.
Trends Pharmacol Sci 39:158–174
35. Marullo S, Bouvier M (2007) Resonance energy transfer approaches in molec-
ular pharmacology and beyond. Trends Pharmacol Sci 28:362–365
36. Pin J-P, Bettler B (2016) Organization and functions of mGlu and GABAB
receptor complexes. Nature 540:60–68
37. Scholler P et al (2017) Allosteric nanobodies uncover a role of hippocampal
mGlu2 receptor homodimers in contextual fear consolidation. Nat Commun
8:1967
38. Pittolo S et al (2014) An allosteric modulator to control endogenous G protein-
coupled receptors with light. Nat Chem Biol 10:813–815
A Decade of GPCR Structure and Signalling in Three Dimensions xi
39. Goudet C, Rovira X, Llebaria A (2018) Shedding light on metabotropic gluta-

mate receptors using optogenetics and photopharmacology. Curr Opin
Pharmacol 38:8–15
40. Font J et al (2017) Optical control of pain in vivo with a photoactive mGlu5
receptor negative allosteric modulator. elife 6:e23545
Contents
Structures of Non-rhodopsin GPCRs Elucidated Through X-Ray

Crystallography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Chady Nasrallah and Guillaume Lebon
NMR Spectroscopy for the Characterization of GPCR Energy
Landscapes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Marina Casiraghi, Jean-Louis Banères, and Laurent J. Catoire
Structure and Activation Mechanism of GPCRs . . . . . . . . . . . . . . . . . . . 53
A. J. Venkatakrishnan
Structure-Based Discovery of GPCR Ligands from Crystal Structures
and Homology Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Anirudh Ranganathan, David Rodríguez, and Jens Carlsson
Integration on Ligand and Structure Based Approaches in GPCRs . . . . 101
Anil K. Saxena, Shome S. Bhunia, and Mridula Saxena
Biased Agonist Pharmacochaperones: Small Molecules in the Toolbox
for Selectively Modulating GPCR Activity . . . . . . . . . . . . . . . . . . . . . . . 163
Bernard Mouillac and Christiane Mendre
Endosomal PTH Receptor Signaling Through cAMP and Its
Consequence for Human Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
Ieva Sutkeviciute, Frederic G. Jean-Alphonse, and Jean-Pierre Vilardaga
Structure and Function Studies of GPCRs by Site-Specific
Incorporation of Unnatural Amino Acids . . . . . . . . . . . . . . . . . . . . . . . . 195
Meilin Tian, Qian Wang, Chonggang Yuan, and Shixin Ye
Fluorescent-Based Strategies to Investigate G Protein-Coupled
Receptors: Evolution of the Techniques to a Better Understanding . . . . 217
Orestis Faklaris, Joyce Heuninck, Amandine Falco, Elise Goyet,
Jurriaan M. Zwier, Jean-Philippe Pin, Bernard Mouillac, Julie Perroy,
and Thierry Durroux
xiii
xiv Contents
Modulation of Metabotropic Glutamate Receptors by Orthosteric,

Allosteric, and Light-Operated Ligands . . . . . . . . . . . . . . . . . . . . . . . . . 253
Cyril Goudet, Xavier Rovira, Philippe Rondard, Jean-Philippe Pin,
Amadeu Llebaria, and Francine Acher
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285
Top Med Chem (2019) 30: 1–26
DOI: 10.1007/7355_2017_28
© Springer International Publishing AG 2017
Published online: 5 October 2017
Structures of Non-rhodopsin GPCRs

Elucidated Through X-Ray
Crystallography
Chady Nasrallah and Guillaume Lebon
Abstract During the last decade, more than 130 structures of G Protein-Coupled
Receptors were solved mostly using X-ray crystallography, either bound to antag-
onist, agonist or in complexes with intracellular partners such as G-proteins and
arrestin. These structures shed light on molecular mechanisms of GPCR ligand
recognition, activation, and allosteric modulation. This is primarily due to tremen-
dous advances in protein engineering and micro-crystallography making GPCRs
structure determination more straightforward. This chapter presents an overview of
the widely used and successful approaches that led to atomic resolution structures
of GPCR. Moreover, we briefly introduce recent technological advancements in the
structural biology field that will undoubtedly accelerate solving GPCRs structure in
the foreseeable future and provide a more complete understanding of GPCR
signaling.
Keywords Detergents solubilization, Diffraction, GPCRs, Micro-crystals,

Stability, X-ray crystallography
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2 Large-Scale Production of GPCRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
3 Solubilization of GPCRs Prior to Crystallization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
4 Enhancing GPCRs Crystallizablity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
4.1 Sample Homogeneity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
4.2 Binding Partners Stabilizing Receptor Conformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
4.3 Increasing Receptor Hydrophilic Area . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
4.4 Increasing Receptor Thermal Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
C. Nasrallah and G. Lebon (*)

Institut de Génomique Fonctionnelle, Centre National de la Recherche Scientifique (CNRS),
Institut National de la Santé et de la Recherche Médicale (INSERM), Université de
Montpellier, Montpellier 34000, France
e-mail: Guillaume.Lebon@igf.cnrs.fr
2 C. Nasrallah and G. Lebon
5 GPCR Sub-Micrometer and Micrometer-Size Crystals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

6 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
1 Introduction
G Protein-Coupled Receptors (GPCRs) are the largest class of human extracellular

cell-exposed receptors (800 members) that recognize a large array of ligands and
mediate diverse intracellular signalling cascades [1]. In the last several years, the
available high-resolution X-ray crystallography structures of GPCRs advanced our
understanding of cell signalling and in particular of the molecular switches that
occur upon receptor activation and the intimate connections that bind these recep-
tors to their natural or synthetic ligands [2, 3]. In addition, these structures gave
insights into the spectrum of conformational states that GPCRs could adapt starting
from the inactive ground state (R) to the fully active state (R*) when coupled to
heterotrimeric G proteins. Indeed, this high inherent conformational flexibility
constitutes a major challenge for obtaining well-ordered crystals required for
structural studies by X-ray crystallography [4]. Here, we give an overview of the
limitations encountered when attempting to crystallize and solve GPCR high-
resolution structures, including expression and purification of large amount of
functional receptors with controlled inherent conformational flexibility. We sum-
marize the methodological advances that helped to overcome these limitations and
enabled GPCR structural determination. This includes a variety of innovative
strategies such as (a) the use of binding partners (e.g. Fabs or nanobodies) to lock
the receptor in a single conformation (b) the replacement of flexible loops by a
stable soluble fusion protein in order to increase the hydrophilic molecular surface
of the receptor (c) the introduction of thermostablizing mutations and/or the
mixture of these strategies (Fig. 1). Finally, we emphasize on how novel structural
developments are likely to change the face of GPCR research in the near future
paving the way for accelerating GPCR structure determination not only for ligand-
bound conformations but also for complexes with different intracellular partners.
2 Large-Scale Production of GPCRs
With the exception of rhodopsin that is expressed at high level and can be extracted
form native tissues, GPCRs are usually expressed at very low levels [5]. In order to
perform crystallization trials, large quantities of proteins need to be produced.
Several strategies can be followed for performing large scale GPCRs expression,
all relying on heterologous expression systems. The choice of the expression
system for the target protein is often empirical, yet it is admitted that the closer
the chosen expression system is to the native environment of the target protein, the
Structures of Non-rhodopsin GPCRs Elucidated Through X-Ray Crystallography 3
Fig. 1 Successful stabilization strategies enhancing GPCRs crystallization. (a) Structures of

transmembrane domain (7TM) GPCR (cartoon mode; grey) crystallized with different binding
partners (cartoon mode; green) are shown: The human β2 adrenergic receptor (β2AR) in complex
with an antibody fragment (Fab) (upper left; PDB code 2R4S); the agonist-bound of the human M2
muscarinic acetylcholine receptor stabilized by a nanobody fragment (Nb) (upper right; PDB code
4MQS) and the adenosine A2A receptor (A2AR) bound to an engineered G protein, mini-Gs
(bottom left; PDB code 5G53). (b) Structures of engineered 7TM domain GPCR crystallized
with different fusion proteins (cartoon mode; cyan) are highlighted: The structure of the A2A
receptor with apocytochrome b(562)RIL (BRIL) fusion protein replacing its third intracellular
loop (upper; PDB code 4EIY) and the structure of the A2A receptor with T4 Lysozyme (T4L)
fusion protein replacing its third intracellular loop (bottom; PDB code 4EML). (c) Structure of
adenosine A2A receptor with 4 thermostabilizing point mutations (sphere mode; red) is shown
(upper; PDB code 2YDO). (d) A mix of different stabilizing strategies (a–c) is highlighted: The
structure of metabotropic glutamate receptor 5 (mGlu5) with 6 thermostabilizing point mutations
and a T4L fusion protein replacing the ICl2 is shown (upper; PDB code 4OO9). The structure of
beta2AR with T4L fusion protein replacing its third intracellular loop and bound to an engineered
nanobody is shown (bottom left; PDB code 4QKX) and the structure of the ternary complex
composed of beta2AR fused to the T4L lysozyme (N), the Nb and nucleotide-free Gs heterotrimer
(bottom right; PDB code 3SN6). See Table 1 for more details
better are the chances to retrieve a well-folded and fully functional receptor. This is
especially true for GPCRs that are difficult to express in prokaryote system in a
functional state [6]. GPCR structures were solved for the majority from receptors
produced using baculovirus expression system in insect cells, while only a minority
were solved using other systems such bacteria (e.g. Escherichia coli) and yeast
(e.g. Pichia Pastoris) (Table 1). Indeed, insect cell expression system has a more
sophisticated cellular and enzymatic machinery than bacteria and yeast ensuring
production of functional protein and performing post-translational modifications.
4
Table 1 Structures of non-rhodopsin GPCRSs solved by X-ray crystallography

GPCRs class Receptors Stabilization Expression Crystallization Ligand (type) Conf. PDB code Ref.
method system method state (Resolution)
Class A Rho- Adenosine A1 receptor BRIL-ICl3 Sf9 Lipid cubic DU172 (ANT) R 5UEN (3.2 Glukhova et al.
dopsin-like phase A) [7]
Adenosine A2A T4L-ICl3 Sf9 Lipid cubic ZM241385 (ANT) R 3EML (2.6 Jaakola et al. [8]
receptor phase A)
T4L-ICl3 Sf9 Lipid cubic UK432097 (AGO) R* int 3QAK (2.7 Xu et al. [9]
phase A)
T.S mutations High five Vapor Adenosine (AGO) R* int 2YDO (3 A) Lebon et al. [10]
diffusion
T.S mutations High five Vapor NECA (AGO) R* int 2YDV (2.6
diffusion A)
T.S mutations Sf9 Vapor ZM241385 (ANT) R 3PWH (3.3 Dore et al. [11]
diffusion A)
T.S mutations Sf9 Vapor XAC (ANT) R 3REY (3.3
diffusion A)
T.S mutations Sf9 Vapor Caffeine (ANT) R 3RFM (3.6
diffusion A)
T.S mutations Sf9 Vapor 6-(2,6-Dimethylpyridin-4-yl)-5-phenyl-1, 2,4-triazin-3- R 3UZA (3.2 Congreve et al.
diffusion amine (ANT) A) [12]
T.S mutations Sf9 Vapor 4-(3-Amino-5-phenyl-1, 2,4-triazin-6-yl)-2-chlorophenol R 3UZC (3.3
diffusion (ANT) A)
T.S mutations Sf9 Vapor ZM241385 (ANT) R 3VG9 (2.7 Hino et al. [13]
diffusion A)
BRIL-ICl3 Sf9 Lipid cubic ZM241385 (ANT) R 4EIY (1.8 Liu et al. [14]
phase A)
Fab P. pastoris Vapor 4-(2-[7-amino-2-(2-furyl)-[1,2,4]triazolo-[2,3-a] [1,3,5] R 3VGA (3.1 Hino et al. [13]
diffusion triazin-5-ylamino]ethyl)-phenol (ANT) A)
T.S mutations High five Vapor CGS21680 (AGO) R* 4UG2 (2.6 Lebon et al. [15]
diffusion A)
T.S mutations High five Vapor CGS21680 (AGO) R* 4UHR (2.6
diffusion A)
T.S mutations High five Vapor ZM241385 (ANT) R 5IU4 (1.7 Segala et al. [16]
diffusion A)
T.S mutations High five Vapor Modified ZM-compound 12 c (ANT) R 5IU7 (1.9
diffusion A)
C. Nasrallah and G. Lebon
T.S mutations High five Vapor Modified ZM-compound 12 f (ANT) R 5IU8 (2 A)
diffusion
T.S mutations High five Vapor Modified ZM-compound 12 b (ANT) R 5IU4 (2.2
diffusion A)
T.S mutations High five Vapor Modified ZM-compound 12 x (ANT) R 5IUB (2.1
diffusion A)
Binding High five Vapor NECA (AGO) R* 5G53 (3.4 Carpenter et al.
miniGs diffusion A) [17]
BRIL-ICl3 Sf9 Lipid cubic ZM241385 (ANT) R 5K2A (2.5 Batyuk et al. [18]
phase A)
BRIL-ICl3 Sf9 Lipid cubic ZM241385 (ANT) R 5K2B (2.5
phase A)
BRIL-ICl3 Sf9 Lipid cubic ZM241385 (ANT) R 5K2C (1.9
phase A)
BRIL-ICl3 Sf9 Lipid cubic ZM241385 (ANT) R 5K2D (1.9
phase A)
Angiotensin receptor BRIL (N) Sf9 Lipid cubic Olmestran (IAG) R* int 4ZUD (2.8 Zhang et al. [19]
(AT1R) phase A)
BRIL (N) Sf9 Lipid cubic ZD7155 (ANT) R 4YAY (2.9
phase A)
Angiotensin receptor BRIL (N) Sf9 Lipid cubic (N-benzyl-N-(2-ethyl-4-oxo-3-{[20 -(2H-tetrazol-5-yl) R 5UNF (2.8 Zhang et al.
(AT2R) phase [1,10 -biphenyl]-4-yl])methyl}-3,4-dihydroquinazolin-6- A) [20, 21]
yl)thiophene-2-carboxamide) (ANT)
BRIL (N) Sf9 Lipid cubic (N-[(furan-2-yl)methyl]-N-(4-oxo-2-propyl-3-{[2- R 5UNH (2.9 Zhang et al.
0
phase -(2H-tetrazol-5-yl)[1,10 -biphenyl]-4-yl]methyl}-3,4- A) [20, 21]
dihydroquinazolin-6-yl)benzamide) (ANT)
Class A rho- Beta-1 adrenergic T.S mutations High five Vapor Cyanopindolol (ANT) R 2VT4 (2.7 Warne et al. [22]
dopsin-like receptor diffusion A)
T.S mutations High five Vapor Dobutamine (PAG) R* int 2Y00 (2.5 Warne et al. [23]
diffusion A)
T.S mutations High five Vapor Dobutamine (PAG) R* int 2Y01 (2.6
Structures of Non-rhodopsin GPCRs Elucidated Through X-Ray Crystallography
diffusion A)
T.S mutations High five Vapor Carmoterol (AGO) R* 2Y02 (2.6
diffusion A)
T.S mutations High five Vapor Isoprenaline (AGO) R* 2Y03 (2.85
diffusion A)
T.S mutations High five Vapor Salbutamol (PAG) R* int 2Y04 (3.05
5
diffusion A)
(continued)
6
Table 1 (continued)
T.S mutations High five Vapor Carazolol (IAG) R* int 2YCW (3.0 Moukhametzianov
diffusion A) et al. [24]
T.S mutations High five Vapor Cyanopindolol (ANT) R 2YCX (3.25
diffusion A)
T.S mutations High five Vapor Cyanopindolol (ANT) R 2YCY (3.15
diffusion A)
T.S mutations High five Vapor Iodocyanopindolol (ANT) R 2YCZ (3.65
diffusion A)
T.S mutations High five Vapor Bucindolol (BAG) R* int 4AMI (3.2 Warne et al. [25]
diffusion A)
T.S mutations High five Vapor Carvedilol (BAG) R* int 4AMJ (2.3
diffusion A)
T.S mutations High five Vapor (APO) R* int 4GPO (3.5 Huang et al. [26]
diffusion A)
T.S mutations High five Vapor 4-Methyl-2-(piperazin-1-yl) quinolone (ANT) R 3ZPR (2.7 Christopher et al.
diffusion A) [27]
T.S mutations High five Vapor 4-(Piperazin-1-yl)-1H-indole (ANT) R 3ZPQ (2.8
diffusion A)
T.S mutations High five Lipid cubic Cyanopindolol (ANT) R 4BVN (2.1 Miller-Gallacher
phase A) et al. [28]
T.S mutations High five Vapor 7-Methylcyanopindolol (IAG) R 5A8E (2.4 Sato et al. [29]
diffusion A)
Beta-2 adrenergic Fab Sf9 Vapor Carazolol (IAG) R 2R4R (3.4 Rasmussen et al.
receptor diffusion A) [30]
Fab Sf9 Vapor Carazolol (IAG) R 2R4S (3.4
diffusion A)
T4L-ICl3 Sf9 Lipid cubic Carazolol (IAG) R 2RH1 (2.4 Cherezov et al.
phase A) [31]
T4L-ICl3 Sf9 Lipid cubic Timolol (IAG) R 3D4S (2.8 Hanson et al.
phase A) [132]
T4L-ICl3 Sf9 Lipid cubic ICI (118,551) (2S, 3S)-1-[(7-methyl-2,3-dihydro-1H- R 3NY8 (2.84 Wacker et al. [33]
phase inden-4-yl) oxy]-3-[(1-methylethyl) amino] butan-2-ol A)
(IAG)
T4L-ICl3 Sf9 Lipid cubic Ethyl-4-[2-hydroxy-3(isopropylamino)propoxy]-3- R 3NY9 (2.84
phase methyl-1-benzofuran-2-carboxylate (IAG) A)
Fab Sf9 Lipid cubic Carazolol (IAG) R 3KJ6 (3.4 Bokoch et al. [34]
phase A)
T4L-ICl3 Sf9 Lipid cubic Alprenolol (ANT) R 3NYA (3.16 Wacker et al. [33]
phase A)
T4L-ICl3 Sf9 Lipid cubic BI-167107 (AGO) R* int 3P0G (3.5 Rasmussen et al.
phase A) [35, 36]
T4L-ICl3 Sf9 Vapor FAUC50 (IAGO) R* int 3PDS (3.5 Rosenbaum et al.
diffusion A) [37]
T4L (N); Sf9 Lipid cubic BI-167107 (AGO) R* 3SN6 (3.2 Rasmussen et al.
Nb35; trimeric phase A) [35, 36]
Gp
T4L (N) Sf9 Lipid cubic Carazolol (IAG) R 4GBR (3.99 Zou et al. [38]
phase A)
T4L (N); Sf9 Lipid cubic BI-167107 (AGO) R* 4LDE (2.79 Ring et al. [39]
Nb6B9 phase A)
T4L (N); Sf9 Lipid cubic Hydroxybenzyl isoproterenol (AGO) R* 4LDL (3.1
Nb6B9 phase A)
T4L (N); Sf9 Lipid cubic Adrenaline (AGO) R* 4LDO (3.2
Nb6B9 phase A)
T4L (N); Sf9 Lipid cubic 4-[(1R)-1-Hydroxy-2-({2-[3-methoxy-4- R* 4QKX (3.3 Weichert et al.
Nb6B9 phase A) [40]
Class A rho- Lipid cubic (2-Sulfanylethoxy) phenyl] ethyl} amino) ethyl] ben-
dopsin- like phase zene-1,2-diol (AGO)
T4L-ICL3 Sf9 Lipid cubic Carazolol (IAG) R 5D5B (3.8 Huang et al. [41]
phase A)
T4L-ICL3 Sf9 Lipid cubic Carazolol (IAG) R 5D5A (2.48
phase A)
Nb60 Sf9 Lipid cubic Carazolol (IAG) R 5JQH (3.2 Staus et al. [42]
phase A)
Cannabinoid receptor Flavodoxin- HEk293F Lipid cubic AM6538 (ANT) R 5TGZ (2.8 Hua et al. [43]
(CB1) ICl3 ; T.S phase A)
mutations
PGS-ICl3 ; T.S Sf9 Lipid cubic Taranabant (ANT) R 5U09 (2.6 Shao et al. [44]
mutation phase A)
CC Chemokine
receptor
(CCR2) T4L-ICL3 Sf9 Lipid cubic BMS-681 (ANT) R 5T1A (2.81 Zheng et al. [45]
phase A)
(CCR5) Rubredoxine Sf9 Lipid cubic Maraviroc (IAG) R 4MBS (2.71 Tan et al. [46]
phase A)
7
(CCR9) T.S mutations Sf9 Lipid cubic Vercirnon (ANT) R 5LWE (2.8 Oswald et al. [47]
phase A)
(continued)
Table 1 (continued) 8
(CXCR4) T.S mutations; Sf9 Lipid cubic IT1t (Isothiourea derivative) (ANT) R 3OE6 (3.2 Wu et al. [48]
T4L-ICL3 phase A)
T.S mutations; Sf9 Lipid cubic IT1t (Isothiourea derivative) (ANT) R 3ODU (2.5
T4L-ICL3 phase A)
T.S mutations; Sf9 Lipid cubic IT1t (Isothiourea derivative) (ANT) R 3OE8 (3.1
T4L-ICL3 phase A)
T.S mutations; Sf9 Lipid cubic IT1t (Isothiourea derivative) (ANT) R 3OE9 (3.1
T4L-ICL3 phase A)
T.S mutations; Sf9 Lipid cubic CVX15r (ANT) R 3OE0 (2.9
T4L-ICL3 phase A)
n.d Sf9 Lipid cubic vMIP-II (ANT) R 4RWS (3.1 Qin et al. [49]
phase A)
Cytomegalovirus Disulfide Sf9 Lipid cubic Fractalkine -CX3CL1 (Ago) R 4XT1 (2.89 Burg et al. [50]
US28 engineering phase A)
Dopamine receptor T.S Mutations; Sf9 Lipid cubic Eticlopride (ANT) R 3PBL (2.89 Chien et al. [51]
3 (D3R) T4L-ICL3 phase A)
Endothelin receptor B T.S mutations; Sf9 Lipid cubic Endothelin 1 (Ago) R* int 5GLH (2.8 Shihoya et al. [52]
T4L-ICL3 phase A)
T.S mutations; Sf9 Lipid cubic Apo Apo 5GLI (2.5
T4L-ICL3 phase A)
T.S mutations; Sf9 Lipid cubic TAK-875 (Fasiglifam) (PAG) R* int 4PHU (2.33 Srivastava et al.
T4L-ICL3 phase A) [53]
Histamine receptor T4L-ICL3 Sf9 Lipid cubic Doxepin (ANT) R 3RZE (3.1 Shimamura et al.
1 (H1R) phase A) [54]
Lysophosphatidic acid BRIL-ICL3 Sf9 Lipid cubic ONO9780307 (ANT) R 4Z34 (3.0 Chrencik et al.
receptor (LPA1) phase A) [55]
BRIL-ICL3 Sf9 Lipid cubic ONO-9910539 (ANT) R 4Z35 (2.9
phase A)
Disulfide engi- Sf9 Lipid cubic ONO-3080573 (ANT) R 4Z36 (2.9A)
neering; BRIL- phase
ICL3
M1 muscarinic acetyl- T4L-ICL3 Sf9 Lipid cubic Tiotropium (IAG) R* int 5CXV (2.7 Thal et al. [56]
choline receptor phase A)
(CHRM1)
Class A rho- M2 muscarinic acetyl- T4L-ICL3 Sf9 Lipid cubic 3-Quinuclidinyl-benzilate (ANT) R 3UON (3.0 Haga et al. [57]
dopsin-like choline receptor phase A)
(CHRM2)
Nb9-8 Sf9 Lipid cubic Iperoxo (AGO) R* 4MQS (3.5 Kruse et al. [58]
phase A)
Nb9-8 Sf9 Lipid cubic Iperoxo +LY2119620p (AGO) R* 4MQT (3.7
phase A)
M3 muscarinic T4L-ICL3 Sf9 Lipid cubic Tiotropium (IAG) R* int 4DAJ (3.4 Kruse et al. [58]
Acetylcholine phase A)
Receptor (CHRM3)
T4L-ICL3 Sf9 Lipid cubic Tiotropium (IAG) R* int 4U14 (3.57 Thorsen et al. [59]
phase A)
T4L-ICL3 Sf9 Lipid cubic Tiotropium (IAG) R* int 4UI5 (2.8
phase A)
T4L-ICL3 Sf9 Lipid cubic N-methylscopolamine NMS (ANT) R 4U16 (3.7
phase A)
M4 muscarinic mT4L-ICL3 Sf9 Lipid cubic Tiotropium (IAG) R* int 5DSG (2.6 Thal et al. [56]
Acetylcholine phase A)
Receptor (CHRM3)
Neurotensin receptor T.S mutations; High five Lipid cubic NTS8-13q (AGO) R* int 4GRV (2.8 White et al. [60]
T4L-ICL3 phase A)
T.S mutations E. coli Vapor NT1 (Neurotensin) (AGO) R* int 3ZEV (3 A) Egloff et al. [6]
diffusion
T.S mutations E. coli Vapor NT1 (Neurotensin) (AGO) R* int 4BUO (2.75
diffusion A)
T.S mutations E. coli Vapor NT1 (Neurotensin) (AGO) R* int 4BV0 (3.1
diffusion A)
T.S mutations E. coli Vapor NT1 (Neurotensin) (AGO) R* int 4BWB
diffusion (3.57 A)
T.S mutations; High five Lipid cubic NTS8-13q (AGO) R* int 4XES (2.6 Krumm et al. [61]
T4L-ICL3 phase A)
T.S mutations; High five Lipid cubic NTS8-13q (AGO) R* int 4XEE (2.9
T4L-ICL3 phase A)
nd nd nd NT1 (Neurotensin) (AGO) R* int 5T04 (3.3 Krumm et al. [62]
A)
Nociceptin/orphanin BRIL (N) Sf9 Lipid cubic 1-Benzyl-N-(3-[spiroisobenzofuran-1(3H),40 -piperidin- R 4EA3 (3.01 Thompson et al.
FQ receptor phase 1-yl]propyl)pyrrolidine-2-carboxamide (ANT) A) [63]

(NOP/ORL-1)
BRIL (N) Sf9 Lipid cubic SB-612111 (ANT) R 5DHH (3.0 Miller et al. [64]
phase A)
BRIL (N) Sf9 Lipid cubic C-35 (ANT) R 5DHG (3.0
phase A)
9
Orexin receptor type PGS domain- Sf9 Lipid cubic Suvorexant (ANT) R 4ZJ8 (2.75 Yin et al. [65]
1 (Ox1) ICL3 phase A)
(continued)
Table 1 (continued)
10
SB-674042 (ANT) R 4ZJC (2.83

A)
Orexin receptor type PGS domain- Sf9 Lipid cubic Suvorexant (ANT) R 4S0V (2.5 Yin et al. [66]
2 (Ox2) ICL3 phase A)
κ-Opioid receptor T4L-ICL3 Sf9 Lipid cubic (3R)-1,2,3,4-tetrahydro-7-hydroxy-N-[(1S)-1-[[(3R, 4R)- R 4DJH (2.9 Wu et al. [67]
phase 4-(3-hydroxyphenyl)3,4-dimethyl-1-piperidinyl] methyl] A)
2-methylpropyl]-3-isoquinolinecarboxamide) (ANT)
μ-Opioid receptor T4L-ICL3 Sf9 Lipid cubic β-Funaltrexamine (β-FNA) (ANT) R 4DKL (2.8 Manglik et al.
phase A) [68]
dsT4L-ICL3 Sf9 Lipid cubic Tiotropium (ANT) R 4U14 (3.57 Thorsen et al. [59]
phase A)
mT4L-ICL3 Sf9 Lipid cubic Tiotropium (ANT) R 4U15 (2.8
phase A)
mT4L-ICL3 Sf9 Lipid cubic NMS (ANT) R 4U16 (3.7 Thorsen et al. [59]
phase A)
δ-Opioid receptor Fab Sf9 Lipid cubic BU72 (AGO) R* 5C1M (2.1 Huang et al. [69]
phase A)
nd Sf9 Lipid cubic DIPP-NH2 (AGO) R* int 4RWD (2.7 Fenalti et al. [70]
phase A)
T4L-ICL3 Sf9 Lipid cubic Naltrindole (ANT) R 4EJ4 (3.4 Granier et al. [71]
phase A)
BRIL (N) Sf9 Lipid cubic Naltrindole (ANT) R 4N6H (1.8 Fenalti et al. [72]
phase A)
n.d Sf9 Lipid cubic DIPP-NH2 (ANT) R 4RWA Fenalti et al. [70]
phase (3.28 A)
Protease-activated T4L-ICL3 Sf9 Lipid cubic Vorapaxar (ANT) R 3VW7 (2.2 Zhang et al. [73]
receptor1 phase A)
Purinoceptor BRIL-ICL3 Sf9 Lipid cubic AZD1283:Ethyl 6-(4-((benzylsulphonyl) carbamoyl) R 4NTJ (2.62 Zhang et al. [74]
12 (P2Y12) phase piperidin-1-yl)-5-cyano-2-methylnicotinate (ANT) A)
BRIL-ICL3 Sf9 Lipid cubic 2MeSADP: 2-methylthio-adenosine-50 -diphosphate R* int 4PXZ (2.5
phase (AGO) A)
BRIL-ICL3 Sf9 Lipid cubic 2MeSATP: methylthio-adenosine-50 -triphosphate (PAG) R* int 4PY0 (3.1
phase A)
Purinoceptor 1 (P2Y1) Rubredoxine- Sf9 Lipid cubic MRS2500 (ANT) R 4XNW (2.7 Zhang et al. [19]
ICL3 phase A)
Rubredoxine- Sf9 Lipid cubic BPTU (ANT) R 4XNV (2.2
ICL3 phase A)
Serotonin BRIL-ICL3; T. Sf9 Lipid cubic Ergotamine (AGO) R* int 4IAR (2.7 Wang et al. [75]
(5-hydroxytryptamine) S mutations phase A)
type 1b receptor
BRIL-ICL3; T. Sf9 Lipid cubic Dihydroergotamine (AGO) R* int 4IAQ (2.8
S mutations phase A)
Serotonin BRIL-ICL3; T. Sf9 Lipid cubic Ergotamine (BAG) R* int 4IB4 (2.7 Wacker et al. [76]
(5-hydroxytryptamine) S mutations phase A)
type 2b receptor
BRIL-ICL3; T. Sf9 Lipid cubic Ergotamine (BAG) R* int 4NC3 (2.8 Liu et al. [77]
S mutations phase A)
T.S mutations Sf9 Lipid cubic LSD (AGO) R* int 5TVN (2.9 Walker et al. [78]
phase A)
Sphingosine-1-phos- T4L-ICL3 Sf9 Lipid cubic ML056: (R)-3-amino-(3-hexyl phenylamino)-4-oxobutyl R 3V2W (3.35 Hanson et al. [79]
phate receptor phase phosphonicacid (ANT) A)
R 3V2Y (2.8
A)
Class B Glucagon receptor BRIL (N) Sf9 Lipid cubic NNC0640: 4-[1-(4-Cyclohexylphenyl) R 4L6R (3.4 Siu et al. [80]
secretin like phase 3-(3-methanesulfonyl phenyl) ureidomethyl]-N- A)
(2H-tetrazol-5-yl) benzamide (ANT)
T4L-ICL2; T.S Sf21 Lipid cubic MK-0893 (ANT) R 5EE7 (2.5 Jazayeri et al. [81]
mutations phase A)
Corticotropin-releasing T4L-ICL2; T.S Sf9 Lipid cubic CP-376395: 3,6-dimethyl-N-pentan-3-yl 2-(2,4,6- R 4K5Y (2.98 Hollenstein et al.
factor receptor 1 mutations phase trimethyl phenoxy)pyridin-4-amine (ANT) A) [82]
Class C Metabotropic gluta- BRIL (N) Sf9 Lipid cubic FITMs: 4-fluoro-N-(4-(6-(isopropylamino)pyrimidin4- R 4OR2 (2.8 Wu et al. [83]
metabotropic mate receptor type 1 phase yl) thiazol-2-yl) N-methylbenzamide (ANT) A)
glutamate Metabotropic gluta- T4L-ICl2; T.S Sf21 Lipid cubic Mavoglurants: Methyl (3aR,4S,7aR)-4-hydroxy-4- R 4OO9 (2.6 Dore et al. [84]
mate receptor type 5 mutations phase [(3-methylphenyl) ethynyl] octahydro-1H-indole A)
1-carboxylate (ANT)
T4L-ICl2; T.S Sf21 Lipid cubic 3-Chloro-4-fluoro-5-[6-(1H-pyrazol-1-yl)pyrimidin-4-yl] R 5CGC (3.1 Christopher et al.
mutations phase benzonitrile (ANT) A) [27]

T4L-ICl2; T.S Sf21 Lipid cubic HTL14242:3-chloro-4-fluoro-5-[6-(5-fluopyridin-2-yl) R 5CGD (2.6
mutations phase pyrimidin-4-yl]benzonitrile (ANT) A)
(continued)
11
Table 1 (continued)
12
Class F Smoothened receptor BRIL (N) Sf9 Lipid cubic LY2940680: 4-fluoro-N-methyl-N-(1-(4-(1-methyl-1H- R 4JKV (2.45 Wang et al. [75]
Frizzeled (SMO) phase pyrazol-5yl)phthalazin-1-yl)piperidin-4-yl)2- A)
(trifluoromethyl)benzamide (ANT)
BRIL (ICl3) Sf9 Lipid cubic Cyclopamine (ANT) R 4O9R (3.2 Weierstall et al.
phase A) [86]
BRIL (N) Sf9 Lipid cubic SANT1 ((N-[(1E)-(3,5-dimethyl-1phenyl-1H-pyrazol-4- R 4N4W (2.8 Wang et al. [87]
phase yl) methylidene]-4(phenylmethyl)-1-piperazinamine) A)
(ANT)
BRIL (ICl3) Sf9 Lipid cubic R 4QIM (2.6
phase A)
Anta XV: (2-(6-(4-(4-benzylphthalazin-1-yl) piperazin-
1-yl)pyridin-3-yl)propan-2-ol) (ANT)
BRIL (ICl3) Sf9 Lipid cubic SAG1.5: (3-chloro-4,7-difluoro-N-[trans-4- R*int 4QIN (2.6
phase (methylamino) cyclohexyl]-N-[[3-(4-pyridinyl) phenyl] A)
methyl]-1-benzothio phene-2-carboxamide) (ANT)
BRIL (ICl3) Sf9 Lipid cubic Vismodegib (ANT) R 5L7I (3.3 A) Byrne et al. [88]
phase
BRIL (ICl3) Sf9 Lipid cubic Vismodegib (ANT) R 5L7D (3.2
phase A)
IAG Inv agonist, ANT antagonist, AGO agonist, PAG partial agonist, APO no ligand, IAGO irreversible agonist, BAG biased agonist, Nb nanobody, Fab
antigen binding fragment of antibody, ICL intracellular loop, PGS Pyrococcus abyssi glycogen synthase, T.S Mutations thermostabilized mutations, T4L T4
lysosyme fusion protein mT4L, dsT4L stabilized versions of T4L, BRIL apo-cytochrome b562RIL fusion protein Sf9, Sf21 Spodoptera frugiperda cell line,
High five Trichoplusia ni cell line
Additionally, the lipidic membrane composition is very different for each cell-type
(bacteria, yeast or mammalian) and can impact the function of expressed receptors
[89]. Finally, post-translational modifications, mentioned above, that including
glycosylations and phosphorylations are essential for obtaining a mature functional
receptor in membrane. For instance, it has been reported that N-linked glycosyla-
tion has a major impact on receptor folding in the endoplasmic reticulum and its
trafficking to the plasma membrane [90] while phosphorylation by G protein
Receptor Kinases is mandatory for receptor internalization and desensitization
[91]. It is worthy to mention that not all GPCRs expressed in insect cells are
properly folded, some receptors show considerable misfolded proportion and inca-
pable of ligand binding as compared to expression in inducible stable mammalian
cell lines [92]. Nevertheless, improvements in recombinant baculovirus have been
made allowing their use for mammalian cell infection and subsequent protein
production (BacMam, Invitrogen) [93]. Although sparsely used for structural
study to date, mammalian expression systems start to emerge as an efficient way
to produce fully functional GPCRs in sufficient amounts for structural studies
[94]. For instance, bovine rhodopsin structure was solved using this approach in
two different cell lines, COS7 [95] and HEK293-GnTi [96, 97]. It is worthy to
mention that ligands complementation during expression often enhances the final
GPCR yield in the membrane, in particular for antagonists or inverse agonists.
Binding of the ligand within the transmembrane domain composed of 7 helices
(7TM) stabilizes the inactive state of the receptor and acts as a chemical chaperone
for improving the quantity of expressed and functional receptor at the cell surface
[98]. In contrast agonist addition could adversely affect receptor expression level by
triggering signalling cascade or internalization [99] (Fig. 1).
3 Solubilization of GPCRs Prior to Crystallization
Before proceeding to purification that is required to set up crystallization trials,

GPCRs need first to be extracted from their natural lipid environment using deter-
gents. These amphiphatic molecules are capable of disrupting the membrane at
concentrations higher than their defined critical micelle concentration (cmc) and of
dispersing its components in the form of detergent-solubilized particles. Despite their
solubilizing efficiency, detergents remain poor substitute to the membrane bilayer
environment. Although a wide panel of synthetic detergents are available commer-
cially, only few of them have been successful to maintain functional and viable
GPCRs enough time to go through purification and/or crystallization trials. For
instance, n-Dodecyl β-D-maltoside (DDM), a mild non-ionic detergent with large
hydrophilic head and 12-carbon long hydrophobic tail was the detergent of choice to
extract a wide variety of well-folded GPCRs. Moreover, different studies show
improvement in stability when DDM was supplemented with additional lipids such
as cholesteryl hemisuccinate (CHS) [100]. This is not surprising since many solved
GPCR structures highlighted specific cholesterol binding sites within helices from
transmembrane domain [32]. Lauryl maltose neopentyl glycol (MNG3), another
detergent synthetized based on DDM scaffold with further constraints on detergent

conformation (i.e. two hydrophilic and two lipophilic subunits linked by a central
quaternary carbon) also showed improved solubility and stability of GPCRs in recent
case studies [101]. For instance, the measured thermal stability for ß2-adrenergic
receptor was higher when solubilized with MNG3 as compared to DDM [101]. Of
interest MNG3 provides stability to solubilized GPCRs even in absence of choles-
terol derivatives. It is important to mention that a detergent with good solubilizing
proprieties is not necessarily suitable for crystallization. Indeed, this depends on the
choice of crystallization methods. In fact, GPCR crystals were obtained mainly from
two approaches: the vapour diffusion method (in surfo) and the lipidic cubic phase
method (LCP) (Table 1). When considering the conventional in surfo method, the
choice of detergent is crucial and working with mild detergents that maintain the
receptor native structure and function is important. Unfortunately, mild detergents
such as DDM and MNG3 form large micelles that often occlude the hydrophilic part
of the receptor making difficult to promote protein–protein interactions in crystal
lattice while detergent forming smaller micelles are often denaturating for GPCRs
[102]. Therefore when balance between shielding the hydrophobic domain of the
target GPCR from the aqueous environment and fostering interactions between the
hydrophilic domains fails to be achieved, other engineering strategies aiming at
enhancing GPCR stability in detergent-solubilized conditions need to be considered
such as increasing receptor thermostability by introducing point mutations (see
Sect. 4.4). For example, the thermostabilized Adenosine (A2A), ß1-adrenergic recep-
tor (ß1AR) and neurotensin (NT1) receptors were solved in detergent micelles
(Table 1).
For LCP crystallization, the detergent-micelles surrounding the receptor are
exchanged with a lipidic matrix providing a more native-like environment
[103]. Briefly, the bicontinuous cubic phase (i.e. both the aqueous and bilayer
compartments are continuous in three dimensions) is formed when lipids such as
monoacylglycerol (MAG) and water are mixed at a given ratio. The highly curved
bilayer of the cubic phase could then host the target receptor. Under conditions
leading to crystallization, the receptor could freely diffuse within the bilayer
promoting nucleation and crystallization. Following this approach, large detergent
micelles including DDM and MNG3 could be accommodated for receptor solubi-
lization and subsequent purification. Although monoolein (MAG9.9) is the main
component of the lipidic matrix for in meso crystallization, other additives includ-
ing cholesterol derivatives have shown utility to furthermore improve GPCR
stability and thereby increasing their probability to crystallize in lipidic environ-
ment. To date, the majority of GPCR structures were successfully solved following
this approach (Table 1). Interestingly, the structure of the ß2-adrenergic receptor in
complex with heterotrimeric Gs protein was crystallized with a short-chain MAG,
the 2,3-dihydroxypropyl-(7Z)-tetradec-7-enoate (MAG7.7) supplemented with
cholesterol [35]. This suggests that several lipid composition for in meso crystalli-
zation should be tested and optimized for each target, especially when considering
GPCR signalling complexes.
4 Enhancing GPCRs Crystallizablity
GPCRs are sophisticated allosteric machineries that signal through multiple path-
ways once interacting with specific ligands. Indeed, the signalling mechanism relies
on the intrinsic dynamic properties of the receptor that oscillates between different
fluctuating conformational states [37, 97, 104]. For X-ray crystallography, the
protein conformation with lower energy (i.e. more stable) is the most likely to be
captured. Therefore, GPCRs are challenging for crystallogenesis and it is difficult to
obtain high-resolution structures of the several conformation state of a receptor
constituting is activation cycle. Accordingly, it is required to develop surrogates
for shifting the energy landscape to the thermodynamically most favourable confor-
mations, i.e. inactive (R) or fully active states (R*) [10]. We will describe the
successful approaches that have been used to solve GPCR structures in the past
decade. Indeed, the first structure of a diffusible ligand GPCR was solved in 2007
[31]. Nowadays, more than 130 non-rhodopsin GPCR structures are available in the
protein data bank (PDB) representing a wide variety of individual receptors that
belong to different GPCR classes (Table 1 and Fig. 2).
4.1 Sample Homogeneity
4.1.1 Ligands Screening
GPCRs are targets for approximately 30–50% of drugs currently on the market. This
in-depth characterization of the binding pocket from X-ray crystallographic GPCR
Fig. 2 A schematic representation of the different GPCR classes highlighting the structural
differences. From the left to the right, class A GPCR family harbours an orthosteric-binding site
within the transmembrane domain (7TM) where endogenous ligand is recognized. For class B, the
ligand (i.e. peptide) binds to both the extracellular (ECD) and 7TM domains. Unlike Class A and B
GPCRs, the orthosteric binding for class C receptors is located in the N-terminal extracellular
bi-lobed domain (VFT) and not within the 7TM. The large VFT is connected via a cysteine-rich
region to the 7TM domain. Class F receptors also possess a cysteine-rich region where endogenous
ligand (lipoprotein) binds. Endogenous ligands are highlighted in red
structures available in the protein data bank (PDB) database facilitates the GPCR
structure-based drug design [105]. To date, all the solved structures of GPCRs have a
ligand bound, either in the orthosteric or allosteric binding sites except for the ligand-
free structure of opsin [106] (Table 1; Fig. 2). This is not surprising, since in the free
state, GPCRs are thought to adopt different conformations ranging from the R to R*
states making them unstable and thus difficult to crystallize. Therefore, ligand
binding is a prerequisite for receptor stabilization, which helps reducing conforma-
tional flexibility by driving receptors to adopt one major population [19]. Indeed, the
ideal ligand should have the highest affinity with an extremely slow dissociation rate,
which reflects a tight binding to the receptor, especially when dealing with the fully
active agonist-bound conformation [35, 36]. Nevertheless, it is often difficult to
obtain the ideal ligand commercially and screening synthetic derivatives need to be
considered. In addition, ligands with different branching substituents may form
additional interactions in the binding pocket making the ligand-receptor complex
more stable thus better for structural studies. For instance, the structure of the A2A
receptor co-crystallized with the high affinity agonist UK-432097 reveals a variety of
molecular interactions such as salt bridges, hydrogen bonding, and aromatic-ring
stacking as well as non-polar interactions [9].
4.1.2 Deglycosylation
GPCRs require post-translational modifications for proper folding and correct traf-
ficking to the plasma membrane. In fact, the sequence prediction for the majority of
GPCRs reveals at least one N-linked glycosylation site. Glycan moieties are hetero-
geneous and flexible in nature, hence hardly compatible with crystallization, and
removing them is often detrimental to successful crystallization. This could be
achieved either by single point mutations or by enzymatic de-glycosylation. Both
approaches do not guarantee the optimal way to deal with the problem, since
mutations might affect the receptor expression and/or folding while enzymatic
digestion may be incomplete due to enzymatic steric inaccessibility. To circumvent
these limitations, N-glycosylation defective cell lines are available, both for HEK293
[107] and CHO [108]. For instance, the HEK293 N-actetylglucosaminetransferase I
(GnTI-) cell line lacking the N-actetylglucosaminetransferase I activity produces
proteins with controlled length sugar unit (GlcNAc2Mn5) [107]. This alternative
expression system ensures a well-folded and homogenous receptor preparation
suitable for crystallization [96, 97].
4.1.3 N and C Termini Domains
GPCRs exhibit remarkably conserved protein architecture with seven transmembrane

helices despite their large structural and pharmacological diversity [109]. Indeed,
receptors from class B and C have a long N-terminal extracellular domain, which
shares (class B) or contains (class C), the orthosteric binding site (Fig. 2). The length
of the intracellular C-terminal tail is also variable ranging from few amino acids to
long variants among receptors belonging to the same class [110]. To date, only few
GPCRs were crystallized with both N- and C-termini domains intact; the human
dopamine D3 [51], the M2 muscarinic acetylcholine [57], GPR40 [53] and P2Y12 [74]
receptors (Table 1). While these domains play an important role in the activation
process, they remain highly flexible and may hinder crystallization. Therefore,
removing such domains by serial truncations while preserving the coupling to G
proteins and ligand-binding activity of the receptor is often considered. Despite that
significant portions of unstructured extracellular and/or intracellular tails were
deleted in the majority of crystallized GPCRs, additional approaches were also
required in order to achieve diffracting quality crystals including the use of binding
partners, fusion proteins and stabilizing point mutations as described below.
4.2 Binding Partners Stabilizing Receptor Conformation
Binding partners with high affinity for GPCRs such as antibody fragments (Fab) or
nanobodies are powerful tools that have shown their efficiency to lock the receptor in
a single conformational state [13, 111]. As a consequence, receptor dynamics is
significantly reduced. Fabs have even been successfully used to reduce the flexibility
of large extracellular domain of class B Glucagon receptor and constrain loop
conformation [112]. Thus, they facilitate well-ordered crystal formation by acting
as crystallogenesis chaperones. Nanobodies present different advantages regarding
conventional monoclonal antibodies [113]. Camelids naturally express a subtype of
antibodies that are devoid of light chains and called heavy chain antibodies (HcAbs).
HcAbs harbour a single variable domain (called VHH) recognizing the epitope on
the target protein. These small domains (13 kDa), also called single domain anti-
bodies (sdAbs) or nanobodies, are easily produced in bacteria or yeast. Moreover,
they have an extensive antigen-binding repertoire, superior stability as compared to
conventional antibodies and epitope recognition even in small cavities of proteins
making them logical candidates for stabilizing specific GPCR conformations
[114]. The structures of both A2A Adenosine and ß2 Adrenergic receptors were
solved in the inactive state bound to Fab fragments [8] while the structure of ß2
Adrenergic receptor was solved in the active state bound to a trimeric G-protein and
nanobodies [35]. It is worthy to mention that nanobodies have shown major utility to
stabilize fully active conformation of M2 muscarinic acetylcholine and ß2 Adrener-
gic receptor, by mimicking the G protein [36, 58]. Indeed the nanobody-stabilized
active conformation is identical to the ß2 Adrenergic receptor conformation solved in
complex with heterotrimeric G protein Gαsß1γ2 (Table 1). In addition, to favour the
production of nanobodies for a given state of the GPCR, the immunization of a
camelid (mostly camels or llamas) should be done with purified target receptor
stabilized in that given state. Thus, optimal condition for nanobody generation will
require stabilizing the selected receptor conformation using, for example, a high
affinity ligands, ideally with a very slow Koff, to prevent dissociation from the
receptor following immunization process and to guarantee optimal receptor confor-

mation stability in its reconstituted form (e.g. liposome) [114]. For instance, the
agonist BI-167107 used to solve the structure of ß2 Adrenergic receptor in active
state, has an affinity of 84 pM and an extremely slow off-rate of 30 h [35] and the
receptor detergent was exchanged to MNG3 prior to nanobodies screening.
4.3 Increasing Receptor Hydrophilic Area
Highly crystallizable soluble proteins replacing disordered segments within

GPCRs have been widely used to obtain well-ordered diffracting crystals
(Table 1). Indeed, among potential fusion partners, T4 lysozyme (T4L) was
mostly used to replace the long unstructured intracellular loop 3 (ICL3) of mainly
class A GPCRs without affecting its relative orientation to the membrane
(Table 1). The T4L consists of two alpha helices perpendicular to each other
and forms crystals under a wide range of conditions. Recently, two versions of
T4L were engineered in order to improve the diffraction quality of GPCR crystals
[59]. This was achieved either by introducing disulphide-stabilized point muta-
tions (dsT4L) or by replacing the flexible N-terminal domain of T4L by a small
linker (mT4L), as highlighted for the structure of muscarinic M3 receptor
[59]. Interestingly, the 7TM domain of both class B corticotropin-releasing factor
1 (CRF1) [82] and class C metabotropic glutamate (mGlu5) [84] receptors was
solved with T4L lysozyme fused to their second intracellular loop. Moreover, the
T4L was also successfully used to replace the truncated N terminus domain as
highlighted for ß2-adrenoreceptor [38]. In addition, this construct helped to solve
the structure of the active conformation of the ß2-Adrenergic receptor in complex
with intracellular binding partners such as Gs protein [36]. Other fusion proteins
were also explored, such as the thermostabilized apocytochrome b562 (BRIL)
replacing either the ICL3 (e.g. the structure of the A2A; 5-HT1B; 5HT2B; P2Y12
and SMO receptors) or the truncated version of N-terminus domain (e.g. the
structure of the NOP; glucagon; SMO; mGlu1 and δ-Opioid receptors) [115]
(Table 1). BRIL consists of two adjacent alpha helices anti-parallel to each
other. It is worthy to mention that other potential fusion partners appeared as
valid alternatives in few GPCR cases such as rubredoxin (Rd) for the CCR5
receptor [46] and the acid catalytic domain of Pyrococcus abysii glycogen
synthase (PGS) for the OX2 receptor [66]. Another advantage for this approach
for conventional vapour diffusion crystallogenesis method is that fusion proteins
could serve as crystallogenesis chaperones by increasing the hydrophilic area of
the target GPCR such that large detergents could still be accommodated. How-
ever, GPCRs are often unstable in detergent-solubilized conditions and it is
required to couple other stabilization approaches, precisely if vapour diffusion
technique is considered, such as increasing the thermostability of the receptor by
single point mutations as described below. Otherwise, crystallization from the
membrane-mimetic environment (LCP), where most GPCR structures to date
were obtained, has proven to be a powerful alternative to in surfo crystallization

(Table 1).
4.4 Increasing Receptor Thermal Stability
Another successful approach to stabilize GPCRs for structural studies consists of

introducing single point mutations (e.g. alanine mutations at first, or leucine if the
residue is already an alanine), within the 7TM in order to increase the receptor
stability in detergent-solubilized conditions [116]. This approach named confor-
mational thermostabilization relies on the availability of a radioligand with high
affinity for the target in order to measure the receptor binding from unpurified
detergent-solubilized conditions. Such assay allows screening mutant libraries for
identifying thermostabilizing mutations. Once identified, thermostable mutants can
be combined in order to generate an optimally stable receptor which enhances the
probability of obtaining high-quality diffracting crystals in both LCP and vapour
diffusion methods. Structures of the inactive-like state GPCRs (e.g. B1-AR; A2AR;
CCR5; FFA1R; CCR5, CCR9, Glucagon and mGlu5) as well as of the active-like
state (e.g. A2AR and NTSR1) were solved following this approach (see Table 1 for
more details). Interestingly structures solved either by the thermostabilizing
approach or by using fusion protein partner were identical with some differences
mainly the orientation of the loops which impact crystal packing [117]. The
radioligands used to measure binding and to screen the mutants bind in the
transmembrane domain. Interestingly, depending on the nature of the radioligand
used, agonist or antagonist, the thermostabilization appears to alter the equilibrium
between the inactive and active-like states such that the receptors will adopt
preferentially either the inactive state upon antagonist binding or the active states
upon agonist binding [11, 22]. Coupled to structure resolution, this strategy may
uncover molecular transitions between inactive and active-like states. For instance,
the conformational thermostabilization of A2A receptor using [3H]-ZM241385
antagonist led to a themostabilized A2A Star® receptor with reduced affinity for
the agonist and no coupling to G-proteins while similar affinities for the antagonist
were observed for both thermostablized and wild-type A2A receptors [11]. In
contrast, when A2A receptor was stabilized bound to [3H]-NECA agonist, the
affinity of the receptor for antagonist was considerably weaker while similar
affinity for the agonist was observed for both thermostablized and wild-type A2A
receptors [11]. High-resolution structure of A2A receptor ligand bound conforma-
tion reveals common features but also specificity for agonist and antagonist bind-
ing. Interestingly, the full-active conformation of the human A2A receptor was
recently reported in complex with an engineered minimal G protein (mini-Gs)
[118]. This structure is in perfect agreement with previous features revealed from
thermostabilized A2A agonist bound conformation [17]. In addition, following this
approach a large array of ligands could be co-crystallized for the same receptor
making it a desirable strategy for drug development. For instance, the
thermostabilized ß1-AR receptor was solved bound to a wide variety of ligands

including antagonists, inverse agonists, partial agonists and full agonists, giving a
detailed insight into the ligand-binding pockets and opening new possibilities for
hit-to-lead drug optimization [22–25, 27]. When required in difficult cases, this
approach could be combined to other stabilization approaches such as protein
fusion as highlighted in the structures of chemokine 4, dopamine D3, free fatty
acid, neurotensin, serotonin, corticotropin-releasing factor, metabotropic glutamate
5 receptors, and most recently the GLP1 structure (Table 1; Fig. 1).
5 GPCR Sub-Micrometer and Micrometer-Size Crystals
The main difficulty to solve GPCR structures is to obtain well-diffracting crystals of

a descent size, in order to collect data from standard source of photon at the
commonly accessible large synchrotron facilities. GPCRs often produce only
sub-micrometer to micrometer-size crystals making data collection another tricky
task to handle with care. In order to collect high-resolution data from GPCR micro-
crystals, microfocus X-ray beams with high intensity and sufficient exposure time
are required. However, such radiation with very high-energy dose induces radiation
damage and thus merging data from multiple micro-crystals is often required to
obtain a complete data set, but still perfectly achievable for descent-size crystals.
With recent technical progress and large effort by several groups, serial femto-
second crystallography (SFX) taking benefit from the free electron laser (FEL)
source has been developed and demonstrated great promise for obtaining high-
resolution data from nano to sub-micrometer-size GPCR crystals [119]. FEL is a
high-energy source (~2 mJ, 1012 photons per pulse) enabling the recording of data
from individual crystals with a specific orientation and minimal radiation damage
(ultrashort pulses <50 fs) [120]. In addition, LCP injector was developed in order to
collect data directly from randomly distributed crystals grown in the LCP. The
viscous phase is injected with a constant flow rate into a vacuum chamber at room
temperature where single-pulse diffraction pattern could be recorded [77]. Struc-
tures of GPCRs including AT2R and 5HT2c were successfully solved using this
setup [20, 77]. Receptor engineering step is still required in order to produce well-
ordered crystals Finally, this approach allowed solving the structure of difficult
complex such as rhodopsin bound to arrestin [121] and is promised to a great future.
6 Conclusion
All the implementation and developments in the area of GPCR crystallography

have opened new perspectives in GPCR structural biology. The large number of
GPCR high-resolution structures also gave access to previously unexploited poten-
tial for structure-based drug design. With continuously increasing number of GPCR
crystal structures since the first non-rhodopsin structure in 2007, the signalling
diversity of these receptors starts to be unveiled at the molecular level. However,
10 years down the line, a lot remains to be learned from these receptors and more
structures need to be solved for a complete understanding of the receptor-effector
coupling. Therefore it is not surprising if novel technological developments such as
smaller microfocus beamlines and serial femtosecond crystallography using free
electron laser will continue to emerge in the years to come, accelerating GPCR
structure solving.
Although the X-ray crystallography technique gives a snapshot view of the
receptor in a defined conformation, which is insightful for target based drug design,
complementary techniques are required to obtain valuable insights in the dynamic
proprieties of GPCRs. Indeed retrieving dynamic information on GPCRs is an
active research area and remains challenging for many reasons, some of which
are cited in this chapter. For instance, NMR studies of GPCRs require the produc-
tion of isotopically labelled receptor with very high amounts (milligrams), which is
challenging especially when the majority of GPCRs requires sophisticated heterol-
ogous systems for expression such as insect or mammalian cells while only a
minority of receptors could be produced in bacteria (see more details could be
found in the chapter describing NMR studies of GPCRs). Finally, single-particle
cryo-electron microscopy (EM) is currently gaining attention for its ability to solve
structures at high-resolution giving access to a wider set of receptor conformations
as compared to X-ray crystallography [122] but also for avoiding structure misin-
terpretation biased due to crystal packing in case of molecular crystallography.
Importantly, cryo-EM data collection requires only a small amount of purified
material without the need for an additional crystallization step. Recently, near
atomic resolution structures were obtained using cryo-EM from purified ternary
complex including calcitonin and GLP1, both class B receptors [21, 123]. Indeed,
this is the first time where the structure of a full-length GPCR was solved by cryo-
EM. Altogether, these implementations will help providing a better understanding
of GPCR biology at the molecular level and eventually accelerate the structure-
based drug design process for numerous human diseases.
Acknowledgments The authors acknowledge program ATIP (2013–2016), CNRS, FRM,

INSERM, and the university of Montpellier for their support.
References
1. Lagerstrom MC, Schioth HB (2008) Structural diversity of G protein-coupled receptors and

significance for drug discovery. Nat Rev Drug Discov 7:339–357
2. Rosenbaum DM, Rasmussen SG, Kobilka BK (2009) The structure and function of G-
protein-coupled receptors. Nature 459:356–363
3. Venkatakrishnan AJ et al (2016) Diverse activation pathways in class A GPCRs converge
near the G-protein-coupling region. Nature 536:484–487
4. Ghosh E, Kumari P, Jaiman D, Shukla AK (2015) Methodological advances: the unsung

heroes of the GPCR structural revolution. Nat Rev Mol Cell Biol 16:69–81
5. Park JH, Scheerer P, Hofmann KP, Choe HW, Ernst OP (2008) Crystal structure of the
ligand-free G-protein-coupled receptor opsin. Nature 454:183–187
6. Egloff P et al (2014) Structure of signaling-competent neurotensin receptor 1 obtained by
directed evolution in Escherichia coli. Proc Natl Acad Sci U S A 111:E655–E662
7. Glukhova A et al (2017) Structure of the adenosine A1 receptor reveals the basis for subtype
selectivity. Cell 168, 867–877 e813
8. Jaakola VP et al (2008) The 2.6 angstrom crystal structure of a human A2A adenosine
receptor bound to an antagonist. Science 322:1211–1217
9. Xu F et al (2011) Structure of an agonist-bound human A2A adenosine receptor. Science
332:322–327
10. Lebon G et al (2011) Agonist-bound adenosine A2A receptor structures reveal common
features of GPCR activation. Nature 474:521–525
11. Dore AS et al (2011) Structure of the adenosine A(2A) receptor in complex with ZM241385
and the Xanthines XAC and caffeine. Structure 19:1283–1293
12. Congreve M et al (2012) Discovery of 1,2,4-triazine derivatives as adenosine A(2A) antag-
onists using structure based drug design. J Med Chem 55:1898–1903
13. Hino T et al (2012) G-protein-coupled receptor inactivation by an allosteric inverse-agonist
antibody. Nature 482:237–240
14. Liu W et al (2012) Structural basis for allosteric regulation of GPCRs by sodium ions.
Science 337:232–236
15. Lebon G, Edwards PC, Leslie AG, Tate CG (2015) Molecular determinants of CGS21680
binding to the human adenosine A2A receptor. Mol Pharmacol 87:907–915
16. Segala E et al (2016) Controlling the dissociation of ligands from the adenosine A2A receptor
through modulation of salt bridge strength. J Med Chem 59:6470–6479
17. Carpenter B, Nehme R, Warne T, Leslie AG, Tate CG (2016) Erratum: structure of the
adenosine A2A receptor bound to an engineered G protein. Nature 538:542
18. Batyuk A et al (2016) Native phasing of x-ray free-electron laser data for a G protein-coupled
receptor. Sci Adv 2:e1600292
19. Zhang X, Stevens RC, Xu F (2015) The importance of ligands for G protein-coupled receptor
stability. Trends Biochem Sci 40:79–87
20. Zhang H et al (2017) Structural basis for selectivity and diversity in angiotensin II receptors.
Nature 544:327–332
21. Zhang Y et al (2017) Cryo-EM structure of the activated GLP-1 receptor in complex with a G
protein. Nature 546:248
22. Warne T et al (2008) Structure of a beta1-adrenergic G-protein-coupled receptor. Nature
454:486–491
23. Warne T et al (2011) The structural basis for agonist and partial agonist action on a beta(1)-
adrenergic receptor. Nature 469:241–244
24. Moukhametzianov R et al (2011) Two distinct conformations of helix 6 observed in antag-
onist-bound structures of a beta1-adrenergic receptor. Proc Natl Acad Sci U S A 108:8228–
8232
25. Warne T, Edwards PC, Leslie AGW, Tate CG (2012) Crystal structures of a stabilized beta
(1)-adrenoceptor bound to the biased agonists Bucindolol and Carvedilol. Structure 20:841–
849
26. Huang J, Chen S, Zhang JJ, Huang XY (2013) Crystal structure of oligomeric beta1-
adrenergic G protein-coupled receptors in ligand-free basal state. Nat Struct Mol Biol
20:419–425
27. Christopher JA et al (2013) Biophysical fragment screening of the beta1-adrenergic receptor:
identification of high affinity arylpiperazine leads using structure-based drug design. J Med
Chem 56:3446–3455
28. Miller-Gallacher JL et al (2014) The 2.1 A resolution structure of cyanopindolol-bound

beta1-adrenoceptor identifies an intramembrane Na+ ion that stabilises the ligand-free recep-
tor. PLoS One 9:e92727
29. Sato T et al (2015) Pharmacological analysis and structure determination of 7-
methylcyanopindolol-bound beta1-adrenergic receptor. Mol Pharmacol 88:1024–1034
30. Rasmussen SG et al (2007) Crystal structure of the human beta2 adrenergic G-protein-
coupled receptor. Nature 450:383–387
31. Cherezov V et al (2007) High-resolution crystal structure of an engineered human beta2-
adrenergic G protein-coupled receptor. Science 318:1258–1265
32. Hanson MA et al (2008) A specific cholesterol binding site is established by the 2.8 angstrom
structure of the human beta(2)-adrenergic receptor. Structure 16:897–905
33. Wacker D et al (2010) Conserved binding mode of human beta(2) adrenergic receptor inverse
agonists and antagonist revealed by X-ray crystallography. J Am Chem Soc 132:11443–
11445
34. Bokoch MP et al (2010) Ligand-specific regulation of the extracellular surface of a G-protein-
35. Rasmussen SG et al (2011) Crystal structure of the beta2 adrenergic receptor-Gs protein
complex. Nature 477:549–555
36. Rasmussen SG et al (2011) Structure of a nanobody-stabilized active state of the beta(2)
adrenoceptor. Nature 469:175–180
37. Rosenbaum DM et al (2011) Structure and function of an irreversible agonist-beta(2)
adrenoceptor complex. Nature 469:236–240
38. Zou Y, Weis WI, Kobilka BK (2012) N-terminal T4 lysozyme fusion facilitates crystalliza-
tion of a G protein coupled receptor. PLoS One 7:e46039
39. Ring AM et al (2013) Adrenaline-activated structure of beta(2)-adrenoceptor stabilized by an
engineered nanobody. Nature 502:575-+
40. Weichert D et al (2014) Covalent agonists for studying G protein-coupled receptor activation.
Proc Natl Acad Sci U S A 111:10744–10748
41. Huang CY et al (2016) In meso in situ In meso in situ serial X-ray crystallography of soluble
and membrane proteins at cryogenic temperatures. Acta Crystallogr D 72:93–112
42. Staus DP et al (2016) Allosteric nanobodies reveal the dynamic range and diverse mecha-
nisms of G-protein-coupled receptor activation. Nature 535:448–452
43. Hua T et al (2016) Crystal structure of the human cannabinoid receptor CB1. Cell 167:750–
762 e714
44. Shao Z et al (2016) High-resolution crystal structure of the human CB1 cannabinoid receptor.
Nature. doi:https://doi.org/10.1038/nature20613
45. Zheng Y et al (2016) Structure of CC chemokine receptor 2 with orthosteric and allosteric
antagonists. Nature 540:458–461
46. Tan Q et al (2013) Structure of the CCR5 chemokine receptor-HIV entry inhibitor maraviroc
complex. Science 341:1387–1390
47. Oswald C et al (2016) Intracellular allosteric antagonism of the CCR9 receptor. Nature
540:462–465
48. Wu B et al (2010) Structures of the CXCR4 chemokine GPCR with small-molecule and
cyclic peptide antagonists. Science 330:1066–1071
49. Qin L et al (2015) Structural biology. Crystal structure of the chemokine receptor CXCR4 in
complex with a viral chemokine. Science 347:1117–1122
50. Burg JS et al (2015) Structural biology. Structural basis for chemokine recognition and
activation of a viral G protein-coupled receptor. Science 347:1113–1117
51. Chien EY et al (2010) Structure of the human dopamine D3 receptor in complex with a D2/
D3 selective antagonist. Science 330:1091–1095
52. Shihoya W et al (2016) Activation mechanism of endothelin ETB receptor by endothelin-1.
Nature 537:363–368
53. Srivastava A et al (2014) High-resolution structure of the human GPR40 receptor bound to
allosteric agonist TAK-875. Nature 513:124–127
54. Shimamura T et al (2011) Structure of the human histamine H1 receptor complex with
doxepin. Nature 475:65–70
55. Chrencik JE et al (2015) Crystal structure of antagonist bound human lysophosphatidic acid
receptor 1. Cell 161:1633–1643
56. Thal DM et al (2016) Crystal structures of the M1 and M4 muscarinic acetylcholine
receptors. Nature 531:335–340
57. Haga K et al (2012) Structure of the human M2 muscarinic acetylcholine receptor bound to an
antagonist. Nature 482:547–551
58. Kruse AC et al (2013) Activation and allosteric modulation of a muscarinic acetylcholine
receptor. Nature 504:101–106
59. Thorsen TS, Matt R, Weis WI, Kobilka BK (2014) Modified T4 lysozyme fusion proteins
facilitate G protein-coupled receptor crystallogenesis. Structure 22:1657–1664
60. White JF et al (2012) Structure of the agonist-bound neurotensin receptor. Nature 490:508–
513
61. Krumm BE, White JF, Shah P, Grisshammer R (2015) Structural prerequisites for G-protein
activation by the neurotensin receptor. Nat Commun 6:7895
62. Krumm BE et al (2016) Structure and dynamics of a constitutively active neurotensin
receptor. Sci Rep 6:38564
63. Thompson AA et al (2012) Structure of the nociceptin/orphanin FQ receptor in complex with
a peptide mimetic. Nature 485:395–399
64. Miller RL et al (2015) The importance of ligand-receptor conformational pairs in stabiliza-
tion: spotlight on the N/OFQ G protein-coupled receptor. Structure 23:2291–2299
65. Yin J et al (2016) Structure and ligand-binding mechanism of the human OX1 and OX2
orexin receptors. Nat Struct Mol Biol 23:293–299
66. Yin J, Mobarec JC, Kolb P, Rosenbaum DM (2015) Crystal structure of the human OX2
orexin receptor bound to the insomnia drug suvorexant. Nature 519:247–250
67. Wu H et al (2012) Structure of the human kappa-opioid receptor in complex with JDTic.
Nature 485:327–332
68. Manglik A et al (2012) Crystal structure of the mu-opioid receptor bound to a morphinan
antagonist. Nature 485:321–U170
69. Huang WJ et al (2015) Structural insights into mu-opioid receptor activation. Nature
524:315-+
70. Fenalti G et al (2015) Structural basis for bifunctional peptide recognition at human delta-
opioid receptor. Nat Struct Mol Biol 22:265–268
71. Granier S et al (2012) Structure of the delta-opioid receptor bound to naltrindole. Nature
485:400–404
72. Fenalti G et al (2014) Molecular control of delta-opioid receptor signalling. Nature 506:191–
196
73. Zhang C et al (2012) High-resolution crystal structure of human protease-activated receptor
1. Nature 492:387–392
74. Zhang K et al (2014) Structure of the human P2Y12 receptor in complex with an
antithrombotic drug. Nature 509:115–118
75. Wang C et al (2013) Structure of the human smoothened receptor bound to an antitumour
agent. Nature 497:338–343
76. Wacker D et al (2013) Structural features for functional selectivity at serotonin receptors.
Science 340:615–619
77. Liu W et al (2013) Serial femtosecond crystallography of G protein-coupled receptors.
Science 342:1521–1524
78. Wacker D et al (2017) Crystal structure of an LSD-bound human serotonin receptor. Cell
168:377–389 e312
79. Hanson MA et al (2012) Crystal structure of a lipid G protein-coupled receptor. Science

335:851–855
80. Siu FY et al (2013) Structure of the human glucagon class B G-protein-coupled receptor.
Nature 499:444–449
81. Jazayeri A et al (2016) Extra-helical binding site of a glucagon receptor antagonist. Nature
533:274–277
82. Hollenstein K et al (2013) Structure of class B GPCR corticotropin-releasing factor receptor
1. Nature 499:438–443
83. Wu H et al (2014) Structure of a class C GPCR metabotropic glutamate receptor 1 bound to
an allosteric modulator. Science 344:58–64
84. Dore AS et al (2014) Structure of class C GPCR metabotropic glutamate receptor 5 trans-
membrane domain. Nature 511:557–562
85. Christopher JA et al (2015) Fragment and structure-based drug discovery for a class C GPCR:
discovery of the mGlu5 negative allosteric modulator HTL14242 (3-chloro-5-[6-(5-
fluoropyridin-2-yl)pyrimidin-4-yl]benzonitrile). J Med Chem 58:6653–6664
86. Weierstall U et al (2014) Lipidic cubic phase injector facilitates membrane protein serial
femtosecond crystallography. Nat Commun 5:3309
87. Wang C et al (2014) Structural basis for smoothened receptor modulation and
chemoresistance to anticancer drugs. Nat Commun 5:4355
88. Byrne EFX et al (2016) Structural basis of Smoothened regulation by its extracellular
domains. Nature 535:517–522
89. Jarvis DL, Finn EE (1995) Biochemical-analysis of the N-glycosylation pathway in
Baculovirus-infected Lepidopteran insect cells. Virology 212:500–511
90. Lanctot PM et al (2005) Importance of N-glycosylation positioning for cell-surface expres-
sion, targeting, affinity and quality control of the human AT1 receptor. Biochem J 390:367–
376
91. Pierce KL, Lefkowitz RJ (2001) Classical and new roles of beta-arrestins in the regulation of
G-protein-coupled receptors. Nat Rev Neurosci 2:727–733
92. Thomas J, Tate CG (2014) Quality control in eukaryotic membrane protein overproduction. J
Mol Biol 426:4139–4154
93. Kost TA, Condreay JP, Jarvis DL (2005) Baculovirus as versatile vectors for protein
expression in insect and mammalian cells. Nat Biotechnol 23:567–575
94. Andrell J, Tate CG (2013) Overexpression of membrane proteins in mammalian cells for
structural studies. Mol Membr Biol 30:52–63
95. Standfuss J et al (2007) Crystal structure of a thermally stable rhodopsin mutant. J Mol Biol
372:1179–1188
96. Standfuss J et al (2011) The structural basis of agonist-induced activation in constitutively
active rhodopsin. Nature 471:656–660
97. Deupi X et al (2012) Stabilized G protein binding site in the structure of constitutively active
metarhodopsin-II. Proc Natl Acad Sci U S A 109:119–124
98. Andre N et al (2006) Enhancing functional production of G protein-coupled receptors in
Pichia pastoris to levels required for structural studies via a single expression screen. Protein
Sci 15:1115–1126
99. Ferguson SS (2001) Evolving concepts in G protein-coupled receptor endocytosis: the role in
receptor desensitization and signaling. Pharmacol Rev 53:1–24
100. Thompson AA et al (2011) GPCR stabilization using the bicelle-like architecture of mixed
sterol-detergent micelles. Methods 55:310–317
101. Chae PS et al (2010) Maltose-neopentyl glycol (MNG) amphiphiles for solubilization,
stabilization and crystallization of membrane proteins. Nat Methods 7:1003–1008
102. Bill RM et al (2011) Overcoming barriers to membrane protein structure determination. Nat
Biotechnol 29:335–340
103. Caffrey M (2009) Crystallizing membrane proteins for structure determination: use of lipidic
mesophases. Annu Rev Biophys 38:29–51
104. Olofsson L et al (2014) Fine tuning of sub-millisecond conformational dynamics controls

metabotropic glutamate receptors agonist efficacy. Nat Commun 5:5206
105. Kumari P, Ghosh E, Shukla AK (2015) Emerging approaches to GPCR ligand screening for
drug discovery. Trends Mol Med 21:687–701
106. Scheerer P et al (2008) Crystal structure of opsin in its G-protein-interacting conformation.
Nature 455:497–502
107. Reeves PJ, Callewaert N, Contreras R, Khorana HG (2002) Structure and function in
rhodopsin: high-level expression of rhodopsin with restricted and homogeneous N-glycosyl-
ation by a tetracycline-inducible N-acetylglucosaminyltransferase I-negative HEK293S sta-
ble mammalian cell line. Proc Natl Acad Sci U S A 99:13419–13424
108. Stanley P, Caillibot V, Siminovitch L (1975) Selection and characterization of eight pheno-
typically distinct lines of lectin-resistant Chinese hamster ovary cell. Cell 6:121–128
109. Venkatakrishnan AJ et al (2013) Molecular signatures of G-protein-coupled receptors. Nature
494:185–194
110. Mirzadegan T, Benko G, Filipek S, Palczewski K (2003) Sequence analyses of G-protein-
coupled receptors: similarities to rhodopsin. Biochemistry 42:2759–2767
111. Steyaert J, Kobilka BK (2011) Nanobody stabilization of G protein-coupled receptor con-
formational states. Curr Opin Struct Biol 21:567–572
112. Koth CM et al (2012) Molecular basis for negative regulation of the glucagon receptor. Proc
Natl Acad Sci U S A 109:14393–14398
113. Hamers-Casterman C et al (1993) Naturally occurring antibodies devoid of light chains.
Nature 363:446–448
114. Pardon E et al (2014) A general protocol for the generation of nanobodies for structural
biology. Nat Protoc 9:674–693
115. Chun E et al (2012) Fusion partner toolchest for the stabilization and crystallization of G
protein-coupled receptors. Structure 20:967–976
116. Magnani F et al (2016) A mutagenesis and screening strategy to generate optimally
thermostabilized membrane proteins for structural studies. Nat Protoc 11:1554–1571
117. Tate CG (2012) A crystal clear solution for determining G-protein-coupled receptor struc-
tures. Trends Biochem Sci 37:343–352
118. Carpenter B, Tate CG (2016) Engineering a minimal G protein to facilitate crystallisation of
G protein-coupled receptors in their active conformation. Protein Eng Des Sel 29:583–593
119. Chapman HN et al (2011) Femtosecond X-ray protein nanocrystallography. Nature 470:73–
77
120. Chapman HN et al (2006) Femtosecond diffractive imaging with a soft-X-ray free-electron
laser. Nat Phys 2:839–843
121. Kang Y et al (2015) Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray
laser. Nature 523:561–567
122. Callaway E (2015) The revolution will not be crystallized. Nature 525:172–174
123. Liang YL et al (2017) Phase-plate cryo-EM structure of a class B GPCR-G-protein complex.
Nature 546:118–123
Top Med Chem (2019) 30: 27–52
DOI: 10.1007/7355_2017_31
Published online: 16 November 2017
NMR Spectroscopy for the

Characterization of GPCR Energy
Landscapes
Marina Casiraghi, Jean-Louis Banères, and Laurent J. Catoire
Abstract G protein-coupled receptor (GPCR)-mediated signal transduction has a

central role in human physiology and implication in many diseases. Despite the
tremendous number of X-ray crystallography structures published in the past
decade, the molecular mechanisms of ligand-dependent signaling remain to be
completed. In particular, very little information is available concerning the impli-
cation of receptor dynamics and conformational changes on GPCR ligand efficiency
and coupling. In this context, mapping the conformational landscape of GPCRs, and
how it is modulated by the membrane environment and allosteric and signaling
partners, is fundamental in order to gain a clear picture of how the signaling
mechanism proceeds. Solution-state nuclear magnetic resonance (NMR) is a pow-
erful technique to study GPCR energy landscapes, i.e., conformational ensembles
along activation and inactivation pathway, and associated kinetic barriers.
Keywords Energy landscape, Escherichia coli, GPCR, Perdeuteration, Solution-

state NMR
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
1.1 What Crystals Tell Us? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
1.2 The Picture Needs to Be Completed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
2 Solution-State NMR as a Powerful Tool to Investigate Biomolecules Energy
Landscapes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
M. Casiraghi and L.J. Catoire (*)

Laboratory of Physical and Chemical Biology of Membrane Proteins, Institut de Biologie
Physico-Chimique (IBPC), UMR 7099 CNRS, Paris, France
Paris Diderot University, Paris, France
PSL Research University, Paris, France
e-mail: catoire@ibpc.fr
J.-L. Banères
Faculté de Pharmacie, Institut des Biomolécules Max Mousseron (IBMM), UMR 5247
CNRS – Université Montpellier – ENSCM, Montpellier, France
28 M. Casiraghi et al.
3 Solution-State NMR Investigations of GPCR Conformational Landscapes: State

of the Art . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
4 An Alternative and Powerful Approach to Study GPCR Energy Landscape by Solution-
State NMR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
5 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
Abbreviations
β-DDM n-Dodecyl-beta-maltoside
BLT2 Leukotriene B4 human receptor 2
DEER Double electron–electron resonance
GPCR G protein-coupled receptor
LMNG Lauryl maltose neopentyl glycol
MD Molecular dynamics
MNG-3 Maltose neopentyl glycol-3
NB Nano-bodies
NMR Nuclear magnetic resonance
SDS Sodium dodecyl sulfate
TET Trifluoroethylthiol
TM Transmembrane
1 Introduction
Countless biological functions are closely linked to changes in spatial and temporal
location of groups of atoms in biomolecules. These conformational transitions
involve the existence of different conformers connected by molecular motions,
global and internal. Associated with an energy description to characterize the
relative probabilities of the different conformations, the kinetics that accompany
these transitions represent an important contribution to the understanding of a
biological mechanism [1, 2]. According to this paradigm, modulation of a protein
function relies on its excursions between ground states and higher energy con-
formers. Therefore, especially in the case of membrane proteins, understanding
how a given protein fulfills its function requires a description of its energy land-
scape and how this landscape can be remodeled by interactions with other proteins
or smaller ligands or the membrane environment. G protein-coupled receptors
(GPCRs) are not exception to the rule, in particular the complex pharmacological
behavior of these receptors indicates that a simple two-state model considering only
an active and an inactive conformation is inadequate to describe their function, i.e.,
suggesting a complex conformational plasticity for these proteins [3].
The conformational space that GPCRs explore can be described with the concept
of energy landscape [4], defined as a multidimensional hypersurface that governs
NMR Spectroscopy for the Characterization of GPCR Energy Landscapes 29
the thermodynamics and kinetics of the conformational transitions [5]. Following

this concept, based on a multitude of biophysical and pharmacological studies,
these receptors are thought to be highly dynamic proteins that can explore a wide
range of conformations, including in the absence of any ligand. In this scenario, the
native or ground state would correspond to the global minimum of energy, an
ensemble of conformers separated by small kinetic barriers that the protein could
easily overcome to switch to another conformation. This would explain why
different ligands can bind to a same GPCR for instance. In other words, these
preexisting conformations or substates would have differential propensities to
interact with one or another ligand. Subsequently, the ligand binding event would
lead to the selection and/or selective stabilization of a certain conformation, thus
causing a population shift in the conformational ensemble that would favor the
interaction with a specific intracellular partner to engage a biological response.
1.1 What Crystals Tell Us?
At the atomic scale, X-ray crystal structures published during the last decade
represent the major breakthrough and contribution in the structural biology of
GPCR receptors. They represent a precious starting point in the understanding of
the mechanism of signal transduction by placing structures in the conformational
ensemble of these receptors along the activation or inactivation pathways. The α
subfamily in class A GPCRs, which contains receptors for the biogenic amines (like
adrenaline and acetylcholine for example), is by far the best characterized subfam-
ily of GPCRs functionally and structurally [6]. Receptors belonging to this group
are essentially the β-adrenergic and muscarinic receptors. These structures reveal
some common and conserved features about the conformational changes during the
activation mechanism that occur in GPCRs, especially at the intracellular side with
various outward and inward movements of transmembrane (TM) helices, associ-
ated also with the description of very conserved intracellular “microswitches” [7–
12]. In addition, thanks to very high-resolved crystals, several potential allosteric
sites have been mapped by crystal structures [6], like the phosphate ion binding site
in the H1 histamine structure [13], a cholesterol binding site in β-AR [14], the
allosteric pocket for Na+ binding in A-AR [15, 16], and δ-opioid receptor [17].
1.2 The Picture Needs to Be Completed
Crystal structures obtained so far have largely contributed to our understanding of

ligand binding specificity and the process of G protein activation, but they still
provide little information on the role of GPCR dynamics in connection with their
complex signaling behavior [18]. To complete the picture and better understand the
function of these complex allosteric machines, we need to characterize more
extensively the energy landscape, by determining the different conformations that

exist or coexist along the activation and inactivation pathways, to define their
relative populations (thermodynamics) and the kinetics barriers that separate the
different conformers and associated energies of activation [5].
In complement to crystals, a large and diversified panel of biophysical
approaches have been used, such as hydrogen/deuterium exchange mass spectrom-
etry (e.g., [19]), double electron–electron resonance (e.g., [20, 21]), single-particle
electron microscopy (e.g., [22]), single molecule approaches (e.g., [23, 24]), molec-
ular dynamics (MD) simulations (e.g., [25]), atomic force microscopy (e.g., [26]),
and fluorescence and infrared spectroscopy (e.g., [27–30]). These different tech-
niques mostly provided information on the evolution of conformational ensembles
in various situations, and sometimes kinetic and thermodynamic parameters.
Among all these techniques, nuclear magnetic resonance (NMR) appears as a
very powerful tool to characterize the energy landscape of biomolecules [31]. In
this chapter we will focus on solution-state NMR studies, despite solid-state was
also successfully applied to some GPCRs, but in a lesser extent. Indeed, rhodopsin
was studied by 2H solid-state NMR combined with MD simulations of the receptor
in lipid membranes [32] and another solid-state NMR/MD study was also devel-
oped to determine the structure of the Chemokine Receptor CXCR1 in phospho-
lipid bilayers, thanks to a backbone model generated by MD based on the
experimental chemical shifts observed [33]. Similarly, the global fold of human
cannabinoid type 2 (CB2) receptor in the agonist-bound active state in lipid bilayers
was investigated by solid-state 13C and 15N magic-angle spinning NMR, in com-
bination with chemical-shift prediction from a structural model of the receptor
obtained by MD simulations [34]. The scenario gets more complicated when
structural data are more scarce and without the help of advanced MD calculations.
A solid-state NMR study conducted on the human growth hormone secretagogue
(GHS) receptor, expressed in Escherichia coli (E. coli) and reconstituted in lipid
bilayers, underlines the difficulty to obtain good spectral resolution when working
with large MPs like GPCRs, which hampered the acquisition of site-specific
dynamics information [35].
Thanks to efficient labeling schemes dedicated to the study of large proteins and
protein complexes in solution [36–40], in addition to the advent of high magnetic
fields and associated NMR methodology, it is possible to study the conformational
space of very large objects with this spectroscopy (e.g. [41–43]). This is typically
the case of GPCRs where NMR can take profit of the large number of available
X-ray crystal structures, as stated by Louis Kay in a recent review [31]: “Although
generating detailed structures of such large complexes is the domain of X-ray
crystallography and cryo-EM, characterization of the dynamics of such machines
and relating such motions to function is an important area that is now accessible to
study via NMR.” This probably explains why, during the past decade, solution-state
NMR spectroscopy has brought major contributions to the field. Indeed, to inves-
tigate the energy landscape of GPCRs, NMR spectroscopy represents a very
powerful technique to (1) determine the conformational ensemble, including
lowly populated and transiently formed conformations, and (2) characterize in
detail the chemical exchanges between the different substates detected.
2 Solution-State NMR as a Powerful Tool to Investigate

Biomolecules Energy Landscapes
NMR spectroscopy represents a powerful technique to investigate biomolecule

properties at equilibrium, thanks to its capacity to evaluate the relative populations
at physiological temperatures in the conformational ensemble and the kinetics
barriers separating the different substates. In the schematic in Fig. 1a, which
describes a simple chemical exchange between a ground (G) and an excited (E)
state, NMR can lead to the determination of populations G and E which follow a
Boltzmann distribution based on their difference in free energy. Because it is
straightforward to accurately control the sample temperature with the spectrometer,
the determination of these populations at different temperatures allows to determine
the enthalpic and entropic contributions to a specific motion (van’t Hoff equation).
Interestingly, the kinetics of exchange can also be determined, the forward and
backward kinetics constant rates kGE and kEG, which, again by varying the temper-
ature, can give access to the determination of forward and backward activation
energies by using a semiempirical Arrhenius law. The latter could play an important
role to explain the ligand efficacy phenomenon encountered with GPCR ligands
[44], that can display an efficacy which depends on the signaling pathway consid-
ered, when the GPCRs can participate to multiple signaling pathways.
Fig. 1 Chemical exchange and NMR spectroscopy. (a) Illustration with a two-site chemical
exchange between a highly populated (pG) and long-lived ground state G and a lowly populated
(pE) and transiently formed excited state E. kGE and kEG are respectively the forward and backward
kinetic rate constants (the exchange rate constant kex ¼ kGE + kEG), EEG and EGE are the forward
and backward energies of activation. (b) Simulated 1D NMR spectra where relative populations G
and E are respectively 75 and 25%. ΔΩ corresponds to the difference in NMR chemical shifts for a
given nucleus between the two state G and E. Three different regimes of exchange are indicated
respectively to ΔΩ (i.e., the magnetic field)
The first step in the characterization of an energy landscape is to determine the

number of conformations that can coexist or exist along the different activation or
inactivation pathways. The number of conformations that can be observed by NMR
will depend on the rate of transition between the different substates versus the
intensity of the magnetic field used (Fig. 1b). In other words, for a given nucleus,
if the exchange is much slower than the difference in NMR chemical shifts
(kex ¼ kGE + kEG versus ΔΩ) between the different states, then these states will
be directly observable under the requirement that they are enough populated to
emerge from the spectral noise. Under the assumption that the nucleus has the same
spin relaxation properties under these different substates, relative populations can
be directly associated with the volume or intensities of the different NMR signals
observed. In the opposite situation, if the chemical exchange is fast compared to
ΔΩ, only one averaged population will be observed. Finally, in the intermediate
regime, the exchange will affect the line width of the signals (Fig. 1b). Regarding
the high magnetic fields used today, the intermediate regime corresponds to molec-
ular motions in the micro- to millisecond timescale.
The second step consists in characterizing the kinetics of the different chemical
exchanges that occur in the energy landscape. NMR can accurately determine the
kinetics of exchange, thanks to the spin relaxation properties. Global and internal
motions in biomolecules locally induce variations in the magnetic field in both
intensity and orientation, and some variations in frequencies correspond to frequen-
cies separating nuclear spin energy levels, leading to spin relaxation. A large panel
of experiments can investigate biomolecule motions from ps/ns (local flexibilities)
to s (collective motions) and beyond [45]. So this includes also kinetics barriers in
the micro- to millisecond timescales which are known to play fundamental roles in
major biological events at the molecular scale like enzymatic catalysis, interactions
between biomolecules, allosteric regulations, protein folding, and so on.
In most cases, beside the characterization of the kinetics of exchange, to
determine the conformational ensemble at equilibrium, spin relaxation measure-
ments are mandatory in order to (1) correctly evaluate relative populations involved
in the chemical exchanges, and thus to access to the thermodynamics of the
exchanges; (2) detect lowly populated and/or transiently formed conformers [46–
48] that are not directly experimentally observed but thermally accessible from
ground states, highly populated and long lived states; and (3) obtain structural
information through variations in the NMR chemical shift ΔΩ (e.g., [49, 50]).
Indeed, in theory, the NMR chemical shift contains all the information on the
structure of the molecules possessing a spin. Thanks to the help of powerful
computational approaches of de novo protein structure determination based on
the use of structural data banks, it is possible to obtain structural information on
protein states that are not directly experimentally visible (e.g., [51]).
One interesting case is the capacity of NMR to detect lowly and transiently
populated conformers. This typically concerns high kinetic barriers (i.e., of several
kT, where k is the Boltzmann constant and T the absolute temperature). In such a
case, if we consider a simple chemical exchange between a native or ground state
G and an excited state E (Fig. 1a), the ground state will be highly populated
compared to state E according to their difference in their free energies (low

probability of transition from state G to E). Moreover, the lifetime of the excited
state will be short because of a fast backward kinetic rate (Fig. 1a). Due to their
transient nature and their sparse population, these excited states are difficult to be
detected with the conventional structural biology techniques that focus on low
energy and highly populated conformations. By looking at the relaxation properties
of the major conformation by NMR, it is possible to detect and characterize
the presence of a chemical exchange towards a second conformation that is not
experimentally directly observable in the NMR spectrum [47]. A number of NMR
approaches has been developed to characterize these invisible states that are all
based on the study of the relaxation properties of the main state from which we can
deduce the chemical exchange rate and the populations involved in the exchange.
3 Solution-State NMR Investigations of GPCR

Conformational Landscapes: State of the Art
During the last decade, most studies on energy landscapes by NMR focused on the
determination of conformational ensembles in various situations (in the presence or
absence of ligands or G peptide mimetic). The vast majority of these studies have
been carried out with GPCRs expressed in the membrane of insect cells. This is
why, except for a few of them, these studies were performed with either fully
protonated or sometimes partially and not homogeneously deuterated receptors.
This inhomogeneity can render the interpretation of the relaxation measurements of
nuclei under investigation difficult. The direct consequence of this is that most
studies were carried out in detergent solutions with sometimes the help of chemi-
cally modified amino acids in order to improve the probability of detecting a signal
by solution-state NMR considering these large tumbling objects. Solution-state
NMR has been used to study several GPCR conformational ensembles, i.e., β2-
adrenergic receptor (β2AR) [16, 21, 52–56], μ-opioid receptor (μOR) [57, 58], β1-
adrenergic receptor (β1AR) [59], and the adenosine A2A receptor (A2AR) [59]
according to different approaches, as reported in Table 1.
In 2010, the group of Kobilka published an analysis on the conformational
ensemble of some extracellular domains of the β2AR by NMR [52] in a detergent
solution. In this study, they showed that the extracellular surface of this GPCR can
be specifically regulated by ligands of various pharmacological profiles. This was
done by following a unique salt bridge between a lysine and an aspartic acid that
connects the extracellular loops 2 and 3, previously observed in crystals. Based on
crystal structures, they speculated that the extracellular region and the associated
salt bridge rearranged upon activation. To confirm this hypothesis, they based their
analysis on the use of 13C-labeled protonated methyl probes attached to lysine
residues. To do so, they performed a reductive methylation reaction on lysine
residues to exploit the sensitivity of methyl groups as NMR probes for analysis of
34
Table 1 Solution-state NMR investigations of GPCR energy landscapes

Receptor Modifications Type of isotope labeling Expression system Environment NMR experiment
β-AR Truncation after Gly365 (13CH3)2 lysines reductive sf9 insect cells Detergent 2D 1H,13C
[52] di-methylation (β-DDM)
13 19
β-AR Thermal stabilizing mutation E122W, C-term C F3 cysteines covalent sf9 insect cells Detergent 1D 19F
[16] truncation at residue 348, Δ245–259 (extra label with TET (β-DDM) NMR + Lorentzian
cellular loop 3) deconvolution
β-AR Receptor sequence Gly 2-Gly 365 ε-13CH3 methionines sf9 insect cells Detergent 2D 1H,13C
[54] (β-DDM)
13 19
β-AR β-AR-Δ4 cysteines deletion C F3 cysteine265 sf9 insect cells Detergent 1D 19F
[53] 3bromo1,1,1-trifluoroacetone (MNG-3) NMR + Lorentzian
(BTFA) labeling deconvolution
β-AR Δ of the 5 extra-membrane met ε-13CH3 met (3 met retained) sf9 insect cells Detergent 2D 1H,13C
[56] (β-DDM)
β-AR Receptor sequence Gly2-Gly365 ε-13CH3 met (5 met retained) sf9 insect cells, Lipid nanodiscs 2D 1H,13C
[55] partially deuterated
medium
13 19
β-AR β-AR-Δ4 cysteines deletion C F3, 19F-BTFA at Cys265 sf9 insect cells Detergent 1D 19F
[21] (MNG-3) NMR + Lorentzian
deconvolution
μOR F158W, Δ6 extra-membrane met ε-13CH3 methionines sf9 insect cells, Detergent 2D 1H,13C
[57] partially deuterated LMNG and lipid
medium nanodiscs
μOR M72T mutation (13CH3)2 lysines reductive sf9 insect cells Detergent 2D 1H,13C
[58] di-methylation (MNG-3)
15
β-AR 9 thermostabilizing mutations, 3 point muta- N valines sf9 insect cells Detergent (DM) 2D 1H,15N
[60] tions, N-term truncation, ICL3 truncation
13 19
A-AR V229C mutation C F3, 19F-BTFA on Yeast, Pichia Detergent 1D 19F
[59] Cys229 pastoris (MNG-3) NMR + Lorentzian
M. Casiraghi et al.
deconvolution
large protein structures and dynamics [61]. This consists in the addition of two
protonated and 13C-labeled methyl groups to the ε-NH2 of lysine side chains and
the α-NH2 at the receptor amino terminus using 13C-labeled formaldehyde. The 1H3
13
C-dimethyllysines served as conformational probes in two-dimensional 1H-13C
correlation NMR experiments. Thanks to this strategy, they could detect three
distinct conformations at the β2AR extracellular surface: one for the unliganded
receptor or in the presence of a neutral antagonist, and one in the presence of an
inverse agonist and an additional one in the presence of an agonist [52]. This
approach was limited to solvent-accessible lysines because of the isotopic labeling
method employed, which requires solvent-accessible residues to perform the chem-
ical reaction. So, it was not possible to correlate these observations to residues that
belong to the TM part of the receptor.
In another study on the conformational ensemble sampled by the β2AR in a
detergent solution, 19F-NMR was chosen to study the subtle conformational
changes involved in β2AR-ligand binding, by observing the line shapes and chem-
ical shifts of strategically located 19F-labeled methyl groups in the cytoplasmic
region of the β2AR, in complex with various ligands [16]. Three native cysteines in
the cytoplasmic region were covalently labeled with 2,2,2-trifluoroethanethiol
(TET) [62], Cys2656.27 and C3277.54 located at the cytoplasmic end of helices VI
and VII, and the extra-membrane Cys341 at the C-term which represents a negative
control relative to the two other cysteines. They observed changes in the 19F-NMR
spectra upon addition of different ligands, demonstrating that the active state
samples a wider range of conformers with slightly different chemical shifts [16].
19
F-NMR data had to be deconvoluted using a double-Lorentzian function to obtain
quantitative information about the conformational equilibrium, confirming that
agonist binding shifts the equilibrium of helices VI and VII from the inactive
state to the active ones [16]. They also suggested that the degree to which agonists
shift the equilibrium towards the active state of helix VI results in different G
protein signaling capacity, what is known as “biased signalization” [16]. In this
study, they observed that the cytoplasmic ends of helices VI and VII adopt two
major conformational states that they attribute to an inactive and an active state.
Their interpretation is that the balance between these two states is ligand dependent,
i.e., agonist binding primarily shifts the equilibrium towards the G protein-specific
active state of helix VI. In contrast, β-arrestin-biased ligands predominantly impact
the conformational states of helix VII.
The group of Kobilka also used 1D 19F-NMR spectroscopy to examine the
functional states associated with the β2AR [53]. This time this was done with the
receptor reconstituted in maltose neopentyl glycol (MNG-3) detergent micelles, a
new generation of detergents which display a very low critical micellar concentra-
tion (cmc) [63]. This study revealed the presence of two distinct inactive states, an
activation intermediate state en route to activation, and, in the presence of a G
protein mimic, a predominant active state [53]. The observation of four distinct
states compared to the only two in the previous study of Liu and collaborators [16]
was possible only with the use of a newly developed detergent, MNG-3, which
compared to n-Dodecyl-beta-maltoside (β-DDM) detergent displays a slower off
rate, that allows to distinguish between different states with an improved resolution
[64]. The study also focused on the temperature dependence of the populations of
the inactive and intermediate states, revealing that activation is enthalpically
unfavorable and entropically favorable, regardless of the ligand [53]. Interestingly,
some experiments were performed with an unliganded receptor and revealed the
coexistence of at least three states, two major ones and an additional one more
sparsely populated, in echo with some results recently obtained with the low affinity
leukotriene B4 receptor BLT2 (vide infra Fig. 4) [65].
These results were confirmed and further completed by other investigations on
the β2AR in MNG-3 micelles, performed by 1D 19F-NMR associated with double
electron–electron resonance (DEER) measurements [21]. DEER experiments were
aimed at accounting the number of existing or coexisting states while 1D fluorine
experiments were used to extract constant rates or substate lifetimes through
relaxation experiments. Manglik and collaborators confirmed the coexistence of
two inactive states for the unliganded receptor that interconvert in the microseconds
regime. The presence of an agonist shifts the equilibrium towards the active state
but in an incomplete manner without the presence of the G protein, resulting in wide
conformational heterogeneity due to the existence of the two inactive, intermediate
and active states [21], as also observed with the BLT2 receptor [65]. The complete
transition to the active state occurs when a G protein or a nanobody mimicking a G
protein is added to the sample. They also mentioned one structural hypothesis
regarding the coexistence of two major inactive conformations in the unliganded
state that would correspond to an intact and broken “ionic lock,” that interconvert
due to the low energy barrier between them. Under this assumption, the break of the
lock seems to be an important step towards the active state, and might be conserved
among other receptors of the family [21] but not in BLT2 for instance [65]. To be
noticed, concerning the use of fluorine nucleus in some studies of GPCR confor-
mational ensembles, expected conformational changes were not detected. This was
associated with possible effects of aromatic ring current fields which could hide
some variations in the chemical environment sampled by some 19F nuclei in β2AR
and mammalian rhodopsin. Indeed, these 19F-labelling sites, that should have
displayed conformational changes, were located near aromatic residues [66].
Beside the use of modified lysines or cysteines, unmodified, naturally methyl-
ated amino acids can also be used to investigate the conformational ensemble of
GPCRs. Indeed, methionines represent ideal NMR probes for high molecular
weight proteins, because of the length and flexibility of methionines side chain
which compensate for the slow tumbling rate of large proteins, improving the
resolution and sensitivity of the spectra [61]. In the case of membrane proteins,
methionines are also very interesting as they are usually located in the TM domain.
Two groups, Kobilka in Stanford and Shimada in Tokyo, used 13CH3-methionines
to investigate the β2AR conformational ensemble in various situations [54, 56] in
0.1% w/v β-DDM detergent solution. 13CH3-methionine-labeled βAR was achieved
by addition of [methyl-13C] methionines to a methionine-deficient medium during
receptor expression in sf9 cells, with a final incorporation yield of ~90% [54]. In
the Japanese study, by following NMR signals in 2D 1H,13C experiments, they
observed the presence of two inactive states at equilibrium, and a third active one in
the presence of the agonist, that can interact with the G-protein. In the case of the
partial agonist, the receptor is in equilibrium between the inactive and active
conformations. They also managed to detect a minor active conformation in the
case of the inverse agonist, in equilibrium with the inactive one, that might reflect
the presence of basal activity [54]. The study by Kobilka and colleagues led to
almost identical NMR data. In this second study, thanks to the use of a G protein-
mimetic nanobody, they also observed that an agonist alone does not stabilize a
fully active conformation [56]. This is in accordance with the capacity of the
receptor to engage several alternative signaling pathways or to bind different
intracellular partners according to the paradigm of biased agonism [67].
Following these studies, the group of Shimada introduced two major break-
throughs: (1) the use of lipid nanodiscs [68, 69] and (2) a partial deuteration of
the receptor, which improves both NMR sensitivity and resolution [55] (vide infra
Sect. 4). Whereas the β2AR in the previous studies was maintained soluble thanks to
detergents, lipid nanodiscs allow the reconstitution of the receptor in a more
membrane-like environment. Partial deuteration was performed on sf9 cells, a
system that is able to grow on minimal media at high concentrations of deuterium
oxide [70]. Fourteen types of amino acids were deuterated based on the NMR and
mass spectrometry studies performed on the test protein thioredoxin (Trx). Selec-
tive amino acid deuteration was necessary, as the growth of insect cell is highly
affected in D2O solutions. The amino acid deficient medium was supplemented
with [methyl-13C]-methionines. Deuteration increased sensitivity as attested by a
direct comparison between spectra of the β2AR in nanodiscs with or without
deuteration (Fig. 2), which clearly shows an enhancement in receptor sensitivity
by more than fivefolds upon partial deuteration. To be noticed, the rate of incorpo-
ration of 2H is amino acid dependent. Serine, glycine, or tyrosine residues have a
Fig. 2 Sensitivity enhancement of the CH-methionine resonances of β-AR in lipid nanodiscs upon
partial deuteration as observed in 2D H,C correlation experiments. (a) Without and (b) with the
presence of deuterons around 13CH3-Met residues (from Kofuku et al. [55]. Copyright Wiley-VCH
Verlag GmbH & Co. KGaA. Reproduced with permission)
rate comprised between 40 and 50%, while for some residues like cysteines,
phenylalanines, threonines, or methionines, the rates are close to 90%. In this
study, β2AR in nanodiscs was observed in equilibrium between two inactive
conformations and one active conformation, as previously observed [54]. However,
in the case of the presence of a partial agonist, only one signal was observed in
β-DDM micelles, while two signals were detected in nanodiscs, suggesting that
exchange rates between inactive and active conformations were lower in nanodiscs
than in β-DDM. Similarly, the active population detected in nanodiscs was higher
than in β-DDM [55].
The same deuteration method for protein expressed in insect cells was repeated
for the μOR [57]. In this study, eight types of amino acids were deuterated, allowing
the detection of the signals corresponding to seven methionines of the receptor.
They observed a conformational equilibrium for this receptor between a closed and
open conformation, and the latter one would be directly linked to the degree of G
protein activation. It is also postulated that the open conformation in the presence of
the full agonist is in turn in equilibrium between multiple open conformations,
including those that activate the G protein-mediated and β-arrestin signaling path-
ways. In the presence of the partial agonist, the receptor would be in equilibrium
between the closed and multiple open conformations. In the presence of biased
ligands, the equilibrium is shifted towards the conformation that preferentially
activates the G protein-mediated signaling or the β-arrestin according to the ligand
pharmacology [57].
More recently, conformational ensemble investigations performed with the same
receptor were conducted with a different approach in MNG-3 micelles, by looking
at dimethylated lysines in the solvent-accessible parts of μOR [58]. Nine
dimethylated extracellular and intracellular lysines were studied by 2D 1H,13C
experiments. The data demonstrated that the binding of the G protein (the nanobody
surrogate in this case) is required to fully stabilize the agonist-bound active
conformation, in agreement with previous studies [21, 56]. This study was aimed
at confirming and completing the findings of a companion paper that described the
crystal structure of the active μ-opioid receptor [8]. In the case of the addition of an
agonist or a nanobody or addition of both, this study suggests an allosteric coupling
between the extracellular μOR binding domain and the G-protein coupling
interface [58].
Another approach following an innovative method based on the use of thermo-
stabilizing mutations [71, 73] was used with the β1AR [60]. This time, the isotope-
labeling scheme concerned labeled 15N-valines. The 26 valines resonances
obtained were analyzed for the receptor in the presence of various ligands and in
the unliganded form. The authors concluded that the response to various ligands is
very heterogeneous in the vicinity of the binding pocket, but homogeneous at the
intracellular side of helix 5 (TM5), which correlates linearly with ligand efficacy for
the G protein pathway. They also reported that the stabilization of the GPCR fully
active conformation requires the binding of an agonist and an intracellular partner,
as already proved for other GPCRs [21]. In spite of the use of a thermostabilized
mutant, data seems to indicate that agonist binding, even in the absence of a G
protein mimic, induces initial changes in the conformational equilibrium of TM5

towards the conformation observed in the presence of G protein. However, a
complete shift towards the active state is only possible when the G protein or its
mimetic NB80 is bound. These observations required the presence of two residues
that were initially mutated, i.e., Y2275.58 and Y3437.53, which significantly reduced
the thermal stability of the receptor.
Finally, a study published in 2016 concerned the A2A receptor investigated by
19
F-NMR. V229C at the cytoplasmic end of TM6 was selected as trifluoromethyl
labeling site to discriminate between active and inactive states identified by
crystallography [59]. The study revealed the presence of four states, two inactive
and two active, for the unliganded receptor, in contrast to what observed in the
case of the β2AR [21]. In this “loosely coupled” ensemble, the two active states
already present for the unliganded receptor see their populations increasing upon
addition of partial or full agonists, consistent with the notion of conformational
selection [59]. They observed an exchange between the two inactive states, in the
millisecond regime, while between the inactive and active states the exchange
occurs at a slower rate, i.e., in the second timescale, probably due to high
activation barriers [59]. The addition of a G protein mimicking peptide shifts the
equilibrium towards the active states. The study shows insights on basal activity
as well, as inverse agonists shift the equilibrium towards the inactive states,
suppressing the active ones [59]. Remarkably different from the case of β-AR,
which is biased towards the inactive state unless the concomitant addition of an
agonist and a nanobody [21], 70% of the unliganded A2AR adopts the active states
[59]. They explain that difference by suggesting that the active state associated
with unliganded β2AR is very weakly or shortly populated, to the point that it
could not be observed in their NMR experiments.
All these studies led to very interesting information and converge to indicate that
GPCRs have a conformational landscape more complicated that initially thought
with just a balance between an inactive and an active conformation. Interestingly,
some studies indicate the presence of several coexisting states without the presence
of ligands and the detection of several distinct active states in some cases, with also
the detection of at least one on-activation pathway or intermediate state. Regarding
the studies briefly described herein, the methods used could be improved consid-
ering the following drawbacks:
1. A fully or partially protonated environment around the spins under investigation
precludes the detection of subtle but important variations in the conformational
landscape. This is due to the large size of GPCR/surfactant or GPCR/nanodisc
complexes. Indeed, even a residual amount of protons will have a dramatic
impact on the quality of NMR signals due to these large tumbling objects (vide
infra Fig. 5).
2. Nine studies on a total of eleven were conducted in detergent solutions in order
to work with size-compatible protonated or partially deuterated complexes with
solution-state NMR. Detergents do not form a lipid bilayer around the receptor
so they do not mimic very well a native environment which undoubtedly affects
the structure and the dynamics of the protein under investigation. In particular,
the relative fast chemical exchange of detergent molecules associated with the
receptor towards free-receptor detergent micelles or free detergent molecules
can have an impact on the conformational landscape [64]. Moreover, detergents
are known to be a quite destabilizing environment for membrane proteins
[74]. However, new promising detergents like maltose neopentyl glycol have
proven to be useful in the study of various membrane proteins [63, 75], but it is
still difficult to estimate the impact of lipid composition on protein function
using for instance detergents in the presence of lipids, because the lipid stoichi-
ometry cannot be strictly controlled in mixed micelles containing the receptor.
3. Most of the studies used chemically modified amino acids. This is again to
compensate the lack of perdeuteration. This has probably an impact on the
conformational equilibrium in the receptor that is difficult to gauge and, impor-
tantly, these modifications concern only amino acids that belong to the extra-
membrane domain of the receptor, so this is not possible to sample the confor-
mational ensemble of the TM domain.
4. One example concerns a thermostabilized receptor [60]. Thermostabilization is a
smart subterfuge to improve the quality of crystals [71, 76–79], but the question
remains whether it is a relevant approach to investigate dynamic issues. In this
study, they needed to revert some mutations to observe a coupling of the receptor
with its cognate G protein, but they needed also to perform this coupling with a
receptor reconstituted in lipid nanodiscs, while the whole NMR study was
carried out in a detergent solution.
4 An Alternative and Powerful Approach to Study GPCR

Energy Landscape by Solution-State NMR
As the molecular weight increases, the quality of NMR signals of the nuclei under
investigation will highly depend on the dipolar interactions with neighboring pro-
tons. In other words, the slower overall tumbling of a large molecule renders the
relaxation properties less favorable and the spectral quality deteriorates, giving rise
to peaks that can become so broad that eventually no signal could be detectable. In
order to improve the quality of NMR signals and to open the possibility to work in
lipid environments, the best labeling scheme to study large protein or protein
complexes by solution-state NMR is 13CH3 methyl probes immersed in a
perdeuterated environment [36, 37, 39, 40]. Methyl groups are ideal probes for
NMR because of their intense and well-resolved NMR signals, that are due to their
multiplicity of protons and the rapid rotation around the threefold methyl symmetry
axis. This contributes to increase the sensitivity, in comparison for instance with the
backbone amide protons, and to slow down the relaxation properties of the NMR
signal. Methyl groups are also of particular interest in NMR studies of proteins
because they occur frequently in the hydrophobic cores of the molecules [80] and
thus often serve as sensitive reporters of molecular structure and dynamics
[81]. Associated with these labeled methyl groups, deuteration of high-molecular-

weight proteins significantly improves the relaxation properties of the remaining
subset of protons that is detected in NMR experiments [37, 38, 40, 82]. The
development of strategies for selective methyl protonation in a deuterated environ-
ment using E. coli has led to the most robust and cost-effective approach
[81]. Thanks to this labeling scheme, associated with advances in NMR technology
(including ultra-high-field magnets and cryogenically cooled probes) and method-
ology [39], high-resolution NMR data can be obtained with proteins with molecular
weights in the range of 0.1–1 MDa, at a level of detail that was previously reserved
for much smaller systems [31, 42, 83, 84].
This strategy implies to use E. coli as expressing host, for its capacity to grow
in very hostile conditions like 100%-D2O solutions, as deuteration dramatically
affects the metabolism of most organisms. Organisms other than E. coli can grow in
100% D2O, like some algae and fungi [85], but the way to produce recombinant
proteins and to incorporate the traditional isotope-labeled precursors or labeled
amino acids to produce labeled membrane proteins is not feasible in practice or
unknown. In such hostile growth conditions, using E. coli, it is better to target the
receptor expression to inclusion bodies in order to greatly improve the yield of
production [86–89].
Adopting this strategy includes at one point a folding step of the receptor. Since
the first trials in the 80s with the bacteriorhodopsin [90–92], in vitro membrane
protein folding does not necessarily represent a bottleneck today. This is attested by
more than 50 α-helical membrane proteins and more than 40 β-barrels, monomeric
or oligomeric, from prokaryotic or eukaryotic organisms, which have been suc-
cessfully folded or refolded [93–96]. This is particularly feasible with class A
GPCRs that do not bear large extra-membrane domains [97]. One of the main
criteria is to fold the protein quickly to avoid the formation of kinetically trapped
states. The precipitation of sodium dodecyl sulfate (SDS) detergent to its potassium
salt is particularly indicated, having in mind that a membrane protein solubilized in
SDS already displays α-secondary structure elements [90, 98].
Interestingly, this approach does not require the use of chemically modified
amino acids and on top of that it gives access to the study of the transmembrane
conformational behavior of GPCR receptors. Of importance, despite the in vitro
folding procedure and the insertion into nanodiscs, the GPCR is fully active and
stable in the conditions of the NMR experiments, based on ligand and GTP-to-G
protein binding experiments. As an example, the spectrum displayed in Fig. 3
describes the conformational ensemble of the low leukotriene B4 receptor BLT2
in lipid nanodiscs in the absence of ligands. The receptor is perdeuterated and
contains protonated and 13C-labeled methyl groups in methionine and isoleucine
residues. The high quality of NMR data allowed the detection of several coexisting
substates including the active or active-like state. This occurrence of several
conformational states observed for this receptor was also in accordance with
some studies performed with the β2AR [21, 53]. In particular, this concerns the
coexistence of two main inactive conformations in the unliganded state and also
the presence of one intermediate state (III in Fig. 4, left). Thanks to receptor
Fig. 3 Functional modulation of BLT2 conformational landscape in a lipid bilayer. BLT2 was
perdeuterated and labeled on the methyl groups of Met (ε-13CH3) and Ile (δ1-13CH3). The choice of
working with Ile and Met residues was particularly recommended in the case of BLT2. First, this
receptor contains only one Ile, Ile229, ideally located in TM helix VI close to the cytoplasmic part of
the receptor, a region that is known to sample large conformational variations upon activation
[9]. Second, it contains only two transmembrane Met located in two different helices, Met105 at
the bottom of the putative orthosteric binding pocket on TM helix III, and Met197 on helix V.
(a) Conformational landscape of unliganded BLT2 in nanodiscs. 2D 1H-13C SOFAST-methyl-
Heteronuclear Multiple-Quantum Correlation (HMQC)/Transverse Relaxation Optimized Spectros-
copY (TROSY) spectrum acquired with [u-2H,12C]Ile-[δ1-13CH3], [u-2H,12C]Met-[ε-13CH3] BLT2
in lipid nanodiscs in the unliganded state. (a) Global view of the [ε-13CH3]-Met and [δ1-13CH3]-
Ile2296.40 regions (boxed in orange); the additional weak peaks correspond to residual lipid signals.
(b, c) From panel (a), close-ups of the [ε-13CH3]-Met region and [δ1-13CH3]-Ile2296.40 region,
respectively. In panel (b), the red spectrum corresponds to the mutant receptor that contains the
transmembrane Met residues 1053.35 and 1975.54 only. The peak labeled V in parentheses in panel (c)
was not included in the present analysis of the BLT2 conformational ensemble (adapted with
permission from Casiraghi et al. [65]. Copyright 2016 American Chemical Society)
S3
S2
S1
–83.2 –83.4 –83.6 –83.8 –84.0 –84.2 –84.4 –84.6

19
F Chemical Shift (ppm)
Fig. 4 Comparison of GPCR conformational ensembles in the unliganded state of protonated

β2AR in a detergent solution and perdeuterated BLT2 in lipid nanodiscs. (Left) 2D 1H,13C
correlation spectrum of Ile229 of BLT2 (from panel c in Fig. 9; adapted with permission from
Casiraghi et al. [65]. Copyright 2016 American Chemical Society) and (right) 1D fluorine NMR
signal of Cys265 in β2AR that carries a fluorinated methyl group (a) on TM helix 6 (adapted with
permission from Kim et al. [53]. Copyright 2013 American Chemical Society). (b) In the
unliganded state both receptors display a slow equilibrium between two main populated inactive
states labeled I and II for BLT2 and S2 and S3 for β2AR, and one intermediate populated state III
(BLT2) and S1 (β2AR). States I and II, or S2 and S3, have been identified as inactive states and III,
or S1, as intermediate conformations on the activation pathway, thanks to the use of various
ligands. In the case of BLT2, a fourth state, lowly populated, corresponding to the active or active-
like state (i.e., in the absence of a G protein) could be observable thanks presumably to a better
resolution compared to the case of the study of β2AR based on 1D NMR experiments carried out
with a fully protonated receptor
perdeuteration, it was possible to detect a fourth state, lowly populated, in the

unliganded state, never observed in previous studies (peak labeled IV in Fig. 4, left).
This fourth state could be assigned to the active or active-like state, thanks to the
subsequent use of agonists, in accordance with G protein activation measurements.
Importantly, in contrast to the description of the conformation ensemble of
unliganded β2AR [21, 53] (Fig. 4, right), population IV already exists in the
conformational landscape of unliganded BLT2, i.e., it does not appear upon the
addition of the agonist only. Considering that BLT2 is 95% active in terms of ligand
binding in the conditions of the NMR experiments [65], this fourth state cannot be
attributed to an isolated kinetically trapped state. This lack of observation of the
active state in the unliganded state for β2AR may be due to investigations conducted
with either 1D fluorine NMR experiments or 2D 1H, 13C correlation experiments

with a partial or totally protonated receptor, which precluded the observation of
lowly populated conformations.
The structure, dynamics, and organization of GPCR are largely determined by
the membrane environment that surrounds the receptor [99]. As GPCRs are embed-
ded in the membrane, they are in strong proximity with lipids, which are one of the
major membrane components and interact with ~40% of the receptor total surface
area [100]. This suggests that the membrane could be an important modulator of
GPCR structure and function. For studying GPCR conformational landscape in the
presence of lipids and the modulation by the membrane environment, nanometric
lipid bilayers, named nanodiscs, are particularly indicated, as they provide a
membrane-like environment where the protein is active and stable, they can be
tuned in size and lipid composition, and they are compatible with solution-
state NMR.
Thanks to high quality NMR data, it was also demonstrated that BLT2 confor-
mational landscape is modulated by lipids, in addition to ligands. In particular, it
has been observed that an increment in the sterol content of the membrane modifies
the distribution of the different conformational states of the receptor in favor of the
active one, indicating a positive allosteric regulation of the sterol on the activation
of this receptor, as confirmed by GTP-to-G protein binding measurements
[65]. That property of the sterol is likely to be important for the control of the
signaling properties of GPCRs and could explain why GPCRs are often found in
cholesterol enriched domains like lipid rafts and caveolae [101].
To highlight the improvements introduced by the use of perdeuteration com-
bined with methyl probes, Fig. 5 shows a comparison of recent 13CH3 NMR signals
of GPCRs in nanodiscs or associated with detergents from the literature, with data
obtained with TM Met residues of BLT2 in nanodiscs. These 2D 1H, 13C correlation
spectra, represented at the same scale in both dimensions, clearly indicate the
substantial improvement in resolution obtained following the E. coli expression
protocol. In particular, the size of the signals obtained without perdeuteration
precludes the observation of several coexisting substates, while the sharpness of
labeled residues in BLT2 allows to distinguish between different conformational
states for the unliganded receptor.
5 Concluding Remarks
The large number of biological functions that GPCRs control and the complex
signaling behavior displayed by these receptors imply the existence of a complex
energy landscape. A single receptor can activate more than one G protein subtype as
well as G protein-independent pathways, without or in the presence of various
ligands. As a consequence, a given ligand can possess distinct intrinsic efficacies
towards these different pathways, a concept described as ligand bias. Receptor
allostery further increases the complexity of GPCR functioning. Indeed, many
Fig. 5 Impact of perdeuteration on the quality of NMR data. (a) 13CH3 methionine region of a
perdeuterated and 13CH3-ε labeled Met residues mutant of the low affinity leukotriene BLT2
receptor in MSP1D1 nanodiscs. The experiment was acquired at 950 MHz 1H Larmor frequency,
298 K, at pH 7.4 [65]. (b) 13CH3 methionine region of partially deuterated β2-adrenergic receptor
(containing 14 types of amino acids deuterated between 50 and 90%) in MSP1 nanodiscs acquired
at 800 MHz 1H Larmor frequency, 298 K, at pH 7.1 (from Kofuku et al. [55]. Copyright Wiley-
VCH Verlag GmbH & Co. KGaA. Reproduced with permission). (c) 13CH3 methionine region of
partially deuterated (8 amino acids deuterated) μ-opioid receptor in LMNG detergent micelles
acquired at 800 MHz 1H Larmor frequency, 298 K, at pH 7.2 (from Okude et al. [57]. Copyright
Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with permission). BLT2 spectrum in red is
displayed at the same scale in both 1H and 13C dimensions than black spectra in panels (b, c)
substances can act as allosteric modulators of GPCR signaling. These include ions,
signaling proteins, lipids, or oligomerization partners. It has been proposed that the
remarkable functional versatility of GPCRs is associated with their intrinsic
dynamic properties. In this model, GPCRs are considered as flexible proteins that
can visit multiple conformational states linked to distinct functional outcomes.
Signaling modulation can then be seen as an alteration of the equilibrium between
such states, with the relative amount of the different populations modulated by
coupling to orthosteric or allosteric ligands, to intracellular protein partners, by
receptor oligomerization, or by alterations of the membrane composition. Although
major progresses have been made in structural biology of GPCRs, we are never-
theless only at the beginning of the study of the role of dynamics in GPCR
signaling. Indeed, a wealth of structural information has been obtained from crystal
structures, but these only offer a limited, but invaluable, number of static snapshots
in a complex dynamics landscape. Hence, answering the question of how GPCR

dynamics can control their downstream signaling pathways still requires a variety
of additional cutting-edge biophysical methods.
In this context, NMR spectroscopy represents a powerful technique to investi-
gate GPCR conformational ensemble and associated kinetics barriers and how they
can be modulated by ligands, signaling proteins, and lipids. So far, solution-state
NMR studies looked at conformational ensembles in various situations. The coex-
istence of more than one population for an unliganded GPCR was observed in the
case of the β2AR [21, 53, 55, 56], A2AR [60] and BLT2 [65]. Two studies also shed
light on the presence of an active or active-like conformation for the unliganded
receptor [59, 65] that could correspond to the receptor basal activity, a key feature
for the fast conformational change within a class of receptors involved in rapid
neurotransmission and sensory perception like GPCRs. In the presence of the
agonist, the conformational heterogeneity decreases, even if the full stabilization
of the active state can be achieved only in the presence of the G protein [21, 56, 102].
Unfortunately, most of these studies have been carried out with receptors
maintained soluble in detergent solutions. Even if detergents cannot necessarily
affect ligand or G-to-GTP binding properties, this does not mean the “natural”
energy landscape is not affected, i.e., that the relative populations, dynamics and
behind thermodynamics parameters, and energies of activation are not modified.
Indeed, there is a close interplay between a membrane protein and lipids. The
dynamics of lipids will be affected around the protein and conversely lipids play a
non-negligible role in the function of the receptor. Thanks to the alternative
approach presented in Sect. 4, it is possible to improve the reading of the confor-
mational ensemble by working with perdeuterated receptors, which, above all, open
the possibility to work with receptors embedded in lipid nanodiscs, even at a better
spectral resolution than those obtained with fully protonated and partially deuter-
ated receptors associated with detergent micelles. This allowed recently to look at a
very fundamental, and difficult, question, which concerns the impact of lipids at the
atomic scale on the conformational ensemble of a GPCR, connected with the
activation of these receptors [65].
Solution-state NMR associated with smart isotope-labeling schemes represents a
very promising technique to go further in the characterization of GPCR energy
landscapes.
In particular, illuminating the dynamic nature of these receptors will offer a key
for the understanding of the fundamental molecular mechanisms governing their
signaling activity.
References
1. Henzler-Wildman K, Kern D (2007) Dynamic personalities of proteins. Nature 450:964–972

2. Smock RG, Gierasch LM (2009) Sending signals dynamically. Science 324:198–203
3. Staus DP, Strachan RT, Manglik A, Pani B, Kahsai AW, Kim TH, Wingler LM, Ahn S,
Chatterjee A, Masoudi A, Kruse AC, Pardon E, Steyaert J, Weis WI, Prosser RS, Kobilka BK,
Costa T, Lefkowitz RJ (2016) Allosteric nanobodies reveal the dynamic range and diverse
mechanisms of G-protein-coupled receptor activation. Nature 535:448–452
4. Frauenfelder H, Sligar SG, Wolynes PG (1991) The energy landscapes and motions of
proteins. Science 254:1598–1603
5. Lazaridis T, Karplus M (2003) Thermodynamics of protein folding: a microscopic view.
Biophys Chem 100:367–395
6. Katritch V, Cherezov V, Stevens RC (2012) Diversity and modularity of G protein-coupled
receptor structures. Trends Pharmacol Sci 33:17–27
7. Choe H-W, Park JH, Kim YJ, Ernst OP (2011) Transmembrane signaling by GPCRs: insight
from rhodopsin and opsin structures. Neuropharmacology 60:52–57
8. Huang W, Manglik A, Venkatakrishnan AJ, Laeremans T, Feinberg EN, Sanborn AL, Kato
HE, Livingston KE, Thorsen TS, Kling RC, Granier S, Gmeiner P, Husbands SM, Traynor
JR, Weis WI, Steyaer J, Dror RO, Kobilka BK (2015) Structural insights into μ-opioid
receptor activation. Nature 524:315–321
9. Katritch V, Cherezov V, Stevens RC (2013) Structure-function of the G-protein-coupled
receptor superfamily. Annu Rev Pharmacol Toxicol 53:531–556
10. Kruse AC (2015) Structural insights into activation and allosteric modulation of G protein-
coupled receptors. Multifaceted roles of crystallography in modern drug discovery. Springer,
Dordrecht, pp 19–26
11. Lee Y, Choi S, Hyeon C (2015) Communication over the network of binary switches
regulates the activation of A2A adenosine receptor. PLoS Comput Biol 11:e1004044
12. Xu F, Wu H, Katritch V, Han GW, Jacobson KA, Gao Z-G, Cherezov V, Stevens RC (2011)
Structure of an agonist-bound human A2A adenosine receptor. Science 332:322–327
13. Shimamura T, Shiroishi M, Weyand S, Tsujimoto H, Winter G, Katritch V, Abagyan R,
Cherezov V, Liu W, Han GW, Takuya K, Stevens RC, Iwata S (2011) Structure of the human
histamine H1 receptor complex with doxepin. Nature 475:65–70
14. Hanson MA, Cherezov V, Griffith MT, Roth CB, Jaakola V-P, Chien EYT, Velasquez J,
Kuhn P, Stevens RC (2008) A specific cholesterol binding site is established by the 2.8 Å
structure of the human β2-adrenergic receptor. Structure 16:897–905
15. Katritch V, Fenalti G, Abola EE, Roth BL, Cherezov V, Stevens RC (2014) Allosteric sodium
in class A GPCR signaling. Trends Biochem Sci 39:233–244
16. Liu JJ, Horst R, Katritch V, Stevens RC, Wuthrich K (2012) Biased signaling pathways in
2-adrenergic receptor characterized by 19F-NMR. Science 335:1106–1110
17. Fenalti G, Giguere PM, Katritch V, Huang X-P, Thompson AA, Cherezov V, Roth BL,
Stevens RC (2014) Molecular control of δ-opioid receptor signalling. Nature 506:191–196
18. Granier S, Kobilka B (2012) A new era of GPCR structural and chemical biology. Nat Chem
Biol 8:670–673
19. Chung KY, Rasmussen SGF, Liu T, Li S, DeVree BT, Chae PS, Calinski D, Kobilka BK,
Woods VL, Sunahara RK (2011) Conformational changes in the G protein Gs induced by the
β2 adrenergic receptor. Nature 477:611–615
20. Kang Y, Zhou XE, Gao X, He Y, Liu W, Ishchenko A, Barty A, White TA, Yefanov O, Han
GW, Xu Q, de Waal PW, Ke J, Tan MHE, Zhang C, Moeller A, West GM, Pascal B, Van
Eps N, Caro LN, Vishnivetskiy SA, Lee RJ, Suino-Powell KM, Gu X, Pal K, Ma J, Zhi X,
Boutet S, Williams GJ, Messerschmidt M, Gati C, Zatsepin NA, Wang D, James D, Basu S,
Roy-Chowdhury S, Conrad C, Coe J, Liu H, Lisova S, Kupitz C, Grotjohann I, Fromme R,
Jiang Y, Tan M, Yang H, Li J, Wang M, Zheng Z, Li D, Howe N, Zhao Y, Standfuss J,
Diederichs K, Dong Y, Potter CS, Carragher B, Caffrey M, Jiang H, Chapman HN, Spence
JCH, Fromme P, Weierstall U, Ernst OP, Katritch V, Gurevich VV, Griffin PR, Hubbell WL,
Stevens RC, Cherezov V, Melcher K, Xu HE (2015) Crystal structure of rhodopsin bound to
arrestin by femtosecond X-ray laser. Nature 523:561–567
21. Manglik A, Kim TH, Masureel M, Altenbach C, Yang Z, Hilger D, Lerch MT, Kobilka TS,
Thian FS, Hubbell WL, Prosser RS, Kobilka BK (2015) Structural insights into the dynamic
process of β2-adrenergic receptor signaling. Cell 161:1101–1111
22. De Zorzi R, Mi W, Liao M, Walz T (2016) Single-particle electron microscopy in the study of
membrane protein structure. Microscopy 65:81–96
23. Gregorio GG, Masureel M, Hilger D, Terry DS, Juette M, Zhao H, Zhou Z, Perez-Aguilar JM,
Hauge M, Mathiasen S, Javitch JA, Weinstein H, Kobilka BK, Blanchard SC (2017) Single-
molecule analysis of ligand efficacy in β2AR–G-protein activation. Nature 547(7661):68–73.
https://doi.org/10.1038/nature22354
24. Tian H, Fürstenberg A, Huber T (2017) Labeling and single-molecule methods to monitor G
protein-coupled receptor dynamics. Chem Rev 117:186–245
25. Dror RO, Dirks RM, Grossman JP, Xu H, Shaw DE (2012) Biomolecular simulation: a
computational microscope for molecular biology. Annu Rev Biophys 41:429–452
26. Zocher M, Zhang C, Rasmussen SGF, Kobilka BK, Muller DJ (2012) Cholesterol increases
kinetic, energetic, and mechanical stability of the human β2-adrenergic receptor. Proc Natl
Acad Sci U S A 109:3463–3472
27. Damian M, Mary S, Maingot M, M’Kadmi C, Gagne D, Leyris J-P, Denoyelle S, Gaibelet G,
Gavara L, Garcia de Souza Costa M, Perahia D, Trinquet E, Mouillac B, Galandrin S,
Galès S, Fehrentz J-A, Floquet N, Martinez J, Marie J, Banères J-L (2015) Ghrelin receptor
conformational dynamics regulate the transition from a preassembled to an active receptor:
Gq complex. Proc Natl Acad Sci U S A 112:1601–1606
28. M’Kadmi C, Leyris J-P, Onfroy L, Galés C, Saulière A, Gagne D, Damian M, Mary S,
Maingot M, Denoyelle S, Verdié P, Fehrentz J-A, Martinez J, Banères JL, Marie J (2015)
Agonism, antagonism, and inverse agonism bias at the ghrelin receptor signaling. J Biol
Chem 290:27021–27039
29. Mary S, Damian M, Louet M, Floquet N, Fehrentz J-A, Marie J, Martinez J, Banères J-L
(2012) Ligands and signaling proteins govern the conformational landscape explored by a G
protein-coupled receptor. Proc Natl Acad Sci U S A 109:8304–8309
30. Vogel R, Mahalingam M, Lüdeke S, Huber T, Siebert F, Sakmar TP (2008) Functional role of
the “ionic lock”-an interhelical hydrogen-bond network in family A heptahelical receptors.
J Mol Biol 380:648–655
31. Kay LE (2016) New views of functionally dynamic proteins by solution NMR spectroscopy.
J Mol Biol 428:323–331
32. Mertz B, Struts AV, Feller SE, Brown MF (2012) Molecular simulations and solid-state
NMR investigate dynamical structure in rhodopsin activation. Biochim Biophys Acta
1818:241–251
33. Park SH, Das BB, Casagrande F, Tian Y, Nothnagel HJ, Chu M, Kiefer H, Maier K,
De Angelis AA, Marassi FM, Opella SJ (2012) Structure of the chemokine receptor
CXCR1 in phospholipid bilayers. Nature 491:779–784
34. Kimura T, Vukoti K, Lynch DL, Hurst DP, Grossfield A, Pitman MC, Reggio PH, Yeliseev
AA, Gawrisch K (2014) Global fold of human cannabinoid type 2 receptor probed by solid-
state 13C-, 15N-MAS NMR and molecular dynamics simulations: NMR studies on CB2
receptor. Proteins: Struct Funct Bioinf 82:452–465
35. Schrottke S, Kaiser A, Vortmeier G, Els-Heindl S, Worm D, Bosse M, Schmidt P, Scheidt
HA, Beck-Sickinger AG, Huster D (2017) Expression, functional characterization, and solid-
state NMR investigation of the G protein-coupled ghs receptor in bilayer membranes. Sci Rep
7:46128
36. Goto NK, Gardner KH, Mueller GA, Willis RC, Kay LE (1999) A robust and cost-effective
method for the production of Val, Leu, Ile (δ1) methyl-protonated 15N, 13C, 2H-labeled
proteins. J Biomol NMR 13:369–374
37. Kay LE (2011) Solution NMR spectroscopy of supra-molecular systems, why bother?
A methyl-TROSY view. J Magn Reson 210:159–170
38. Rosenzweig R, Kay LE (2014) Bringing dynamic molecular machines into focus by methyl-
TROSY NMR. Annu Rev Biochem 83:291–315
39. Tugarinov V, Hwang PM, Kay LE (2004) Nuclear magnetic resonance spectroscopy of high-
molecular-weight proteins. Annu Rev Biochem 73:107–146
40. Tugarinov V, Kanelis V, Kay LE (2006) Isotope labeling strategies for the study of high-
molecular-weight proteins by solution NMR spectroscopy. Nat Protoc 1:749–754
41. Huang R, Ripstein ZA, Augustyniak R, Lazniewski M, Ginalski K, Kay LE, Rubinstein JL
(2016) Unfolding the mechanism of the AAA+ unfoldase VAT by a combined cryo-EM,
solution NMR study. Proc Natl Acad Sci U S A 113:4190–4199
42. Ruschak AM, Religa TL, Breuer S, Witt S, Kay LE (2010) The proteasome antechamber
maintains substrates in an unfolded state. Nature 467:868–871
43. Sekhar A, Rosenzweig R, Bouvignies G, Kay LE (2016) Hsp70 biases the folding pathways
of client proteins. Proc Natl Acad Sci U S A 113:2794–2801
44. Kenakin T (2002) Drug efficacy at G protein-coupled receptors. Annu Rev Pharmacol
Toxicol 42:349–379
45. Kleckner IR, Foster MP (2011) An introduction to NMR-based approaches for measuring
protein dynamics. Biochim Biophys Acta 1814:942–968
46. Baldwin AJ, Kay LE (2009) NMR spectroscopy brings invisible protein states into focus. Nat
Chem Biol 5:808–814
47. Sekhar A, Kay LE (2013) NMR paves the way for atomic level descriptions of sparsely
populated, transiently formed biomolecular conformers. Proc Natl Acad Sci U S A
110:12867–12874
48. Sekhar A, Vallurupalli P, Kay LE (2013) Defining a length scale for millisecond-timescale
protein conformational exchange. Proc Natl Acad Sci U S A 110:11391–11396
49. Shen Y, Lange O, Delaglio F, Rossi P, Aramini JM, Liu G, Eletsky A, Wu Y, Singarapu KK,
Lemak A, Ignatchenko A, Arrowsmith CH, Szyperski T, Montelione GT, Baker D, Bax A
(2008) Consistent blind protein structure generation from NMR chemical shift data. Proc Natl
Acad Sci U S A 105:4685–4690
50. Shen Y, Bax A (2015) Homology modeling of larger proteins guided by chemical shifts. Nat
Methods 12:747–750
51. Korzhnev DM, Religa TL, Banachewicz W, Fersht AR, Kay LE (2010) A transient and
low-populated protein-folding intermediate at atomic resolution. Science 329:1312–1316
52. Bokoch MP, Zou Y, Rasmussen SGF, Liu CW, Nygaard R, Rosenbaum DM, Fung JJ, Choi
H-J, Thian FS, Kobilka TS, Puglisi JD, Weis WI, Pardo L, Prosser RS, Mueller L, Kobilka
BK (2010) Ligand-specific regulation of the extracellular surface of a G-protein-coupled
53. Kim TH, Chung KY, Manglik A, Hansen AL, Dror RO, Mildorf TJ, Shaw DE, Kobilka BK,
Prosser RS (2013) The role of ligands on the equilibria between functional states of a G
protein-coupled receptor. J Am Chem Soc 135:9465–9474
54. Kofuku Y, Ueda T, Okude J, Shiraishi Y, Kondo K, Maeda M, Tsujishita H, Shimada I (2012)
Efficacy of the β2-adrenergic receptor is determined by conformational equilibrium in the
transmembrane region. Nat Commun 3:1045
55. Kofuku Y, Ueda T, Okude J, Shiraishi Y, Kondo K, Mizumura T, Suzuki S, Shimada I (2014)
Functional dynamics of deuterated β2-adrenergic receptor in lipid bilayers revealed by NMR
spectroscopy. Angew Chem Int Ed Engl 53:13376–13379
56. Nygaard R, Zou Y, Dror RO, Mildorf TJ, Arlow DH, Manglik A, Pan AC, Liu CW, Fung JJ,
Bokoch MP, Thian FS, Kobilka TS, Shaw DE, Mueller L, Prosser RS, Kobilka BK (2013)
The dynamic process of β(2)-adrenergic receptor activation. Cell 152:532–542
57. Okude J, Ueda T, Kofuku Y, Sato M, Nobuyama N, Kondo K, Shiraishi Y, Mizumura T,
Onishi K, Natsume M, Maeda M, Tsujishita H, Kuranaga T, Inoue M, Shimada I (2015)
Identification of a conformational equilibrium that determines the efficacy and functional
selectivity of the μ-opioid receptor. Angew Chem Int Ed 54:15771–15776
58. Sounier R, Mas C, Steyaert J, Laeremans T, Manglik A, Huang W, Kobilka BK, Déméné H,
Granier S (2015) Propagation of conformational changes during μ-opioid receptor activation.
Nature 524:375–378
59. Ye L, Van Eps N, Zimmer M, Ernst OP, Prosser RS (2016) Activation of the A2A adenosine
G-protein-coupled receptor by conformational selection. Nature 533:265–268
60. Isogai S, Deupi X, Opitz C, Heydenreich FM, Tsai C-J, Brueckner F, Schertler GFX,
Veprintsev DB, Grzesiek S (2016) Backbone NMR reveals allosteric signal transduction
networks in the β1-adrenergic receptor. Nature 530:237–241
61. Tugarinov V, Hwang PM, Ollerenshaw JE, Kay LE (2003) Cross-correlated relaxation
enhanced 1H[bond]13C NMR spectroscopy of methyl groups in very high molecular weight
proteins and protein complexes. J Am Chem Soc 125:10420–10428
62. Klein-Seetharaman J, Hwa J, Cai K, Altenbach C, Hubbell WL, Khorana HG (1999) Single-
cysteine substitution mutants at amino acid positions 55–75, the sequence connecting the
cytoplasmic ends of helices I and II in rhodopsin: reactivity of the sulfhydryl groups and their
derivatives identifies a tertiary structure that changes upon light-activation. Biochemistry
38:7938–7944
63. Chae PS, Rasmussen SGF, Rana RR, Gotfryd K, Chandra R, Goren MA, Kruse AC, Nurva S,
Loland CJ, Pierre Y, Drew D, Popot JL, Picot D, Fox BG, Guan L, Gether U, Byrne B,
Kobilka B, Gellman SH (2010) Maltose-neopentyl glycol (MNG) amphiphiles for solubili-
zation, stabilization and crystallization of membrane proteins. Nat Methods 7:1003–1008
64. Chung KY, Kim TH, Manglik A, Alvares R, Kobilka BK, Prosser RS (2012) Role of
detergents in conformational exchange of a G protein-coupled receptor. J Biol Chem
287:36305–36311
65. Casiraghi M, Damian M, Lescop E, Point E, Moncoq K, Morellet N, Levy D, Marie J,
Guittet E, Banères J-L, Catoire JL (2016) Functional modulation of a G protein-coupled
receptor conformational landscape in a lipid bilayer. J Am Chem Soc 138:11170–11175
66. Liu D, Wüthrich K (2016) Ring current shifts in (19)F-NMR of membrane proteins. J Biomol
NMR 65:1–5
67. Urban JD, Clarke WP, von Zastrow M, Nichols DE, Kobilka B, Weinstein H, Javitch JA,
Roth BL, Christopoulos A, Sexton PM, Miller JK, Spedding M, Mailman RB (2006)
Functional selectivity and classical concepts of quantitative pharmacology. J Pharmacol
Exp Ther 320:1–13
68. Bayburt TH, Grinkova YV, Sligar SG (2002) Self-assembly of discoidal phospholipid bilayer
nanoparticles with membrane scaffold proteins. Nano Lett 2:853–856
69. Ritchie TK, Grinkova YV, Bayburt TH, Denisov IG, Zolnerciks JK, Atkins WM, Sligar SG
(2009) Reconstitution of membrane proteins in phospholipid bilayer nanodiscs. Methods
Enzymol 464:211–231
70. O’Reilly DR, Miller L, Luckow VA (1994) Baculovirus expression vectors: a laboratory
manual. Oxford University Press, New York
71. Lebon G, Warne T, Edwards PC, Bennett K, Langmead CJ, Leslie AGW, Tate CG (2011)
Agonist-bound adenosine A2A receptor structures reveal common features of GPCR activa-
tion. Nature 474:521–525
72. Serrano-Vega MJ, Magnani F, Shibata Y, Tate CG (2008) Conformational thermostabilization
of the β1-adrenergic receptor in a detergent-resistant form. Proc Natl Acad Sci U S A
105:877–882
73. Warne T, Chirnside J, Schertler GFX (2003) Expression and purification of truncated,
non-glycosylated turkey beta-adrenergic receptors for crystallization. Biochim Biophys
Acta 1610:133–140
74. Popot J-L (2010) Amphipols, nanodiscs, and fluorinated surfactants: three nonconventional
approaches to studying membrane proteins in aqueous solutions. Annu Rev Biochem
79:737–775
75. Zhang Q, Tao H, Hong W-X (2011) New amphiphiles for membrane protein structural
biology. Methods 55:318–323
76. Carpenter B, Tate CG (2016) Engineering a minimal G protein to facilitate crystallisation of

G protein-coupled receptors in their active conformation. Protein Eng Des Sel 29:583–594
77. Carpenter B, Tate CG (2017) Active state structures of G protein-coupled receptors highlight
the similarities and differences in the G protein and arrestin coupling interfaces. Curr Opin
Struct Biol 45:124–132
78. Magnani F, Serrano-Vega MJ, Shibata Y, Abdul-Hussein S, Lebon G, Miller-Gallacher J,
Singhal A, Strege A, Thomas JA, Tate CG (2016) A mutagenesis and screening strategy to
generate optimally thermostabilized membrane proteins for structural studies. Nat Protoc
11:1554–1571
79. Tate CG, Lebon G (2015) Purification and crystallization of a thermostabilized agonist-bound
conformation of the human adenosine A(2A) receptor. Methods Mol Biol 1335:17–27
80. Janin J, Miller S, Chothia C (1988) Surface, subunit interfaces and interior of oligomeric
proteins. J Mol Biol 204:155–164
81. Tugarinov V, Kay LE (2005) Methyl groups as probes of structure and dynamics in NMR
studies of high-molecular-weight proteins. Chembiochem 6:1567–1577
82. Rosenzweig R, Kay LE (2016) Solution NMR spectroscopy provides an avenue for the study
of functionally dynamic molecular machines: the example of protein disaggregation. J Am
Chem Soc 138:1466–1477
83. Kay LE (2005) NMR studies of protein structure and dynamics. J Magn Reson 173:193–207
84. Religa TL, Sprangers R, Kay LE (2010) Dynamic regulation of archaeal proteasome gate
opening as studied by TROSY NMR. Science 328:98–102
85. Katz JJ, Crespi HL (1966) Deuterated organisms: cultivation and uses. Science 151
(3715):1187–1194
86. Arcemisbehere L, Sen T, Boudier L, Balestre M-N, Gaibelet G, Detouillon E, Orcel H,
Mendre C, Rahmeh R, Granier S, Vivès C, Fieschi F, Damian M, Durroux T, Banères JL,
Mouillac B (2010) Leukotriene BLT2 receptor monomers activate the Gi2 GTP-binding
protein more efficiently than dimers. J Biol Chem 285:6337–6347
87. Banères J-L, Martin A, Hullot P, Girard J-P, Rossi J-C, Parello J (2003) Structure-based
analysis of GPCR function: conformational adaptation of both agonist and receptor upon
leukotriene B4 binding to recombinant BLT1. J Mol Biol 329:801–814
88. Banères J-L, Popot J-L, Mouillac B (2011) New advances in production and functional
folding of G-protein-coupled receptors. Trends Biotechnol 29:314–322
89. Mouillac B, Banères J-L (2010) Mammalian membrane receptors expression as inclusion
bodies in Escherichia coli. Methods Mol Biol 601:39–48
90. Huang KS, Bayley H, Liao M-J, London E, Khorana HG (1981) Refolding of an integral
membrane protein. Denaturation, renaturation, and reconstitution of intact bacteriorhodopsin
and two proteolytic fragments. J Biol Chem 256:3802–3809
91. Lind C, H€ ojeberg B, Khorana HG (1981) Reconstitution of delipidated bacteriorhodopsin
with endogenous polar lipids. J Biol Chem 256:8298–8305
92. London E, Khorana HG (1982) Denaturation and renaturation of bacteriorhodopsin in
detergents and lipid-detergent mixtures. J Biol Chem 257:7003–7011
93. Pocanschi CL, Dahmane T, Gohon Y, Rappaport F, Apell H-J, Kleinschmidt JH, Popot J-L
(2006) Amphipathic polymers: tools to fold integral membrane proteins to their active form.
Biochemistry 45:13954–13961
94. Popot J-L, Althoff T, Bagnard D, Banères J-L, Bazzacco P, Billon-Denis E, Catoire LJ,
Champeil P, Charvolin D, Cocco MJ, Crémel G, Dahmane T, de la Maza LM, Ebel C,
Gabel F, Giusti F, Gohon Y, Goormaghtigh E, Guittet E, Kleinschmidt JH, Kühlbrandt W,
Le Bon C, Martinez KL, Picard M, Pucci B, Sachs JN, Tribet C, van Heijenoort C, Wien F,
Zito F, Zoonens M (2011) Amphipols from A to Z. Annu Rev Biophys 40:379–408
95. Popot J-L (2014) Folding membrane proteins in vitro: a table and some comments. Arch
Biochem Biophys 564:314–326
96. Zoonens M, Popot J-L (2014) Amphipols for each season. J Membr Biol 247:759–796
97. Dahmane T, Damian M, Mary S, Popot J-L, Banères J-L (2009) Amphipol-assisted in vitro
folding of G protein-coupled receptors. Biochemistry 48:6516–6521
98. Muller I, Sarramégna V, Renault M, Lafaquière V, Sebai S, Milon A, Talmont F (2008) The
full-length μ-opioid receptor: a conformational study by circular dichroism in trifluoroethanol
and membrane-mimetic environments. J Membr Biol 223:49–57
99. Pucadyil TJ, Chattopadhyay A (2006) Role of cholesterol in the function and organization of
G-protein coupled receptors. Prog Lipid Res 45:295–333
100. Huber T, Botelho AV, Beyer K, Brown MF (2004) Membrane model for the G-protein-
coupled receptor rhodopsin: hydrophobic interface and dynamical structure. Biophys J
86:2078–2100
101. Chini B, Parenti M (2004) G-protein coupled receptors in lipid rafts and caveolae: how, when
and why do they go there? J Mol Endocrinol 32:325–338
102. Han DS, Wang SX, Weinstein H (2008) Active state-like conformational elements in the β2-
AR and a photoactivated intermediate of rhodopsin identified by dynamic properties of
GPCRs. Biochemistry 47:7317–7321
Top Med Chem (2019) 30: 53–64
DOI: 10.1007/7355_2018_62
© Springer Nature Switzerland AG 2019
Published online: 5 June 2019
Structure and Activation Mechanism

of GPCRs
A. J. Venkatakrishnan
Abstract G-protein-coupled receptors (GPCRs) mediate a wide variety of physio-

logical functions and are a rich source of drug targets. In response to activation by
extracellular stimuli, GPCRs trigger cytoplasmic signalling pathways through intra-
cellular partners such as G-proteins and arrestins. This chapter provides a general
overview of the molecular mechanisms of GPCR activation gleaned from crystal
structures, biophysical experiments, and computational analyses. Furthermore,
existing challenges and unresolved mechanistic questions about GPCR signalling
are highlighted.
Keywords Activation, Allostery, Dynamics, GPCR, G-protein binding, Ligand

binding, Structure
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
2 GPCR Signalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
3 Advances in GPCR Structural Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
4 Structural Features and Activation Mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
5 Allosteric Modulation of GPCRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
6 Structural Ensemble of GPCRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
7 Future Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
A. J. Venkatakrishnan (*)
Department of Computer Science, Stanford University, Stanford, CA, USA
Department of Molecular and Cellular Physiology, Stanford University School of Medicine,
Stanford, CA, USA
Institute for Computational and Mathematical Engineering, Stanford University, Stanford, CA,
USA
e-mail: ajvenkat@stanford.edu
54 A. J. Venkatakrishnan
1 Introduction
Membrane proteins expressed at the cell surface act as a communication interface

between the external and internal environments of a cell. There exist a variety of
membrane proteins such as receptors, channels, pumps, and transporters [1]. Among
them, G-protein-coupled receptors (GPCRs) constitute one of the largest and most
diverse membrane protein families; over 800 genes in the human genome encode
GPCRs [2]. GPCRs are organized into different classes (classes A–F) based on their
evolutionary relationships. As a family, GPCRs are modulated by a wide variety of
extracellular signals, encompassing photons, ions, amines, nucleosides, lipids, pep-
tides, and entire proteins. Upon activation by extracellular ligands, GPCRs bind and
activate intracellular signalling partners that in turn relay the signal further down-
stream, generating a cellular response. Standard treatments for numerous ailments
such as cardiac malfunction, asthma, and migraine exploit the ability of certain
medications to alter GPCR activity, including by reducing it (using antagonists or
inverse agonists) or augmenting it (using agonists). Given the tremendous diversity
of GPCRs, there remains enormous potential for the development of new drugs to
ameliorate neurological, oncological, and metabolic disorders. Thus, a mechanistic
understanding of how GPCRs function is not only of fundamental biological interest
but also holds great potential for improving human health.
This chapter is intended as a general introduction to GPCR structure and activa-
tion mechanism. First, I provide a brief overview of GPCR signalling. Next, I
discuss molecular aspects of GPCR structure and activation mechanisms. Finally, I
highlight both existing challenges associated with GPCRs at a molecular level as
well as unresolved mechanistic questions in GPCR signalling.
2 GPCR Signalling
GPCR-mediated signalling can occur via G-protein-dependent and G-protein-inde-

pendent pathways [3]. The classical signalling through GPCRs is mediated by
heterotrimeric G-proteins, comprised of α, β, and γ subunits. In the inactive state,
the Gα subunit of the heterotrimer is bound to GDP. Upon activation by extracellular
stimuli, GPCRs activate their G-protein partners by initiating nucleotide exchange,
i.e., exchange of GDP by GTP, and the dissociation of the trimer into subunits of
GαGTP and Gβγ. The subunits independently initiate downstream signalling cas-
cades through proteins such as adenylyl cyclase, phospholipases, MAP kinases, or
ion channels. Finally, cytosolic proteins that engage in posttranslational modification
and internalization of GPCRs regulate the signalling events [4]. Recent studies also
indicate that GPCR-mediated G-protein signalling occurs not only from the plasma
membrane but also from intracellular organelles such as the endosome and Golgi
membranes [5].
Structure and Activation Mechanism of GPCRs 55
GPCRs can also interact with other intracellular signalling partners such as
β-arrestin and G-protein receptor kinases [6]. Certain ligands are able to preferen-
tially trigger or suppress some of these signalling pathways, a phenomenon known
as “biased agonism” or “functional selectivity.” Biased ligands that can selectively
stimulate signalling through beneficial pathways while reducing signalling through
deleterious pathways may have a high therapeutic potential as drug candidates.
3 Advances in GPCR Structural Biology
GPCR structure determination had remained a formidable challenge for several

decades, owing to the receptor’s overall conformational flexibility and ability to
adopt multiple discrete conformations that readily exchange among each other.
However, in recent years, the combination of innovative protein engineering tech-
niques and developments in crystallography [7] resulted in an exponential growth
in the number of GPCR structures [8]. Protein engineering techniques include
designing chimeric receptors with T4 lysozyme, apocytochrome, BRIL, and co-
crystallization with antibody fragments or nanobody (single-domain antibody) and
thermostabilization by alanine scanning or engineering disulfide bridges. In addition
to protein engineering techniques, usage of high-affinity/low off-rate ligands com-
bined with advances in micro-crystallography and room-temperature crystallogra-
phy led to more structures. While determining crystal structures in an inactive state
has become increasingly tractable for many GPCRs, determining crystal structures
of active-state complexes, useful for identifying binding modes of GPCR agonists,
remains difficult for most receptors due to challenges in obtaining stable complexes
in homogenous conformations. So far, crystal structures of GPCR-transducer com-
plexes have been obtained for a very few GPCRs such as β2 adrenergic receptor
(β2AR) with G-protein [9], A2A receptor with engineered G-protein [10], and
rhodopsin with arrestin [11, 12]. However, recent advances in cryo-EM are driving
an increase in the structures of active-state complexes [13–18]. Overall, there are
over 250 structures of over 50 unique GPCRs (Fig. 1). While a major portion of these
structures are of class A GPCRs, an increasing number of structures of class B,
class C, and class F GPCRs are also being determined.
4 Structural Features and Activation Mechanism
GPCRs share a conserved structural fold (Fig. 2), and the structure of a GPCR can be
divided into three parts: (1) the extracellular region, consisting of the N-terminus and
three extracellular loops, (2) the transmembrane (TM) region consisting of seven
helices (TMs 1–7), and (3) the intracellular region consisting of three intracellular
loops, an intracellular amphipathic helix (helix 8), and the C-terminus. In a broad
sense, the extracellular region modulates ligand access; the transmembrane region
Class A (Rhodopsin) Class B1 (Secretin) Class B2 (Adhesion)

50 Class C (Glutamate) Class F (Frizzled) Taste 2 50
45 45
The number of unique crystallized receptors available
40 40
35 35
30 30
25 25
20 20
15 15
10 10
5 5
0 0
2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018
year
Fig. 1 Bar plot showing the growth of the number of GPCR structures (as of April 2018). The
y-axis denotes the number of unique GPCR structures determined and the x-axis denotes time
(year). The different GPCR classes are denoted using different colors as described in the color
legend. Source: http://www.gpcrdb.org
forms the structural core, binds ligands, and transduces the signal to the intracellular
region through conformational changes; and the intracellular region interfaces with
cytosolic signalling proteins.
The activation process of a GPCR involves multiple molecular process: ligand
binding at the extracellular interface, signal transduction through the transmembrane
region, and binding of transducers at the intracellular interface. The availability of
structures of different members of the GPCR family has helped us understand both
the structural properties shared by different GPCRs and the structural nuances of
individual GPCRs.
Most of GPCR structures have been determined in complex with an orthosteric
ligand, providing insights into properties of ligand binding and ligand action. More
than half of the receptor-ligand interactions involve residues present in the trans-
membrane region, and there is extensive diversity in the receptor-ligand interactions
across class A GPCRs [20]. Despite this diversity, many receptors share conserved
receptor-ligand interactions involving residues on transmembrane helices (TM) 3,
5, 6, and 7, which constitute a “ligand-binding cradle” [20]. Computational struc-
tural analyses focusing on shared receptor-ligand interactions and ligand similarity
Fig. 2 Structure of a GPCR shown using β2AR/G-protein complex as an example. Proteins are
shown using the ribbon representation, and the co-crystallized ligand is shown as spheres. The
receptor is shown in light brown, and the intracellular G-protein trimer is shown in green, cyan, and
magenta. The horizontal lines schematically mark the extracellular (EC) region, the transmembrane
(TM) region, and the intracellular (IC) region. The most conserved positions in transmembrane
helices and helix 8 can be referred to using Ballesteros-Weinstein numbers [19]
are currently being exploited to identify ligands for orphan receptors, which are
GPCRs whose endogenous ligands have not yet been identified [21, 22].
Crystal structures of different class A GPCRs have been determined in both
inactive state and active state (with intracellular binders): β2 adrenergic receptor
[9, 23], M2 muscarinic receptor (M2R) [24, 25], A2A receptor [14, 26], rhodopsin
[27, 28], μ-opioid receptor (μ-OR) [29, 30], and k-opioid receptor (k-OR)
[31, 32]. These pairs of structures are enabling us to understand the mechanisms
of receptor activation and transducer binding. In the binding pockets, one common
feature of activation is the contraction of the binding pocket [33, 34]. However, the
Fig. 3 Structural changes during GPCR activation shown using β2AR as an example. Inactive
(light pink) and active (dark purple) conformations of the β2AR show differences in helix position
and side-chain orientation in three different regions: the binding pocket (top, left); the connector
region, or conserved core triad (bottom, left); and the intracellular coupling site (top and bottom,
right). Figure source: Latorraca et al. [35]
binding pockets of agonists across the different receptors are markedly different
across the different GPCRs. In β2AR, polar interactions between the agonist and the
receptor stabilize a 2 Å inward movement of TM5 [9], and in M2R, polar interac-
tions with the agonist stabilize a 2 Å inward movement of TM6 [25]. On the other
hand, in μ-OR and k-OR, the agonists appear to be mediating their action through
TM3 and TM6 [29, 31]. Taken together, the differences in the agonist-receptor
interactions in different GPCRs suggest that the trigger points of agonist-associated
activation can be present on different helices in the GPCR fold.
These agonist-associated changes in the ligand-binding pocket are coupled to
structural changes in the transmembrane core of the receptor (Fig. 3). In the
transmembrane core, for a subset of class A GPCRs, there is a rearrangement of a
highly conserved triad of structurally equivalent residues on TM3 (Ile), TM5 (Pro),
and TM6 (Phe) [35, 36]. These changes in the transmembrane core are transmitted
through the helices resulting in changes in the cytoplasmic side. There is a large
outward movement of TM6 (5–14 Å) and smaller inward movement of TM7, which
opens up a cleft in the TM bundle where the C-terminus of the G-protein can bind
[20]. The range of motion in TM6 motion may partly be due to the diversity of the
co-crystallized intracellular binders such as nanobodies, G-protein, and engineered
G-protein.
The inactive and active states of class A GPCRs are stabilized by conserved
networks of interatomic interactions formed by residues present in the cytoplasmic
half of the transmembranes helices [37]. A salt bridge between TM6 and the DRY
motif of TM3 and a network of contacts involving residues on TM3, TM5, and TM6
contribute to stabilizing the inactive state [37]. On the other hand, the active state is
stabilized by direct polar interactions and water-mediated polar interactions between
the DRY motif, NPXXY motif, TM3, and TM5 and a network of contacts involving
TM3, TM6, and TM7 and the G-protein [37, 38].
Taken together, while the agonists and the agonist-binding interactions are
different among GPCRs, the activation pathways they initiate nonetheless converge
near the transducer-binding region [37]. Thus, from an evolutionary perspective,
GPCRs appear to have evolved varying ligand-binding pockets and mechanisms for
triggering activation while preserving similar conformational changes upon activa-
tion in the intracellular side in order to couple to a common set of cytoplasmic
proteins.
5 Allosteric Modulation of GPCRs
While most of the ligands modulating GPCR activity bind at the same site as the
endogenous ligand (“orthosteric site”), certain ligands can bind at other sites and still
alter GPCR function [39]. Such ligands are termed as “allosteric ligands.” Such
ligands typically act by modulating an orthosteric ligand’s pharmacological proper-
ties such as binding affinity and efficacy. Some allosteric ligands can also affect
receptor activation in the absence of orthosteric ligands. Structures of GPCRs
determined in complex with allosteric compounds show that allosteric sites can be
present in different parts of the GPCR fold (Fig. 4). Many allosteric regulators act at
the extracellular region, such as in the case of M2R, where the action of allosteric
ligands occurs through electrostatic repulsion and structural changes [41, 42].
Recently, allosteric modulators have been identified to bind at the intracellular
Fig. 4 Allosteric modulation sites in a GPCR. Allosteric modulation can be achieved from
different parts of the GPCR fold, as shown using class A GPCRs as an example. Structures
of allosteric modulators (colored spheres) mapped onto a representative class A GPCR
(M2 muscarinic receptor, PDB ID: 4MQT). The names of the receptors and the co-complexed
allosteric ligands are labelled. Dashed lines indicate the boundary of the lipid bilayer. Figure source:
Thal et al. [40]
region, as seen in the case of glucagon receptor [43, 44], CCR2 [44], CCR9 [45], and
β2AR [46]. These allosteric modulators act by stabilizing the conformations of
transmembrane helices [46]. Similar to small molecules, ions can have allosteric
modulation effects, as seen in the case of a conserved sodium ion buried in the
transmembrane bundle [47]. Overall, allosteric modulation in GPCRs can be
achieved from different sites on the GPCR fold, and the allosteric modulation sites
present new avenues for exploring druggable sites, particularly for achieving selec-
tivity between receptor subtypes.
6 Structural Ensemble of GPCRs
It is important to note that GPCRs are not bimodal with static inactive and active
states. In fact, they are dynamic structural entities that exist in a multitude of
conformations, whether bound to antagonists, to agonists, or to no ligand at all.
Even within a given conformational state, there are conformational fluctuations and
sub-states. For example, in the antagonist-bound crystal structures of inactive β1AR,
two different conformations of TM6 were observed, one with a TM3-TM6 ionic-
lock intact and the other one with this lock broken [48]. The presence of the ionic-
lock intact and ionic-lock broken states has also been observed in antagonist-bound
β2AR in double electron-electron resonance (DEER) spectroscopy experiments [49]
and molecular dynamics (MD) simulations [50]. The binding of an agonist to the
receptor increases the probability of formation of a fully active state, which is then
stabilized by binding of a transducer such as a G-protein. Binding of agonists alone
can favor the transition to “intermediate states” as seen, for example, in the agonist-
bound structures of A2A receptor [33] and serotonin receptors [51, 52]. The complete
transition of the receptor to the active conformation requires interaction of the
G-protein-coupling region with a G-protein or an intracellular G-protein mimetic,
as suggested by the DEER spectroscopy and (19)F-fluorine NMR studies on β2AR
[49]. Overall, the allosteric coupling between the ligand-binding site and the
G-protein-coupling region is loose rather than concerted [53, 54].
7 Future Outlook
While recent studies have led to a rapid increase in our understanding of the structure
and function of GPCRs, several important questions are yet to be answered. One
question is that of the molecular basis of biased signalling. A long-standing chal-
lenge was determining the structure of the GPCR-arrestin complex as it was
expected to provide an understanding of biased signalling. Recently this structure
was solved; however, it turned out to be very similar to the G-protein-interacting
conformation [11, 12]. Studies investigating the structure as well as dynamics of the
same receptor in complex with biased ligands and the corresponding transducer
proteins may help us understand molecular mechanisms of biased signalling.
In parallel to excellent advances in biophysical techniques such as crystallogra-
phy and cryo-EM, there have also been advances in computational methods and
increase in computational power. These developments have been playing a signif-
icant role in improving our understanding of the GPCR structure-function relation-
ship. MD simulations, for instance, have played an extensive role in understanding
GPCR functional properties such as allosteric modulation, receptor activation mech-
anism, mechanisms of activation of cytosolic proteins by GPCRs, ligand-binding
kinetics, and ligand-binding pathways [35, 55–57]. Similarly, computational ana-
lyses of structures have enabled the identification of evolutionarily conserved
properties in ligand binding, receptor activation, G-protein-coupling, and
G-protein activation [20, 37, 38]. In addition, structure-based computational docking
approaches are enabling the virtual screening of large compound libraries [58].
Looking forward, there is tremendous scope for developing new computational
methods that take advantage of our understanding of the GPCR structure-function
relationship. These include predicting thermostabilizing mutations [59], predicting
ligands with desired biased signalling properties, and predicting pathogenicity of
mutations.
The main focus of GPCR structural biology thus far has been on the structurally
ordered regions of the GPCR fold. However, large portions of the extramembrane
regions in GPCRs are predicted to be intrinsically disordered and are typically
removed to facilitate structural studies [60]. These intrinsically disordered regions
are typically in the N-terminus, C-terminus, or the third intracellular loop and have
important functional roles. For example, the C-terminus contains functional sites that
are implicated in activation of transducer proteins such as arrestin [55, 61]. The
mechanistic links between these disordered regions and the functional sites such as
the ligand-binding pocket and the G-protein-coupling region are yet to be fully
understood. With increasing efforts in studying the structure, disorder, and dynamics
of GPCRs, it is anticipated that many of the unanswered mechanistic questions will
be tackled in the near future. Overall, a comprehensive mechanistic understanding of
GPCRs holds promise for the development of safer and more effective drugs.
Acknowledgments The author acknowledges Stanford ChEM-H seed grant, Dror Lab, and
Kobilka Lab at Stanford for supporting his research and Guillaume Lebon, Naomi R. Latorraca,
Siri van Keulen, and Jonas Kaindl for critically reading the manuscript. The author has no conflicts
of interests.
References
1. Vinothkumar KR, Henderson R (2010) Structures of membrane proteins. Q Rev Biophys

43(1):65–158
2. Fredriksson R, Lagerström MC, Lundin L-G, Schiöth HB (2003) The G-protein-coupled

receptors in the human genome form five main families. Phylogenetic analysis, paralogon
groups, and fingerprints. Mol Pharmacol 63(6):1256–1272
3. Hilger D, Masureel M, Kobilka BK (2018) Structure and dynamics of GPCR signaling
complexes. Nat Struct Mol Biol 25(1):4–12
4. Ritter SL, Hall RA (2009) Fine-tuning of GPCR activity by receptor-interacting proteins. Nat
Rev Mol Cell Biol 10(12):819–830
5. Eichel K, von Zastrow M (2018) Subcellular organization of GPCR signaling. Trends
Pharmacol Sci 39(2):200–208
6. Rajagopal S, Rajagopal K, Lefkowitz RJ (2010) Teaching old receptors new tricks: biasing
seven-transmembrane receptors. Nat Rev Drug Discov 9(5):373–386
7. Tate CG, Schertler GFX (2009) Engineering G protein-coupled receptors to facilitate their
structure determination. Curr Opin Struct Biol 19(4):386–395
8. Pándy-Szekeres G, Munk C, Tsonkov TM, Mordalski S, Harpsøe K, Hauser AS, Bojarski AJ,
Gloriam DE (2018) GPCRdb in 2018: adding GPCR structure models and ligands. Nucleic
Acids Res 46(D1):D440–D446
9. Rasmussen SGF, DeVree BT, Zou Y, Kruse AC, Chung KY, Kobilka TS, Thian FS et al (2011)
Crystal structure of the β2 adrenergic receptor-Gs protein complex. Nature 477(7366):549–555
10. Carpenter B, Nehmé R, Warne T, Leslie AGW, Tate CG (2016) Structure of the adenosine A
(2A) receptor bound to an engineered G protein. Nature 536(7614):104–107
11. Kang Y, Zhou XE, Gao X, He Y, Liu W, Ishchenko A, Barty A et al (2015) Crystal structure of
rhodopsin bound to arrestin by femtosecond X-ray laser. Nature 523(7562):561–567
12. Zhou XE, He Y, de Waal PW, Gao X, Kang Y, van Eps N, Yin Y et al (2017) Identification
of phosphorylation codes for arrestin recruitment by G protein-coupled receptors. Cell
170(3):457–469.e13
13. Draper-Joyce CJ, Khoshouei M, Thal DM, Liang Y-L, Nguyen ATN, Furness SGB, Venugopal
H et al (2018) Structure of the adenosine-bound human adenosine A receptor-G complex.
Nature 558(7711):559–563
14. García-Nafría J, Lee Y, Bai X, Carpenter B, Tate CG (2018) Cryo-EM structure of the
adenosine A receptor coupled to an engineered heterotrimeric G protein. elife 7:e35946.
https://doi.org/10.7554/eLife.35946
15. Koehl A, Hu H, Maeda S, Zhang Y, Qu Q, Paggi JM, Latorraca NR et al (2018) Structure of the
μ-opioid receptor-G protein complex. Nature 558(7711):547–552
16. Liang Y-L, Khoshouei M, Radjainia M, Zhang Y, Glukhova A, Tarrasch J, Thal DM et al
(2017) Phase-plate cryo-EM structure of a class B GPCR-G-protein complex. Nature
546(7656):118–123
17. Thal DM, Vuckovic Z, Draper-Joyce CJ, Liang Y-L, Glukhova A, Christopoulos A, Sexton PM
(2018) Recent advances in the determination of G protein-coupled receptor structures. Curr
Opin Struct Biol 51:28–34
18. Zhang Y, Sun B, Feng D, Hu H, Chu M, Qu Q, Tarrasch JT et al (2017) Cryo-EM structure of
the activated GLP-1 receptor in complex with a G protein. Nature 546(7657):248–253
19. Ballesteros JA, Weinstein H (1995) [19] Integrated methods for the construction of
three-dimensional models and computational probing of structure-function relations in
G protein-coupled receptors. Methods Neurosci 25:366–428
20. Venkatakrishnan AJ, Deupi X, Lebon G, Tate CG, Schertler GF, Babu MM (2013) Molecular
signatures of G-protein-coupled receptors. Nature 494(7436):185–194
21. Ngo T, Ilatovskiy AV, Stewart AG, Coleman JLJ, McRobb FM, Riek RP, Graham RM,
Abagyan R, Kufareva I, Smith NJ (2017) Orphan receptor ligand discovery by pickpocketing
pharmacological neighbors. Nat Chem Biol 13(2):235–242
22. Wacker D, Stevens RC, Roth BL (2017) How ligands illuminate GPCR molecular pharmacol-
ogy. Cell 170(3):414–427
23. Cherezov V, Rosenbaum DM, Hanson MA, Rasmussen SGF, Thian FS, Kobilka TS, Choi H-J
et al (2007) High-resolution crystal structure of an engineered human beta2-adrenergic G
protein-coupled receptor. Science 318(5854):1258–1265
24. Haga K, Kruse AC, Asada H, Yurugi-Kobayashi T, Shiroishi M, Zhang C, Weis WI et al (2012)
Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist. Nature
482(7386):547–551
25. Kruse AC, Ring AM, Manglik A, Hu J, Hu K, Eitel K, Hübner H et al (2013) Activation and
allosteric modulation of a muscarinic acetylcholine receptor. Nature 504(7478):101–106
26. Jaakola V-P, Griffith MT, Hanson MA, Cherezov V, Chien EYT, Robert Lane J, Ijzerman AP,
Stevens RC (2008) The 2.6 angstrom crystal structure of a human A2A adenosine receptor
bound to an antagonist. Science 322(5905):1211–1217
27. Choe H-W, Kim YJ, Park JH, Morizumi T, Pai EF, Krauss N, Hofmann KP, Scheerer P,
Ernst OP (2011) Crystal structure of metarhodopsin II. Nature 471(7340):651–655
28. Palczewski K, Kumasaka T, Hori T, Behnke CA, Motoshima H, Fox BA, Le Trong I
et al (2000) Crystal structure of rhodopsin: a G protein-coupled receptor. Science
289(5480):739–745
29. Huang W, Manglik A, Venkatakrishnan AJ, Laeremans T, Feinberg EN, Sanborn AL, Kato HE
et al (2015) Structural insights into μ-opioid receptor activation. Nature 524(7565):315–321
30. Manglik A, Kruse AC, Kobilka TS, Thian FS, Mathiesen JM, Sunahara RK, Pardo L, Weis WI,
Kobilka BK, Granier S (2012) Crystal structure of the μ-opioid receptor bound to a morphinan
antagonist. Nature 485(7398):321–326
31. Che T, Majumdar S, Zaidi SA, Ondachi P, McCorvy JD, Wang S, Mosier PD et al
(2018) Structure of the nanobody-stabilized active state of the kappa opioid receptor. Cell
172(1–2):55–67.e15
32. Wu H, Wacker D, Mileni M, Katritch V, Han GW, Vardy E, Liu W et al (2012) Structure of the
human κ-opioid receptor in complex with JDTic. Nature 485(7398):327–332
33. Lebon G, Warne T, Edwards PC, Bennett K, Langmead CJ, Leslie AGW, Tate CG (2011)
Agonist-bound adenosine A2A receptor structures reveal common features of GPCR activation.
Nature 474(7352):521–525
34. Manglik A, Kruse AC (2017) Structural basis for G protein-coupled receptor activation.
Biochemistry 56(42):5628–5634
35. Latorraca NR, Venkatakrishnan AJ, Dror RO (2017) GPCR dynamics: structures in motion.
Chem Rev 117(1):139–155
36. Deupi X, Standfuss J (2011) Structural insights into agonist-induced activation of G-protein-
coupled receptors. Curr Opin Struct Biol 21(4):541–551
37. Venkatakrishnan AJ, Deupi X, Lebon G, Heydenreich FM, Flock T, Miljus T, Balaji S et al
(2016) Diverse activation pathways in class A GPCRs converge near the G-protein-coupling
region. Nature 536(7617):484–487
38. Flock T, Ravarani CNJ, Sun D, Venkatakrishnan AJ, Kayikci M, Tate CG, Veprintsev DB,
Babu MM (2015) Universal allosteric mechanism for Gα activation by GPCRs. Nature
524(7564):173–179
39. Wootten D, Christopoulos A, Sexton PM (2013) Emerging paradigms in GPCR allostery:
implications for drug discovery. Nat Rev Drug Discov 12(8):630–644
40. Thal DM, Glukhova A, Sexton PM, Christopoulos A (2018) Structural insights into G-protein-
coupled receptor allostery. Nature 559(7712):45–53
41. Dror RO, Green HF, Valant C, Borhani DW, Valcourt JR, Pan AC, Arlow DH et al (2013)
Structural basis for modulation of a G-protein-coupled receptor by allosteric drugs. Nature
503(7475):295–299
42. Hertig S, Latorraca NR, Dror RO (2016) Revealing atomic-level mechanisms of protein
allostery with molecular dynamics simulations. PLoS Comput Biol 12(6):e1004746
43. Jazayeri A, Doré AS, Lamb D, Krishnamurthy H, Southall SM, Baig AH, Bortolato A et al
(2016) Extra-helical binding site of a glucagon receptor antagonist. Nature 533(7602):274–277
44. Zheng Y, Qin L, Zacarías NVO, de Vries H, Han GW, Gustavsson M, Dabros M et al (2016)
Structure of CC chemokine receptor 2 with orthosteric and allosteric antagonists. Nature
540(7633):458–461
45. Oswald C, Rappas M, Kean J, Doré AS, Errey JC, Bennett K, Deflorian F et al (2016)
Intracellular allosteric antagonism of the CCR9 receptor. Nature 540(7633):462–465
46. Liu X, Ahn S, Kahsai AW, Meng K-C, Latorraca NR, Pani B, Venkatakrishnan AJ et al (2017)
Mechanism of intracellular allosteric βAR antagonist revealed by X-ray crystal structure. Nature
548(7668):480–484
47. Katritch V, Fenalti G, Abola EE, Roth BL, Cherezov V, Stevens RC (2014) Allosteric sodium
in class A GPCR signaling. Trends Biochem Sci 39(5):233–244
48. Moukhametzianov R, Warne T, Edwards PC, Serrano-Vega MJ, Leslie AGW, Tate CG,
Schertler GFX (2011) Two distinct conformations of helix 6 observed in antagonist-bound
structures of a beta1-adrenergic receptor. Proc Natl Acad Sci U S A 108(20):8228–8232
49. Manglik A, Kim TH, Masureel M, Altenbach C, Yang Z, Hilger D, Lerch MT et al (2015)
Structural insights into the dynamic process of β2-adrenergic receptor signaling. Cell
161(5):1101–1111
50. Dror RO, Arlow DH, Borhani DW, Jensen MØ, Piana S, Shaw DE (2009) Identification of two
distinct inactive conformations of the beta2-adrenergic receptor reconciles structural and bio-
chemical observations. Proc Natl Acad Sci U S A 106(12):4689–4694
51. Wacker D, Wang C, Katritch V, Han GW, Huang X-P, Vardy E, McCorvy JD et al (2013)
Structural features for functional selectivity at serotonin receptors. Science 340(6132):615–619
52. Wang C, Jiang Y, Ma J, Huixian W, Wacker D, Katritch V, Han GW et al (2013) Structural
basis for molecular recognition at serotonin receptors. Science 340(6132):610–614
53. Dror RO, Arlow DH, Maragakis P, Mildorf TJ, Pan AC, Xu H, Borhani DW, Shaw DE
(2011) Activation mechanism of the β2-adrenergic receptor. Proc Natl Acad Sci U S A
108(46):18684–18689
54. Weis WI, Kobilka BK (2018) The molecular basis of G protein-coupled receptor activation.
Annu Rev Biochem 87:897–919
55. Latorraca NR, Wang JK, Bauer B, Townshend RJL, Hollingsworth SA, Olivieri JE, Xu HE,
Sommer ME, Dror RO (2018) Molecular mechanism of GPCR-mediated arrestin activation.
Nature 557(7705):452–456
56. Marino KA, Shang Y, Filizola M (2017) Insights into the function of opioid receptors from
molecular dynamics simulations of available crystal structures. Br J Pharmacol 175:2834–2845.
https://doi.org/10.1111/bph.13774
57. Vaidehi N, Bhattacharya S (2016) Allosteric communication pipelines in G-protein-coupled
receptors. Curr Opin Pharmacol 30:76–83
58. Rodríguez D, Ranganathan A, Carlsson J (2015) Discovery of GPCR ligands by molecular
docking screening: novel opportunities provided by crystal structures. Curr Top Med Chem
15(24):2484–2503
59. Vaidehi N, Grisshammer R, Tate CG (2016) How can mutations thermostabilize G-protein-
coupled receptors? Trends Pharmacol Sci 37(1):37–46
60. Venkatakrishnan AJ, Flock T, Prado DE, Oates ME, Gough J, Madan Babu M (2014)
Structured and disordered facets of the GPCR fold. Curr Opin Struct Biol 27:129–137
61. Sente A, Peer R, Srivastava A, Baidya M, Lesk AM, Balaji S, Shukla AK, Babu MM, Flock T
(2018) Molecular mechanism of modulating arrestin conformation by GPCR phosphorylation.
Nat Struct Mol Biol 25(6):538–545
Top Med Chem (2019) 30: 65–100
DOI: 10.1007/7355_2016_25
Published online: 25 May 2017
Structure-Based Discovery of GPCR

Ligands from Crystal Structures
and Homology Models
Anirudh Ranganathan, David Rodrı́guez, and Jens Carlsson
Abstract The G protein-coupled receptor (GPCR) superfamily constitutes the

largest group of human membrane proteins and plays key roles in diverse cellular
processes. Major advances in structural biology for GPCRs have provided invalu-
able insights into ligand recognition and signaling for these important drug targets.
Access to high-resolution crystal structures also enables rational ligand design and
in silico methods based on atomic-resolution models are likely to play an increas-
ingly important role in future drug development. In this chapter, examples of ligand
discovery efforts based on molecular docking screening against GPCR crystal
structures will be presented first. Results from these studies suggest that crystal
structures can not only guide discovery of ligands but also predict their selectivity
and signaling properties. As experimental structures are not available for a large
fraction of the superfamily, methods for atomic-resolution modeling of GPCR–
ligand complexes could make important contributions to drug discovery. In the
second part of the chapter, the state-of-the-art in this area is discussed in light of
three community-wide assessments, which have challenged the modeling commu-
nity to blindly predict GPCR structures. Finally, several recent examples of suc-
cessful ligand discovery efforts utilizing atomic-resolution models for GPCRs of
unknown structure are summarized.
The authors “Anirudh Ranganathan” and “David Rodrı́guez” contributed equally to this work.
A. Ranganathan and D. Rodrı́guez
Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm
University, SE-10691 Stockholm, Sweden
J. Carlsson (*)
Science for Life Laboratory, Department of Cell and Molecular Biology, Biomedical Center,
Uppsala University, SE-75124 Uppsala, Sweden
e-mail: jens.carlsson@icm.uu.se
66 A. Ranganathan et al.
Keywords Agonist, Antagonist, Comparative modeling, Drug discovery,

Fragment-based lead discovery, G protein-coupled receptor, Homology modeling,
Molecular docking, Structure-based drug design, Virtual screening
Contents
1 Introduction: A New Era in Structural Biology and Drug Design for GPCRs . . . . . . . . . . . . . 67
1.1 Advances in GPCR Structural Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
1.2 GPCR Structure Prediction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
1.3 Molecular Docking Screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
2 Molecular Docking Screening for GPCR Ligands Using Crystal Structures . . . . . . . . . . . . . . 74
2.1 Adrenergic Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
2.2 Adenosine Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
2.3 Dopamine Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
2.4 C-X-C Chemokine Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
2.5 Histamine Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
2.6 Opioid Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
2.7 Muscarinic Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
2.8 Serotonin Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
2.9 Docking Screens Against GPCR Crystal Structures: Opportunities and Limitations 83
3 Modeling GPCRs of Unknown Structure and Their Complexes with Ligands . . . . . . . . . . . . 84
3.1 Community-Wide Assessments for Prediction of GPCR–Ligand Complexes . . . . . . . 85
3.2 Docking Screens Using Homology Models of GPCRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
4 Conclusions and Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Abbreviations
5-HT 5-Hydroxytryptamine, serotonin

ADR Adrenergic receptor
AR Adenosine receptor
CNS Central nervous system
CXCR C-X-C chemokine receptor
DR Dopamine receptor
EL Extracellular loop
FBLD Fragment-based lead discovery
FLAP Fingerprints for ligands and proteins
HR Histamine receptor
HTS High-throughput screening
IFP Interaction fingerprint
IL Intracellular loop
MC Monte Carlo
MD Molecular dynamics
mGluR Metabotropic glutamate receptor
MR Muscarinic receptor
Structure-Based Discovery of GPCR Ligands 67
MW Molecular weight
NAM Negative allosteric modulator
NECA 50 -N-ethylcarboxamidoadenosine
OR Opioid receptor
PAINS Pan-assay interference compounds
PAM Positive allosteric modulator
PD Parkinson’s disease
RMSD Root-mean-square deviation
SMO Smoothened receptor
TAAR1 Trace amine-associated receptor 1
TINS Target immobilized NMR screening
TM Transmembrane helix
1 Introduction: A New Era in Structural Biology and Drug

Design for GPCRs
G protein-coupled receptors (GPCRs) are ubiquitous membrane proteins with a

conserved topology consisting of seven transmembrane helices (TMs) connected
by three intracellular and three extracellular loops (ILs, ELs). The approximately
800 members of the superfamily are responsible for the transduction of signals into
the cytoplasm. Human GPCRs are divided into five main classes: Rhodopsin-like
(class A), Secretin (class B), Adhesion, Glutamate (class C), and Frizzled/Smooth-
ened (class F) families, of which the first is by far the most populated [1, 2]. These
receptors have evolved to act in response to extracellular stimuli from diverse
sources, including neurotransmitters, peptides, proteins, lipids, amino acids, or even
light. Binding of the endogenous ligand to a GPCR leads to the activation of
intracellular signaling pathways, mediated by G proteins, β-arrestins, and other
effectors [3]. There are multiple GPCRs within each class that recognize the same
endogenous compound, which are grouped together to constitute a receptor family.
Each unique family member is referred to as a receptor subtype, which can have
varying levels of expression across different tissue types and produce specific
downstream signaling effects. Medicinal chemistry and pharmacology efforts have
resulted in the discovery of compounds that activate GPCRs (agonists or partial
agonists) or block (antagonists) the action of the endogenous compound. The basal
activity of a GPCR, i.e., the level of signaling achieved by the receptor in absence of
the natural agonist, can also be reduced by compounds referred to as inverse ago-
nists. These three classes of compounds occupy a binding (orthosteric) site that is
located in the extracellular half of the receptor (Fig. 1). In addition, GPCR signaling
can be modulated by allosteric ligands, which bind to non-orthosteric sites. The suc-
cessful identification of potent GPCR ligands has led to the development of numerous
drugs. Remarkably, GPCRs account for the action of approximately 30% of the drugs
currently on the market [5]. Drug discovery programs continue to focus on the
Fig. 1 Structure of a typical class A (rhodopsin-like) GPCR, exemplified by the A2AAR (PDB
code 4EIY) [4]. The seven transmembrane (TM) receptor helices are shown as gray cartoon and
the orthosteric binding pocket as a blue mesh
identification of GPCR ligands for new indications and, in particular, on receptors

that have not yet been explored as therapeutic targets.
During the last decade, high-throughput approaches for experimental testing of
compound activities have been developed to increase the efficiency of the drug
discovery process. High-throughput screening (HTS), which can be used to assay
chemical libraries of several hundred thousand compounds against a drug target, is
now widely applied to identify starting points for lead development. Although HTS
has been successful in many cases, this technique is costly and sometimes returns
few hits for challenging targets [6]. An alternative approach is to utilize virtual
screening methods. These rely on computer algorithms to search for compounds
with a desired bioactivity in large chemical libraries and prioritize only a small
number of molecules for experimental testing [7, 8]. One class of virtual screening
approaches employs information from atomic-resolution structures of the target
protein to identify ligands. The molecular docking method can be used to screen
millions of compounds in the search for molecules that complement the structure of
a receptor binding site [9]. However, it has been challenging to use this technique to
discover GPCR ligands due to the paucity of available receptor structures. Break-
throughs in membrane protein crystallography during the last 8 years have led to the
determination of the first crystal structures for drug targets from the GPCR super-
family, which makes it possible to apply structure-based methods to discover
ligands [10–12]. The main focus of this chapter will be the impact of the increase
in structural knowledge for GPCRs on the use of molecular docking. Prospective
applications of virtual screening using experimental and computationally modeled

GPCR structures will be discussed.
1.1 Advances in GPCR Structural Biology
GPCRs are highly flexible proteins that exist in a dynamic conformational equilib-
rium between different functional states [13]. The structural plasticity of GPCRs, as
well as their instability under standard conditions for protein crystallization, has
hampered the determination of atomic-resolution structures [14]. In 2000, the first
GPCR crystal structure was determined for the light-sensing bovine rhodopsin. It
took another 7 years before the first high-resolution structure of a GPCR recognizing
a diffusible ligand was determined, which revealed the 3D coordinates of the human
β2 adrenergic receptor (β2ADR) in complex with the inverse agonist carazolol
[15, 16]. This breakthrough was a result of several important advances in membrane
protein structural biology, which have enabled the subsequent determination of
structures for 32 unique GPCRs belonging to 20 different receptor families
[11, 14]. GPCR crystal structures have had a staggering impact on our understanding
of ligand recognition and receptor signaling at the atomic level. Receptors have been
crystallized bound to ligands that stabilize different functional states, revealing
details of the conformational changes involved in receptor activation [10, 17]. An
important milestone was the crystallization of a ternary complex for the β2ADR
with an agonist and intracellular Gs protein, which captured the structural changes
associated with the signal transduction across the membrane mediated by this
receptor [18]. The diversity in the shape of orthosteric sites and location of allosteric
pockets for class A, B, C, and F GPCRs is remarkable [10, 12, 19] and provides new
insights into how safer and more efficacious drugs can be developed. Crystal
structures of receptors from the same receptor family open up opportunities to design
subtype-selective drugs and the identification of allosteric pockets has allowed for an
exploration of alternative approaches for target modulation. Although only a small
number of GPCRs have been crystallized, access to atomic-level information has
already enabled successful discovery of ligands from virtual screens and guided
ligand optimization efforts, suggesting that we are approaching a new era in GPCR
drug discovery [11, 12]. Recent studies in this area will be summarized in Sect. 2.
1.2 GPCR Structure Prediction
Despite the rapid increase in the number of available GPCR crystal structures in
recent years, the structural knowledge within the superfamily is still very limited.
Experimental structures for around 300 druggable GPCRs are still unknown [1],
and at the current rate of crystal structure determination, the shortfall will continue
well into the foreseeable future. To bridge this gap, computational methods for
protein structure prediction could be used to model the many pharmaceutically

relevant receptors that have not yet been crystallized.
Due to the lack of structural information, ab initio methods were first applied to
model GPCRs, as such approaches only require the sequence of the target protein.
These methods have occasionally been tailored for GPCRs, where the 7TM bundle
is constructed from the constituent helices using Monte Carlo (MC) or Molecular
Dynamics (MD) sampling [20–22]. After the atomic-resolution structure of rho-
dopsin was determined, comparative modeling approaches became widely used in
GPCR structure prediction [23]. These methods can be divided into fold recognition
(threading) and homology modeling approaches [24]. Both techniques require
structural templates and are based on the principles that the number of protein
folds appears to be finite (fold recognition) and that homologous proteins have
similar structures (homology modeling). Fold recognition approaches are primarily
used when there are no known structures with significant sequence similarity to the
target. Initial models are generated by threading its sequence onto experimental
protein structures in a database. Homology modeling instead relies on the sequence
alignment of the target to one or several templates and requires significant sequence
similarity to result in reliable results. The target structure is then modeled based on
the alignment, e.g., in the widely used program Modeller [25], this is achieved by
optimizing spatial restraints derived from the template structure(s) and the sequence
alignment. Refinement of the comparative models is often performed using con-
formational sampling approaches such as normal modes, MD, or MC simulations
and is ideally also guided by experimental data [24]. This step is especially
important for protein regions that are inherently flexible and structurally divergent
from the template(s). Loop modeling for GPCRs is very challenging, which has led
to the development of strategies dedicated to tackling this problem [26–29]. Com-
parative modeling is often an iterative process, which can be guided by the
stereochemical quality of predicted structures and statistical potentials for recog-
nition of native-like protein conformations [30, 31]. In virtual screening applica-
tions, models are often also benchmarked for their ability to recognize known
ligands of the target [32] (see Sect. 1.3).
Several online servers have been developed specifically for GPCR structure
prediction [33–37] and homology modeling has been the most widely used tech-
nique for this purpose [23]. The strongly conserved topology of GPCRs makes this
approach particularly suitable for modeling of these targets, even in cases with low
sequence identity (<20–30%) to available templates. Despite the relatively low
number of GPCR structures that could be predicted with high confidence when only
rhodopsin was available as a template, receptor models from this time did guide the
discovery of ligands [38–44]. In this book chapter, particular focus will be put on
the GPCR Dock assessments, which are community-wide competitions for blind
prediction of crystal structures of GPCRs in complex with ligands [45–47]. The
lessons learnt from benchmarking GPCR structure and ligand binding mode pre-
dictions, together with examples of ligand discovery using homology models, will
be discussed in Sect. 3.
1.3 Molecular Docking Screening
Molecular docking is a computational technique that aims to predict the binding

mode of a ligand in a protein structure and the affinity of the resulting complex
(Fig. 2). Docking algorithms consist of two steps: (1) sampling of ligand confor-
mations in the binding pocket, and (2) estimation of the binding energy of these
using a scoring function assessing the complementarity of the predicted ligand–
receptor complexes [9]. A requirement for docking is the tertiary structure of the
target protein, which could either be obtained experimentally (e.g., by X-ray
crystallography or NMR) or modeled computationally (e.g., by homology model-
ing). Several approximations are typically introduced in the sampling and scoring
steps to achieve sufficient computational efficiency. For instance, the search space
is restricted to a specific region of the receptor, e.g., the orthosteric site of a GPCR,
Fig. 2 Overview of a protocol for molecular docking screening against GPCR crystal structures or
models. In the lower right corner, the orthosteric site of the A2AAR is shown here as gray cartoon.
Key residues and the predicted binding mode of a ligand discovered from docking screens [48] are
shown in sticks
and the binding site is often held rigid. The binding site also has to be prepared by
assigning protonation states to ionizable residues and inclusion of potential cofac-
tors. If high-resolution structures are available, consideration of conserved water
molecules can also be important to enhance docking performance [49, 50]. Several
types of sampling techniques and scoring functions have been developed during the
last 30 years, resulting in numerous docking programs, e.g., DOCK, ICM,
AutoDock, GLIDE, PLANTS, and GOLD [51–56] (further details regarding this
topic can be found in dedicated reviews [9, 57, 58]). The performance of docking
protocols can be assessed prior to carrying out screens for novel ligands. A common
evaluation metric is to test if the binding modes for ligands that have been
co-crystallized with the target structure can be reproduced (referred to as “re-
docking”). Another standard benchmarking test is to challenge the docking pro-
gram to prioritize known ligands of the receptor over decoy molecules
(non-binders) [59]. The latter approach has been shown to be particularly useful
for assessment of GPCR models, which is often referred to as ligand-guided or
ligand-steered homology modeling [32, 60, 61]. Based on the results of such
retrospective assessments, the parameters of the docking program can be refined.
This can include optimization of the sampling settings, use of additional steps such
as pose rescoring, or evaluation of multiple receptor conformations (e.g., crystal
structures or homology models) [32, 62].
Access to efficient algorithms and super computer clusters have made it possible
to use molecular docking to screen chemical libraries containing millions of com-
pounds. The screening library can be selected from in-house sources or commercial
vendors. In the latter case, web resources for searching the commercially available
chemical space (e.g., ZINC [63] and eMolecules [64]) have facilitated access to
large databases of compounds. Physicochemical filters based on drug-likeness such
as Lipinski’s rule of five [65], substructures known for pan-assay interference
compounds (PAINS) [66], or target-based features (e.g., formal charge, functional
groups, and pharmacophores) can be applied to enrich the library with molecules
possessing desirable properties. The protonation, tautomerization, and enantiomeric
possibilities for each compound at the relevant pH should also be considered. After
the docking screen has been carried out, the molecules in the library are ranked
based on their predicted binding affinity. The list of top-ranked compounds can also
be clustered for chemical diversity and compared to known ligands to assess novelty.
In addition, the top-ranked molecules are visually inspected to select candidates for
experimental evaluation. In this step, knowledge about the receptor (e.g., key
interactions for ligand recognition), approximations in the docking algorithm (e.g.,
missing energy terms in the scoring function), the composition of the screening
library, and project-specific considerations can be incorporated into the selection
criteria. Finally, the selected molecules are tested experimentally for activity. The
success of a docking screen is quantified by the hit-rate, which is the percentage of
identified ligands from those predicted and evaluated experimentally. Hits from a
screen can be further optimized guided by the predicted binding modes. In the
following sections, examples of successful docking screens for GPCRs will be
presented and the results of these are summarized in Table 1.
Table 1 Summary of results from selected prospective docking screens against GPCRs
Efficacy
Family Receptor PDB code(s)a crystal ligand Results vs. Stated aims Reference Section
β-ADR β2ADR 2RH1 Inverse Hits: 31/150 (21%) [67] 2.1
agonist Hits: 6/25 (24%) [68]
β2ADR 3P0G, 2RH1b Agonist Hits: 6/22 (27%) [69] 2.1
Agonists: 6/22 (27%)
AR A2AAR 3EML Antagonist Hits: 23/56 (41%) [70] 2.2
Hits: 7/20 (35%) [48]
Hits: 14/22 (64%) [71]
A2AAR 2YDO, 2YDV Agonist Hits: 9/20 (45%) [72] 2.2
3QAK, 3EMLb Agonists: 0/20 (0%)
A1AR 3EML Antagonist A1AR hits: 8/39 (21%) [73] 3.2
(template) A1AR selective: 1/39
(3%)
A2AAR 2VT4 Antagonist Hits: 20/230 (9%) [74] 3.2
(template)
A3AR 4EIY Antagonist A3AR hits: 8/21 (38%) [75] 3.2
(template) A3AR selective: 6/21
(29%)
DR D3DR 3PBL Antagonist Hits: 5/25 (20%) [76] 2.3
Hits: 25/92 (27%) [77]
D3DR 3PBL Antagonist Bitopic hits: 14/25 [78] 2.3
(56%)
Allosteric hits: 8/25
(32%)
NAMs: 4/25 (16%)
PAMS: 2/25 (8%)
D3DR 2VT4, 2RH1 Antagonist Hits: 6/26 (23%) [76] 3.2
(templates)
D2DR 3P0G Agonist Hits: 3/15 (20%) [69] 3.2
(template) Antagonist Agonists: 2/15 (13%)
3PBLb,d
CXCR CXCR4 3ODU Antagonist Hits: 4/23 (17%) [79] 2.4
CXCR4 3ODU Antagonist CXCR3 hits: 4/7 (57%) [80] 3.2
CXCR3 3ODU CXCR3 selective: 4/7
(template)c (57%)
Hits: 3/4 (75%)
Dual binders: 2/4
(50%)
CXCR4 hits: 3/6
(50%)
CXCR4 selective: 3/6
(50%)
CXCR4 1U19, 2VT4, Antagonist Hits: 1/23 (4%) [79] 3.2
2RH1, 3EML
(templates)
(continued)
Table 1 (continued)
Efficacy
Family Receptor PDB code(s)a crystal ligand Results vs. Stated aims Reference Section
HR H1HR 3RZE Antagonist Hits: 19/26 (73%) [81] 2.5
H3HR 3RZE Antagonist Hits: 18/29 (62%) [82] 3.2
(template)
H4HR 3RZE, 2RH1 Antagonist Hits: 9/37 (24%) [83] 3.2
(templates) Inverse Hits: 15/85 (18%) [77]
agonist
OR κ-OR 4DJH Antagonist κ-OR hits: 4/22 (18%) [84] 2.6
κ-OR selective: 1/22
(5%)
MR M2MR 3UON Antagonist Hits: 11/18 (61%) [85] 2.7
M3MR 4DAJ Antagonist M3MR hits: 8/16 (50%) 2.7
M2MRc 3UON M3MR selective: 1/16
(6%)
5-HT 5-HT1B 4IAR Agonist 5-HT1B hits: 11/22 [86] 2.8
5-HT2Bc 4IB4 Biased (50%)
agonist 5-HT1B selective: 9/22
(41%)
TAAR TAAR1 2RH1 Inverse Hits: 9/42 (21%) [87] 3.2
(template) agonist
a
PDB codes for the crystal structures used in the docking screens. If homology models were used,
the PDB codes of the templates are shown
b
Selection of predicted agonists considered poor ranks in docking screens against antagonist-
bound structures
c
Selection of ligands with specific selectivity profiles was based on screens against several
structures, e.g. the target and antitarget
d
The crystal structure of the D3DR (3PBL) was used as a model of the inactive D2DR
2 Molecular Docking Screening for GPCR Ligands Using

Crystal Structures
2.1 Adrenergic Receptors
Adrenergic receptors (ADRs) recognize epinephrine (adrenaline) and noradrenaline

as their endogenous ligands. They are classified into α- and β-adrenergic receptors,
which are further subdivided into six (α1A, α1B, α1D; α2A, α2B, and α2C) and three
(β1, β2, and β3) receptor subtypes, respectively. The determination of the first crystal
structure of the β2ADR bound to the inverse agonist carazolol [15, 88] quickly
attracted attention for the opportunities it offered to carry out virtual screening
studies against this drug target, which is relevant for indications such as asthma,
hypertension, and cardiac failure [89].
Sabio et al. [67] and Kolb et al. [68] explored screens of chemical libraries
against the crystal structure of this receptor using molecular docking in two separate
studies. The former study used the docking program GLIDE [54] and screened a
library of 4.4 million compounds, whereas the latter was performed with the pro-
gram DOCK3.5.54 [51] using 1 million compounds from the ZINC database [63].
Both efforts achieved similar and high hit-rates: 21% and 24% of the predicted
ligands were confirmed by radioligand binding assays, respectively. The discovered
ligands also had high affinities and represented novel scaffolds. One of the ligands
that emerged from the screen by Kolb et al. had an affinity of 9 nM, and encour-
agingly, its predicted binding pose was confirmed by a subsequently solved crystal
structure [90]. The structure used for the virtual screens was determined in complex
with an inverse agonist and interestingly all compounds identified from the two
screens were also inverse agonists or antagonists, suggesting that the docking result
was biased by the crystallized functional state of the receptor.
Agonist-bound structures of the β2ADR were determined in 2011, which repre-
sented a breakthrough considering the challenges associated with crystallizing the
receptor in this inherently flexible state [18, 91, 92]. These revealed the conforma-
tional changes associated with β2ADR activation at the atomic level and showed
that, whereas there were large structural rearrangements in the intracellular regions,
the orthosteric site itself was only subtly different from the inactive state. Weiss
et al. investigated if structure-based virtual screening against an active-like struc-
ture of the β2ADR (PDB code 3P0G) [91] could be used to identify agonists for this
receptor [69]. The ZINC database (3.4 million fragment- and lead-like compounds)
was screened against the orthosteric site using DOCK3.6 [51, 93]. The main criteria
used for compound selection were that these should be ranked higher for the active-
compared to an inactive-like structure and form polar interactions to three β2ADR
residues linked to agonist recognition: Ser2035.42, Ser2045.43, and Ser2075.46 (the
Ballesteros–Weinstein residue numbering for GPCRs [94] is indicated in super-
script). Six compounds out of a total of 22 were found to be ligands and all of them
behaved as agonists, activating either the G protein or β-arrestin-mediated path-
ways. Four ligands contained a catechol moiety, which is a known β2ADR agonist
chemotype. The remaining two discovered agonists were dissimilar to previously
known ligands of the β2ADR and could potentially be used to develop new scaf-
folds. These results suggested that the conformational state in which a GPCR is
crystallized could determine the efficacy of ligands from docking screens.
2.2 Adenosine Receptors
Adenosine receptors (ARs) are a group of purinergic GPCRs that have received
significant attention from both academia and pharmaceutical industry. In fact, over
50 compounds targeting an AR subtype (A1, A2A, A2B, and A3) have been tested in
(pre)clinical trials for indications such as cardiovascular diseases, cancer, and
Parkinson’s Disease (PD) [95, 96]. Despite these efforts, few drug candidates that
act through these receptors have reached the market. The first crystal structure of
the A2AAR was released in 2008 and was solved in complex with the potent an-
tagonist ZM241385 (PDB code 3EML), which revealed the molecular details of
ligand binding to this receptor [97].
Three successful docking screens have been performed using the antagonist-
bound crystal structure of the A2AAR. The first two were published shortly after the
release of the crystallographic coordinates. Katritch et al. [70] performed docking
screens of 4.3 million molecules with the software ICM [52]. In the first step, the
receptor was prepared for virtual screening by optimizing side chain rotamers and
consideration of crystallographic waters, which improved enrichment of known
ligands. The top-ranked compounds from the prospective docking screen were
clustered based on their 2D structures and 56 diverse molecules were selected for
experimental testing. From this set of compounds, 23 had Ki values ranging from
32 nM to 2.9 μM, corresponding to an impressive hit-rate of 41%. Carlsson et al.
[48] used DOCK3.5.54 [51] to screen 1.4 million commercially available lead-like
molecules from the ZINC database. Preparation of the receptor for docking in-
cluded the increase of the side chain polarity for residue Asn2536.55, as this was
found to improve the recognition of known A2AAR ligands. From the top-ranked
500 molecules, 20 were selected for testing in radioligand binding assays. Seven of
the predicted ligands were experimentally verified, resulting in a hit-rate of 35%
and the most potent compound had a Ki value of 200 nM. Overall, these two pio-
neering studies exemplify how docking screens against a GPCR crystal structure
can identify ligands with novel scaffolds.
Another example of successful use of crystal structures of the A2AAR was
provided by the work of Chen et al., which explored the possibility of combining
experimental and computational screening in a fragment-based lead discovery
(FBLD) campaign [71]. In the first step, the ability of molecular docking to predict
the outcome of an experimental fragment screen was evaluated. A set of 500 frag-
ments was screened in parallel with experimental and computational approaches,
using the target immobilized NMR screening (TINS) method and molecular dock-
ing to an A2AAR crystal structure, respectively. Interestingly, docking could predict
several of the ligands from the TINS screen and even detected three false negatives
in the fragment library (Fig. 3). However, it should also be noted that the experi-
mental screen identified allosteric modulators, which were not high ranked in the
docking screens, probably due to the restriction of search space to the orthosteric
site. Subsequently, another 22 compounds were selected from a docking screen of
328,000 commercially available fragments. Of these, 14 molecules were confirmed
to be ligands in binding assays (Ki < 250 μM), which corresponded to a remarkable
hit-rate of 64%. These results suggest that a combination of biophysical and mo-
lecular docking screens could be a powerful approach to discover starting points for
lead development.
Ligands discovered from the docking screens against the A2AAR were predicted
to occupy the same site as the co-crystallized antagonist and formed interactions
with residues Asn2536.55, Glu1695.30, and Phe1685.29. An interesting finding was
that all identified ligands were found to behave as antagonists, i.e., matching the
Fig. 3 A timeline for prospective docking screens carried out against crystal structures or
homology models of GPCRs after the release of the first β2ADR crystal structure. Examples of
predicted complexes for two discovered ligands from screens against the D3DR and A2AAR
[71, 76] are shown to the left of the timeline. The orthosteric sites of the A2AAR and D3DR are
shown as gray cartoons. Key residues and the predicted binding modes of the ligands are shown in
sticks
efficacy of the co-crystallized ligand. This was in line with the results for the
β2ADR (see Sect. 2.1), further supporting the hypothesis that ligand efficacy
could be encoded in the binding site of GPCR crystal structures. In 2011, agonist-
bound complexes of the A2AAR were determined for the ligands adenosine, 50 -N-
ethylcarboxamidoadenosine (NECA), and UK-432097 (PDB codes 2YDO, 2YDV,
and 3QAK, respectively) [98, 99]. The adenine group of these ligands formed similar
interactions with the receptor as antagonists, whereas the ribosyl moiety established
additional polar contacts with residues Ser2777.42 and His2787.43, leading to a small
contraction of that region of the binding site relative to the inactive-like receptor
structures. To explore the possibility of identifying novel non-nucleoside A2AAR
agonists, Rodrı́guez et al. performed docking screens of 6.7 million lead-like mole-
cules from the ZINC database against three agonist- and one antagonist-bound A2AAR
crystal structures [72]. In order to bias the screen towards the discovery of A2AAR
agonists, compounds were required to have better database ranking for the active-like
structures compared to the inactive-like conformation. The 20,000 top-ranked mole-
cules from each screen were filtered based on predicted polar contacts to residues
Asn2536.55, Ser2777.42, and His2787.43. After visual inspection of docking poses
fulfilling these criteria, 20 compounds were selected for experimental testing. Nine
had Ki values better than 10 μM for the A2AAR, which corresponded to a hit-rate of
45%. However, none of the discovered ligands were able to activate the A2AAR in
functional assays. Rodrı́guez et al. hypothesized that these results could be influenced
by several factors, including the limited understanding of the structural determinants
of AR activation and a potential ligand efficacy bias in the screening library. In
support of the latter explanation, 3D similarity searches suggested that the chemical
library was biased towards antagonist- over agonist-like chemotypes of the A2AAR.
The authors also showed that a significantly larger number of compounds resembling
adrenaline and serotonin were present in chemical libraries compared to adenosine
[72]. This result provided an explanation as to why Weiss et al. [69] and Rodrı́guez
et al. [86] identified agonists in the screens against active-like conformations of the
β2ADR and the 5-HT1B receptor (see Sects. 2.1 and 2.8), but that no ligands with this
efficacy emerged using the active-like A2AAR crystal structure.
An illustrative example of the use of molecular docking in ligand optimization is
the study of Tosh et al. [100]. An agonist-bound A2AAR structure (PDB code
3QAK [98]) was used to guide the design of 50 -carboxamide analogs of adenosine.
A computationally generated library of compounds was screened against the
A2AAR binding site using molecular docking. Derivatives with favorable scores
were synthesized, and several of these were confirmed to be A2AAR ligands.
Interactions predicted for the substituents of these ligands also provided a rationale
for their efficacy and A2A/A1 selectivity profiles.
2.3 Dopamine Receptors
The five human dopamine receptor (DR) subtypes (D1–D5) have been studied
intensively as targets for the development of drugs against schizophrenia and PD
[101]. To date, the only crystal structure available for members of this family is that
of the D3DR [102], which was released at the end of 2010 and has subsequently
been used in several prospective molecular docking screens.
In the first screen against the D3DR crystal structure, Carlsson et al. docked
3.6 million compounds from the ZINC lead-like library (MW < 350 Da) to the
orthosteric site of the receptor [76]. Of the 25 top-ranked compounds that were
selected for experimental evaluation, five had Ki values lower than 10 μM, corre-
sponding to a hit-rate of 20%. The most potent of the discovered ligands had an
affinity of 300 nM (Fig. 3). The selected compounds were predicted to form a salt
bridge with Asp1103.32, a common interaction for ligands of the aminergic receptor
family. Four out of the five ligands were confirmed to be antagonists and the fifth
was a weak partial agonist. As both potent and novel ligands were obtained, these
results were similar to screens carried out against the β2ADR (see Sect. 2.1). This
suggested that the group of aminergic receptors, which are responsible for the
action of a large number of drugs, would be fruitful targets for structure-based
ligand design.
Vass et al. compared docking screens against the crystal structure of the D3DR
and snapshots from MD simulations of the same receptor [77]. Starting from the
crystallographic coordinates, a simulation of 20 ns was carried out for the D3DR in
an explicit membrane environment. Twenty-seven representative conformations of
the binding site were obtained from clustering of snapshots from the simulation. An
in-house library of 12,905 fragment-like compounds (MW < 300 Da) was screened
against the crystallographic receptor coordinates and the 27 structures obtained
from the MD simulation. The 50 top-ranked compounds from the screen against the
crystal structure were prioritized for experimental evaluation. Another 56 com-
pounds were selected based on their mean rank against the MD ensemble. For the
compounds selected from the crystal structure screen, nine had significant activity
(>20% ligand displacement at 10 μM) whereas 18 molecules from the screens
against MD snapshots displayed the same level of activity. The hit-rates for the
crystal structure and MD ensemble of the D3DR were thus 18% and 32%, respec-
tively. The three most potent compounds had submicromolar binding affinities
and emerged from both the crystal (one compound) and the MD ensemble (three
compounds). Compounds were predicted to occupy the same space as the
co-crystallized ligand and formed a salt bridge to Asp1103.32. An interesting finding
was that only two out of the 27 discovered ligands overlapped between the two sets
of tested compounds, suggesting that using MD snapshots can increase the diversity
of hits from structure-based screens.
Lane et al. carried out molecular docking screens against the D3DR crystal
structure with the goal to identify orthosteric and allosteric modulators of this
receptor [78]. In the first screen, 4.1 million lead- and drug-like molecules
(MW < 500 Da) were docked to the orthosteric site of the D3DR and 25 top-ranked
molecules were prioritized for experimental evaluation. In the second screen, the
endogenous ligand (dopamine) was first docked to the orthosteric site, and then
compounds from the library were screened against a proximal allosteric pocket
facing the extracellular medium. Of the 25 molecules that were predicted to bind to
the orthosteric site, 14 had affinities better than 10 μM (hit-rate: 56%) and all
ligands tested in functional assays acted as antagonists. A majority of the ligands
were predicted to anchor aromatic groups deep in the orthosteric site and formed
salt bridges to Asp1103.32. The second set of predicted compounds, which were
selected from a docking screen against an allosteric site, did not have a basic amine
(a characteristic feature of orthosteric D3DR ligands). Instead, the compounds were
predicted to form extensive contacts with EL2, including interactions with the
backbone amide atoms of residues Cys181 and Ile183. Interestingly, several of
these showed displacement of the bitopic radioligand UNC9994, which likely
occupies both the orthosteric and allosteric sites. Eight out of 25 evaluated com-
pounds displayed Ki values better than 10 μM (hit-rate: 32%). In functional assays,
two compounds displayed clear allosteric modulation of dopamine signaling. The
two virtual screens thus illustrated that docking could guide the discovery of both
orthosteric and allosteric ligands.
2.4 C-X-C Chemokine Receptors
Among peptide-binding GPCRs, one group of receptors has evolved to recognize

chemotactic cytokines, or chemokines. This receptor family is further classified
based on their endogenous chemokine(s), of which the C-X-C chemokine receptors
(CXCRs) are a subfamily consisting of seven subtypes (CXCR1–7). The crystallo-
graphic coordinates for CXCR4, a subtype studied as a drug target for its role in
cancer and HIV infection [103], were publicly released in late 2010 [104].
Mysinger et al. used DOCK3.6 to screen the ZINC lead-like database (4.2 million
compounds) against the orthosteric site of the CXCR4 structure [79]. From the top
0.03% of the ranked list of compounds, 23 were selected for experimental testing.
Four were found to be negative allosteric modulators (NAMs) of the receptor and
all of these were predicted to form salt bridges with Asp1122.63 and Glu2887.39. The
potencies of the discovered ligands were relatively low and ranged from 55 to
76 μM, which likely reflected the challenges of targeting a protein–protein inter-
face. One of the four compounds had a ligand efficiency in the same range as
approved drugs targeting this receptor and appeared to be a promising scaffold for
development of novel lead candidates.
2.5 Histamine Receptors
The histamine receptors (HRs) are divided into four subtypes (H1–H4). Among
these, the H1HR has been most intensively studied for its role in allergic responses,
which led to the development of the widely used “antihistamine” drugs [105]. The
crystal structure of the H1HR in complex with the antagonist doxepin (PDB code
3RZE) was published in 2011 [106]. Subsequently, de Graaf et al. developed a
customized structure-based screening approach that combined molecular docking
and interaction fingerprints (IFPs) for compound rescoring [81]. A library of
108,790 fragment-like compounds (heavy atom count 22) was docked to the
binding site of the crystal structure using the program PLANTS [55]. Only the
compounds that formed a salt bridge to the conserved Asp1073.32, a key residue
for recognition of HR ligands, were considered. The PLANTS docking energy
and IFP similarity score, which quantified the agreement between the interactions
made by doxepin (the co-crystallized ligand) and the docked compounds, were
used to rank the remaining compounds in the library. Twenty-six compounds from
the top-ranked fragments were selected for experimental evaluation in radioligand
assays. Remarkably, 19 compounds displayed affinities better than 10 μM (hit-rate:
73%) and the most potent compound had a Ki value of 6 nM. The high hit-rate
partly reflected the druggability of the H1HR binding pocket but also supported that
fragment libraries tend to yield a large number of starting points for lead develop-
ment, in agreement with the results of Chen et al. (see Sect. 2.2) [71].
2.6 Opioid Receptors
Opioids, a drug class that largely mediates their effects through the opioid receptors
(ORs), are widely used as analgesics and antidepressant agents [107]. Addiction is a
common side effect of opioids and this is often due to activation of the μ-OR, one
of three subtypes in this family of receptors. The other members are the κ-OR and
δ-OR, of which the former is being actively investigated as a possible target for the
development of nonaddictive analgesic drugs [108].
Antagonist-bound crystal structures for the three OR subtypes were first deter-
mined in 2012 (PDB codes 4DJH, 4DKL, and 4EJ4 for κ-, μ-, and δ-OR, respec-
tively) [109–111], providing structural details to understand ligand selectivity.
Negri et al. [84] utilized the structure of the κ-OR receptor to perform large-scale
virtual screens of 4.5 million lead-like compounds from the ZINC database using
DOCK3.6 [51, 93]. The authors incorporated information from the binding modes
of ligands crystallized with the μ-OR and δ-OR to steer the predictions towards
discovery of selective compounds for the κ-OR. The top-ranked 500 molecules
from the docking screen were visually inspected. Compound selection was guided
by multiple criteria, including interaction with Asp1383.32, proximity to residues
unique to the κ-OR among the three subtypes, diversity, and purchasability.
Twenty-two compounds were experimentally evaluated, out of which four were
identified as weak κ-OR ligands, with Ki values ranging from 120 to 450 μM. While
three of the four compounds recapitulated the efficacy of the co-crystallized
antagonist, one of the ligands behaved as an agonist of the G protein pathway.
The isolation of the corresponding S-isomer from this racemic mixture yielded a
κ-OR ligand with >83-fold subtype selectivity.
2.7 Muscarinic Receptors
The muscarinic receptor family has five members (M1–M5) that recognize the neu-
rotransmitter acetylcholine. Muscarinic receptors (MRs) have been extensively
targeted by the pharmaceutical industry for the treatment of disorders such as
type 2 diabetes, PD, and chronic obstructory pulmonary disease [112]. However,
the therapeutic applicability of drugs acting via interactions with these receptors has
been limited by lack of selectivity and the associated risks of causing adverse drug
reactions. The release of the structures of the M2MR and M3MR bound to antag-
onists (PDB codes 3UON and 4DAJ, respectively) [113, 114] revealed strong
similarities between the binding sites of these subtypes. Kruse et al. [85] used
DOCK3.6 [51, 93] to carry out screens of 3.1 million commercially available
chemicals against the M2MR crystal structure. From the top-ranked 500 com-
pounds, 18 were tested in radioligand assays. Eleven novel M2MR ligands achieved
Ki values better than 40 μM, corresponding to a hit-rate of 61%. Six ligands were
fragment-sized molecules, which is in line with the fact that the orthosteric site of
the muscarinic receptors has evolved to recognize the small endogenous agonist
acetylcholine. Encouraged by the successful discovery of novel M2MR chemo-
types, the authors attempted to use crystal structures to bias the screens towards the
discovery of ligands selective for the M3MR over the M2 subtype. This selectivity
profile could be advantageous in the clinic, as activation of the M2MR can lead to
undesired cardiac effects [115]. To investigate this, the same chemical library was
docked to both M2MR and M3MR structures. From the 5,000 top-ranked com-
pounds from the screen against the M3MR structure, 500 molecules with the largest
rank differences between screens were visually inspected. Apart from good com-
plementary to the M3MR binding site, typically involving a salt bridge to the
conserved residue Asp1473.32, compounds were required to exploit interactions
with Leu2255.33. This was the only residue difference in the orthosteric sites bet-
ween the M3 and M2 receptors (the bulkier residue Phe1885.33 was found in the
equivalent position of the antitarget). Of the 16 predicted ligands, eight were found
to bind to the M3 receptor, corresponding to a hit-rate of 50%. One ligand was able
to achieve >fivefold M3/M2 selectivity ratio, which highlights the difficulties in
developing subtype-selective ligands for receptor families with strongly conserved
orthosteric sites.
2.8 Serotonin Receptors
Serotonin (5-hydroxytryptamine, 5-HT) is a key neurotransmitter acting in func-

tions concerning the gastrointestinal, cardiovascular, and central nervous (CNS)
systems [116]. The function of several membrane proteins involves the recognition
of this molecule, of which 13 are GPCRs [117]. Interactions with serotonin receptor
subtypes are responsible for the mechanism of action of several widely used drugs
such as atypical antipsychotics, antiobesity, and migraine medications. A drug class
belonging to the latter category is the triptans, which are selective ligands for the
5-HT1B and 5-HT1D receptors [118]. Selectivity is a highly desired feature of sero-
tonergic drugs given the potential adverse effects produced by cross-reactivity
among the 5-HT receptor family. One example is the drug combination fen-phen,
which was retracted from the market after causing cardiac disorders trigged by
interactions with the 5-HT2B receptor [116]. Crystal structures of the 5-HT1B and
5-HT2B receptors bound to the agonist ergotamine were released in 2013 (PDB
codes 4IAR and 4IB4, respectively) [119, 120]. These structures revealed atomic-
level details of the structural determinants for ligand recognition and receptor se-
lectivity. As for other aminergic receptor structures, the co-crystallized ligand
established a salt bridge with the conserved Asp3.32 residue. Docking of triptans
to the two structures suggested that the impaired binding of triptans to the 5-HT2B
receptor is due to sterical hindrance caused by an inward kink of TM5 and a bulkier
side chain of residue Met2185.39 (Thr2095.39 in 5-HT1B) [120]. Based on these
structural differences in the binding sites, Rodrı́guez et al. examined the possibility
of using the crystal structures to discover 5-HT1B selective ligands [86]. Docking
screens of 1.3 million commercially available molecules were carried out with
DOCK3.6 against these two structures. Compounds were required to achieve
significantly better docking ranks for the target compared to the antitarget. From
the top-ranked 4,000 molecules in the screen against the 5-HT1B receptor, the
500 compounds with the worst ranks for the 5-HT2B subtype were visually
inspected. The 22 compounds that were selected for experimental evaluation
formed a salt bridge to Asp1353.32, as well as hydrogen bonds to non-conserved
5-HT1B residues (e.g., Ser3346.55 and Thr2095.39) in the region where the binding
site of 5-HT2B was relatively contracted. Eleven of the selected molecules achieved
Ki values <10 μM for the 5-HT1B receptor, corresponding to a hit-rate of 50%, and
included novel ligands with affinities as high as 29 nM. Moreover, nine of the
discovered 5-HT1B ligands displayed affinities at least fivefold higher than for the
off-target, with a maximum selectivity ratio over 300-fold. Three discovered
5-HT1B-selective ligands were shown to activate the receptor in functional assays,
matching the efficacy of the co-crystallized ligand ergotamine. Finally, predicted
binding modes were supported by structure–activity relationships and enabled the
identification of an analogue with improved potency and 5-HT1B selectivity. These
results suggested that GPCR crystal structures can guide the discovery of subtype-
selective ligands.
2.9 Docking Screens Against GPCR Crystal Structures:

Opportunities and Limitations
The recent explosion of available atomic-level information from GPCR structures

has enabled numerous successful virtual screens, leading to the discovery of lead
candidates from chemical libraries. GPCRs have been crystallized in conformations
corresponding to different functional states. Remarkably, ligands identified from
docking screens against GPCR crystal structures have typically matched the effi-
cacy of the co-crystallized compound, e.g., for the β2ADR and 5-HT1B receptors
(Sects. 2.1 and 2.8). However, the transferability of these results to other GPCRs is
unclear as other factors such as target-specific mechanisms for receptor activation
and the screening library composition may also influence screening results, as dem-
onstrated for the A2AAR (Sect. 2.2). Recent applications of docking screens also
investigated the possibility to discover ligands displaying subtype selectivity. Many
GPCRs have evolved to recognize the same endogenous compound, resulting in
similar orthosteric sites, which leads to a high risk for off-target interactions in drug
development. Docking screens against target and antitarget crystal structures have
enabled the discovery of subtype-selective ligands for muscarinic and serotonin
receptor subtypes (Sects. 2.7 and 2.8). An alternative approach to achieve selectiv-
ity is to develop allosteric ligands that target binding pockets that are less conserved
than the orthosteric site [121], a possibility that has already been successfully ex-
ploited for the D3DR (Sect. 2.3). Recently determined crystal structures of the
M2MR and metabotropic Glutamate Receptors (mGluRs) 1 and 5 have revealed
additional allosteric pockets that now can be targeted using molecular docking
screening [122–124]. As ORs are the only family with available crystal structures
for all subtypes, it is difficult to use structure-based screens to identify subtype-
selective GPCR ligands. Homology modeling can be used to predict structures of
subtypes related to those experimentally solved, which may enable prediction of
selective and multi-target GPCR ligands (Sect. 3.2). An important aspect to con-
sider while working with crystal structures is that they represent a single snapshot of
an ensemble of thermally accessible conformations. Large-scale docking screens
are often carried out against rigid receptors structures, and more extensive sampling
of receptor conformations using MD simulations can increase the diversity of the
discovered ligands (Sect. 2.3). Similarly, approaches for rescoring docking solu-
tions, such as the use of IFPs, have proven to be useful in ligand discovery efforts
(Sect. 2.5) and this approach could also be valuable in predictions of ligands with
specific signaling properties [125].
Structure-based virtual screens against GPCRs have been remarkably successful.
Many of the screened targets have enclosed and hydrophobic orthosteric binding
sites with a small number of polar receptor–ligand interactions, which are expected
to be very druggable. These features are also particularly suitable for docking algo-
rithms, which in part explains the high hit-rates observed for GPCRs. Another
contributing factor is that due to the medicinal chemistry focus on GPCRs, chem-
ical libraries are likely biased towards these targets [126]. Currently, no in silico
screens have been reported for 17 GPCR crystal structures, which include important
drug targets such as purinergic, metabotropic glutamate, and several peptide-
binding receptors. Structure-based screening protocols could thus already be used
for a large number of additional targets to identify starting points for development
of novel pharmaceuticals.
3 Modeling GPCRs of Unknown Structure and Their

Complexes with Ligands
In the absence of experimental coordinates for many pharmaceutically relevant

GPCRs, protein structure prediction methods could make important contributions
to ligand discovery and design. However, this is not devoid of challenges and it has
been unclear if GPCR modeling has the required accuracy for this purpose. In this
section, we summarize the state of GPCR structure prediction with particular focus
on ligand discovery.
3.1 Community-Wide Assessments for Prediction

of GPCR–Ligand Complexes
Community-wide assessments involving blind predictions provide invaluable

benchmarks of methods in computational chemistry and biology. The flagship of
such assessments is the CASP initiative, which has evaluated the state and progress
of protein structure prediction for more than 20 years [127]. More recently, similar
assessments for prediction of protein–protein interactions (CAPRI) [128], protein–
ligand binding modes and affinities (CSAR) [129], and small-molecule solvation
energies (SAMPL) [130] have been organized. In a similar vein, the “GPCR Dock”
assessments have challenged the molecular modeling community to predict the
structures of GPCR–ligand complexes before the release of the crystallographic
coordinates. Based on the sequence of the target GPCR and the 2D structure of a
ligand, research groups were encouraged to predict: (1) the receptor structure and
(2) the binding mode of the ligand. Participants were typically given 30 days to
generate their predictions and could submit from five to ten unique models ranked
by their expected accuracy. The predicted complexes were then compared with
the experimental structure, starting with a structural superimposition of the TM
regions. The accuracy of the models was evaluated based on the ligand root-mean
square deviation (RMSD) and the predicted ligand–receptor atomic contacts. The
three editions of GPCR Dock held in 2008, 2010, and 2013 have involved pre-
dictions for eight crystal structures of six unique receptors [45–47]. The main
results from the assessments are summarized in Table 2. Five targets were members
of class A GPCRs: the A2AAR, CXCR4, D3DR, 5-HT1B, and 5-HT2B serotonin
receptors. The smoothened receptor (SMO) was the only nonclass A GPCR con-
sidered in these assessments. The different challenges posed by each case provided
valuable information on the state of molecular modeling for GPCRs.
3.1.1 GPCR Dock 2008: The A2A Adenosine Receptor
GPCR Dock 2008 [45] inaugurated the series of assessments by challenging the
molecular modeling community to predict the structure of the A2AAR bound to the
potent antagonist ZM241385 [97]. A total of 29 participant groups submitted
206 solutions and the most used approach for protein structure prediction was
homology-based modeling. Rhodopsin (PDB codes 1U19 and 2Z73) [131, 132]
and β1- and β2-ADRs (PDB codes 2VT4 and 2RH1, respectively) [15, 133] were the
only available templates for modeling. The two latter structures, which shared
~35% sequence identity to the target, were the main templates used. These provided
good starting points to model the backbone of the TM helices of the A2AAR, re-
sulting in that the pool of submitted models had an average Cα RMSD of 2.8 Å for
this region of the protein. Approaches enabling more extensive backbone sampling,
such as consensus multiple-template modeling or structure optimization accounting
for the membrane environment, appeared to have a positive impact on TM bundle
modeling and contributed to increased accuracy of the orthosteric binding site. In
86
Table 2 Summary of the most accurate results submitted to the GPCR Dock assessments
Closest TM Top-ranked predicted complexa

PDB templates (seq. Number of participant groups Ligand Pocket RMSD/Å Contact
Assessment Receptor Ligand code id.) (submitted models) RMSD/Å (Number of residues) accuracyb
GPCR Dock A2AAR ZM241385 3EML β1ADR (35%) 29 (206) 2.8 3.4 (9) 45%
2008 [45]
GPCR Dock D3DR Eticlopride 3PBL β1ADR (41%) 32 (117) 1.0 1.5 (15) 58%
2010 [46] CXCR4 IT1t 3ODU β1ADR (25%) 25 (103) 4.9 4.9 (13) 36%
CVX15 3OE0 19 (55) 8.9 6.7 (26) 6%
GPCR Dock 5-HT1B Ergotamine 4IAR β1ADR (45%) 40 (181) 1.5 1.4 (19) 47%
2013 [47] 5-HT2B Ergotamine 4IB4 β1ADR (41%) 39 (171) 1.1 2.7 (23) 50%
SMO LY- 4JKV M3MR (13%) 20 (88) 4.4 13.9 (12) 2%
2940680
SANT-1 4N4W 20 (88) 4.3 5.6 (19) 7%
a
Models were ranked with a combined Z-score reflecting the average of ligand RMSD and fraction of correctly predicted contacts compared to the
experimental structure
b
Contact accuracy quantifies the agreement between predicted and experimental ligand–receptor contacts at a relevant distance cutoff (4–5 Å). See
corresponding references for details on these calculations
A. Ranganathan et al.
Fig. 4 Summary of top-ranked complexes from the three GPCR Dock assessments [45–47], in
chronological order from top left to bottom right. The PDB codes of the target complexes released
after the assessments are provided in each panel along with the name of the receptor and the three-
letter residue name of the co-crystallized ligand in the respective PDB entries. Carbon atoms from
crystallographic and predicted ligand–receptor complexes are colored in white and gold, respectively
contrast to the overall receptor topology, ligand recognition proved to be more

challenging to predict accurately. In the crystal structure, the ligand displayed an
extended perpendicular orientation with respect to the membrane plane. The main
interactions in the binding site of the A2AAR involved hydrogen bonds of the
adenine-like ligand scaffold with residues Asn2536.55 and Glu1695.30 (Fig. 4).
Additional interactions include π-stacking of the triazolotriazine core with
Phe1685.29 and van der Waals interactions with residues Leu2496.51, Ile2747.39, and
Met2707.35. According to the assessment criteria, the most accurate model of the
A2AAR-ZM241385 complex achieved a ligand RMSD of 2.8 Å and recapitulated
45% (34/75) of the experimental receptor–ligand contacts. The methodology utilized
to achieve this solution combined homology modeling, energy optimization of EL2,
induced-fit docking for ligand binding prediction, and selection of complexes based
on mutagenesis data. The mutagenesis data used clearly made important contribu-
tions to the accuracy of this model, but such data should be interpreted cautiously. In
fact, only four out of ten A2AAR residues that significantly reduced ligand binding in
mutagenesis experiments were found to be in contact with the co-crystallized ligand
[134]. Other strategies that gave rise to top-ranked solutions incorporated ligand
binding data in a different fashion. This included selection and optimization of
receptor models on the basis of enrichment of known ligands in retrospective docking
screens [134]. Such ligand-guided homology modeling approaches appeared to
improve the prediction of native contacts and have the advantage of providing
receptor models validated for prospective lead discovery applications [32]. Overall,
the best solutions reproduced the most relevant receptor–ligand interactions in the
crystal structure, but the binding pose of the ligand was slightly deeper than the
experimental binding mode, which was due to difficulties in modeling regions
structurally divergent from available templates. This involved residues Glu1695.30
and Met1775.38, which were located in protein regions (EL2 and TM5) with unex-
pected secondary structures that were hard to predict also for de novo modeling
approaches. Finally, it should be noted that a crystal structure obtained for the same
complex under different conditions (PDB code 3PWH) [135] has structural differ-
ences both in EL2 and the ligand binding mode, illustrating that the conformation
captured by crystallography represents only one of many accessible states.
3.1.2 GPCR Dock 2010: D3 Dopamine and CXCR4 Receptors
The GPCR Dock 2010 assessment [46] involved predictions for the D3DR and
CXCR4 receptors. For the D3DR, the challenge was to predict the structure of a
complex with the antagonist eticlopride (Fig. 4) [102]. Two separate structures of
CXCR4 in complex with either a small-molecule ligand (IT1t, Fig. 4) or a peptide
analog (CVX15) were the second part of the assessment [104]. Thirty-five research
groups submitted a total of 275 models of the three GPCR complexes. Compared to
the GPCR Dock 2008 assessment, the A2AAR crystal structure was the only
additional available template, but it did not have the highest sequence identity to
any of the target receptors. The β1- and β2-ADRs provided excellent starting points
for modeling of the D3DR, with up to 41% sequence identity in the TM region.
These structures were also the best available templates for CXCR4, but with a TM
sequence identity of only 26%. The two receptors thus provided completely differ-
ent levels of difficulty, which also was reflected in the results. The median Cα
RMSD for the D3DR was 1.7 Å whereas the same number for the CXCR4-IT1t
complex was 2.8 Å. One particular challenge for modeling the structure of CXCR4
was a proline-induced helix constriction of TM2 [46], which was captured by 50%
of the submitted models by including an appropriate gap in the sequence alignment.
Encouragingly, a few groups were able to predict the β-hairpin fold of EL2, which
appears to be a characteristic feature of peptide-binding GPCRs. Ab initio methods
were also used by several groups for the case of CXCR4, but the predicted
structures were found to be less accurate than those generated with homology-
based modeling approaches. The top-ranked model in the assessment involving the
D3DR crystal structure had a ligand RMSD of 1.0 Å and captured 58% of the
receptor–ligand contacts. In the case of the CXCR4-IT1t complex, the most accu-
rate model achieved an RMSD of 4.9 Å and captured 36% of the contacts. Finally,
the best model of the CXCR4–CVX15 complex correctly predicted 6% of the
contacts and had an RMSD of 8.8 Å. There was a clear correlation between
prediction accuracy and modeling difficulty. For the D3DR, 23 of the submitted
models had a ligand RMSD <2.5 Å. Only one prediction was able to reach this
threshold for the CXCR4-IT1t complex. As in the case of the A2AAR in the GPCR
Dock 2008 assessment, not a single model of the CXCR4-CVX15 complex was
close to this level of accuracy. In the case of the D3DR, information from crystal
structures of other aminergic receptors helped to identify a conserved contact (with
residue Asp1103.32) present in all crystallized members of this family. This data was
indeed considered to constrain the docking search in the protocol used to generate
the top-ranked solution [136]. In contrast, available experimental data in the form
of mutagenesis and known ligands was significantly less abundant for CXCR4. As
expected, it was also very difficult to predict the binding mode of the large peptide
analog compared to the small-molecule ligand of CXCR4.
3.1.3 GPCR Dock 2013: The Serotonin 5-HT1B, 5-HT2B,

and Smoothened Receptors
The GPCR Dock 2013 assessment [47] consisted of modeling challenges of varying
difficulty. The competition itself could be subdivided into two categories: the
serotonin 5-HT1B and 5-HT2B receptor subtypes bound to ergotamine [119, 120]
(good templates available, >40% sequence identity) and two SMO structures
[137, 138] (no readily suitable templates, ~15% sequence identity) bound to the
small molecules SANT1 and LY-2940680. A total of 181 and 171 models were
submitted by 40 and 39 research groups for ergotamine bound to the serotonin
5-HT1B and 5-HT2B subtypes, while 88 models from 20 participants were submitted
for the SMO receptors in complex with SANT1 and LY-2940680.
Excellent templates were available for homology modeling of the 5-HT1B and
5-HT2B receptors. The β1ADR had the highest sequence identity in the TM region
(45% and 41%) to both target receptors [133]. This resulted in that a large number
of models achieved high accuracy in terms of TM backbone RMSD, with median
values of 1.9 and 2.1 Å for the 5-HT1B and 5-HT2B subtypes, respectively. How-
ever, difficulties in the prediction of EL2, coupled with the large size and higher
number of conformational degrees of freedom of ergotamine (e.g., compared to the
D3DR ligand eticlopride) made accurate prediction of the complexes challenging.
This was reflected in the results where only 16 out of the 352 complexes (4.5%)
achieved ligand RMSDs of <2.5 Å compared to 20% in the case of the D3DR–
eticlopride complex. Nevertheless, the best submissions for the 5-HT1B and 5-HT2B
receptor complexes with ergotamine achieved very low ligand RMSDs (1.5 and
1.05 Å) and high contact accuracies (47% and 50%) (Fig. 4). In both cases, the
structure of the thermostabilized turkey β1ADR was used as the primary template
for homology modeling, and one of these research groups also used a chimeric
template based on several aminergic GPCR structures. In particular, it was also
noted that the use of multiple templates could contribute to increased accuracy in
modeling of the protein backbone and binding site for the 5-HT2B receptor
[46]. This is exemplified by the successful prediction of a helical extension of the
extracellular tip of TM5 and the portion of the ECL2 from TM5 to the conserved
disulfide bridge, which is part of the binding site [139]. As pointed out by the
organizers of the assessment, there was reasonable agreement between binding site
accuracy and correctness of predicted receptor–ligand contacts. On the other hand,
the crystal structures revealed that neither of the highest ranked solutions managed
to capture the depth of the ergoline core in the binding site, the hydrogen bond
established with residue Thr3.37, or the conformational changes associated with the
β-arrestin biased state of the 5-HT2B subtype. As expected, the models were
significantly less accurate for the much more difficult case of the two SMO receptor
complexes. Since the closest template only shared 15% sequence identity, the use of
profile–profile comparisons, threading or combined methodologies provided the
best alignments. Among the submitted models of the complexes of SMO, the most
accurate in terms of TM backbone had RMSDs of 2.8 and 3.0 Å, respectively, while
the best ligand binding mode solutions for SANT1 and LY-2940680 achieved
RMSDs of 4.3 and 4.4 Å, respectively (Fig. 4). In the most accurate submissions,
only 9% and 12% of the receptor–ligand contacts were correctly predicted for the
complexes with LY-2940680 and SANT1, respectively.
3.1.4 Summary of GPCR Dock Assessments
The GPCR Dock assessments have provided valuable insights into the possibilities
for GPCR structure prediction and the use of models in ligand discovery. Access to
templates with high sequence identity to the target has had a positive impact on
modeling accuracy. For the D3DR, 5-HT1B, and 5-HT2B receptors, several research
groups were able to achieve extremely accurate predictions [120]. Modeling of the
A2AAR and CXCR4 structures was more challenging and the cases involving SMO
illustrated how the lack of structurally related templates could severely restrict
prediction accuracy. A common area for improvement identified from all GPCR
Dock assessments was that participating groups could not blindly rank their sub-
mitted models by accuracy. In other words, the modeling community can generate
models that are close to the experimental coordinates, but it is still challenging to
identify the best one from a pool of solutions.
The results of the three GPCR Dock assessments have demonstrated the utility of
homology modeling and also highlighted the potential of innovative approaches
such as multiple-template and ligand-guided homology modeling. However, the
performance of these methods is strongly dependent on the availability of suitable
templates and ligand binding data. Modeling of receptors distantly related to any
solved GPCR structure will require the use of approaches with a significant de novo
component. These methods must simultaneously achieve conformational sampling
and accurate selection of the resulting receptor conformations. In the following
sections, we will discuss how GPCR drug discovery can benefit from the lessons
learnt from GPCR Dock assessments, including examples of prospective structure-
based screens using receptor models.
3.2 Docking Screens Using Homology Models of GPCRs
The results from the GPCR Dock assessment can be clustered into groups of high,
intermediate, and low accuracy. The level of accuracy should correlate with the success
of applying models in prospective structure-based drug discovery. In this section,
examples of the use of homology models and molecular docking to discover GPCR
ligands will be highlighted.
3.2.1 High Accuracy Modeling
The predicted structures for the D3DR, 5-HT1B, and 5-HT2B receptors are close to
experimental accuracy in terms of ligand and binding pocket RMSD. Models with
this level of detail could likely be used to guide optimization of lead compounds
and provide opportunities to identify novel ligands using molecular docking screen-
ing, which is supported by several recent publications:
• During the GPCR Dock 2010 assessment, Carlsson et al. [76] carried out
molecular docking screens against a homology model of the D3DR prior to the
release of the crystal coordinates. Of 26 predicted ligands, six were found to
display significant binding, with Ki values ranging from 0.2 to 3.1 μM, corres-
ponding to a hit-rate of 23%. When the crystal structure was released, the
docking screen was repeated (see Sect. 2.3) and resulted in a similar hit-rate
and ligand affinity range for the discovered ligands (20% and 0.3–3.0 μM,
respectively). This study demonstrated that virtual screens against an accurate
homology model and crystal structure of the D3DR could be equally successful.
• Three studies using histamine receptor homology models based on the closely
related H1HR (PDB code 3RZE) [106] and β2ADR crystal structures (PDB code
2RH1) [15] have illustrated how access to suitable templates can enable ligand
discovery for receptors of unknown structure. Sirci et al. generated models of the
H3HR and refined these with MD simulations [82]. Around 156,000 compounds
from the ZINC database were evaluated using field-based fingerprints for ligands
and proteins (FLAP) in both ligand- and structure-based modes. Twenty-nine
compounds were selected for experimental evaluation and 18 of these were
ligands of the H3HR, corresponding to an impressive hit-rate of 62%. Istyastono
et al. modeled the H4HR, using the H1HR and β2ADR as templates and per-
formed structure-based virtual screens of a fragment library using molecular
docking with PLANTS and IFPs for compound rescoring. Nine ligands were
identified out of 37 evaluated fragments (hit-rate: 24%) [83]. Vass et al. modeled
the H4HR using the H1HR as template and generated an ensemble of 27 struc-
tures from MD simulations of the homology model [77]. A fragment library of
12,095 compounds was screened using GLIDE, resulting in hit-rates of 22% and
16% for the single model and MD ensemble of the H4HR, respectively.
• Weiss et al. [69] used homology models of the D2DR to identify novel agonists
of this drug target. The active conformation of the D2DR was predicted based on
a β2ADR structure in an active conformation (PDB code 3P0G) [91] and a D3DR
crystal structure (PDB code 3PBL) [102] was utilized to represent an inactive-
like receptor state. A prospective docking screen against both the active and
inactive models of the D2DR was carried out and only compounds that were high
ranked for the active conformation were selected. Fifteen compounds were
selected for testing, out of which three were ligands (hit-rate: 20%) and two
were indeed agonists of the D2DR. In another recent study, Lam et al. carried
out a docking screen against a homology model of the human Trace Amine-
Associated Receptor 1 (TAAR1), which belongs to the group of aminergic receptors
and is a potential target for drug development against schizophrenia and
PD [140]. A total of 42 compounds were predicted from a docking screen of 3 million
compounds, which led to the discovery of nine agonists (hit-rate: 21%) [87].
• Access to accurate homology models of GPCRs could provide opportunities to

design ligands with tailored pharmacological properties. Subtype-selective and
multi-target ligands hold promise for development of therapeutics with
increased efficacy and reduced toxicity [141]. However, it has been unclear if
GPCR models can achieve the required accuracy to guide the design of ligands
with this level of specificity. This was illustrated in a follow-up paper to the
GPCR Dock 2013 assessment by Rodrı́guez et al., where it was demonstrated
that the crystal structures of 5-HT1B and 5-HT2B receptors could be used to
identify selective leads, but top-ranked models from the assessment did not
capture the main structural features that determine ligand selectivity between
the two serotonin receptor subtypes [86]. Several studies have explored the
possibilities of discovering or designing ligands with subtype-selective or
polypharmacological profiles for closely related GPCRs using docking screen-
ing against homology models. Kolb et al. performed docking screens of 2.2 mil-
lion compounds against an A1AR homology model (40% sequence identity to
the A2AAR crystal structure) with the aim of discovering ligands selective for
that AR subtype [73]. The challenges involved in using homology models for
this purpose was evidenced by higher hit-rates obtained for the antitargets
A2AAR (38%) and A3AR (36%) than for the targeted A1AR (21%). More
recently, docking screens using crystal structures and homology models of
related subtypes led to the identification of ligands with tailored pharmacolog-
ical properties. In order to achieve this, Schmidt et al. [80] utilized the crystal
structure of CXCR4 (37% sequence identity) to discover subtype-selective and
multi-target ligands for the CXCR4 and CXCR3 pair. Since the structure of the
latter was not available, CXCR3 homology models were generated using the
CXCR4 structure as template, and 2 million compounds from the ZINC database
were screened against both receptors using DOCK3.5.54 [51]. To bias the screen
towards selective ligands, compounds were required to have higher docking
ranks for the target receptor over the antitarget. Three out of six predicted
CXCR4-selective ligands were found to be selective NAMs or positive allosteric
modulators (PAMs) of the CXCR4. Interestingly, the homology model of

CXCR3 also guided the discovery of four selective NAMs. Furthermore, four
compounds were predicted to be dual ligands based on high docking ranks in
both screens. Two of these molecules were found to modulate the effect of the
endogenous agonist at both CXC receptor subtypes. Vass et al. modeled the
D2DR based on the D3DR crystal structure, with the goal of probing the
structural determinants of subtype selectivity using a fragment-based approach
[142]. Docking screens against the primary (orthosteric) site and a secondary
pocket facing the extracellular medium were carried out with GLIDE [54].
Top-ranked compounds with basic amines that formed the conserved salt bridges
to Asp3.32 in the primary sites of the D3 and D2DR were linked via synthesis to
fragments that predicted to form favorable interactions in the secondary site. The
three synthesized compounds were evaluated in binding assays and displayed
subnanomolar Ki values at the D3DR, achieving between nine- and 55-fold
selectivities for this receptor over the D2 subtype. Ranganathan et al. used a
fragment-based approach to identify subtype-selective AR ligands [75]. The
ZINC fragment library was screened against the A3AR, with an aim of discov-
ering scaffolds selective over the A1 subtype. A multistage homology modeling
procedure was employed for both the target and antitarget subtypes, using the
closely related A2AAR as template. In order to bias compound selection towards
selective ligands, the screen was performed against one homology model of the
A3AR and an ensemble of ten A1AR models representing the antitarget. A list of
top-ranked molecules from the A3AR screen with a significant difference in rank
with the antitarget ensemble were further visually inspected for interactions in
regions of the orthosteric site that differed between the two subtypes. Twenty-
one fragments were experimentally evaluated, yielding eight ligands of the
A3AR (hit-rate: 38%) and six of these were also selective over the A1AR. The
two most promising scaffolds were further optimized using structural informa-
tion from the predicted complexes to yield high-affinity leads for the A3AR with
100-fold selectivity over the A1AR.
• To summarize, homology models based on closely related templates can per-

form as well as crystal structures in prospective molecular docking screening.
More challenging problems such as identifying ligands with specific selectivity
or efficacy have also been accomplished in several cases. In addition to gener-
ation of accurate models, careful selection of compounds that exploit structural
motifs responsible for ligand selectivity and receptor activation is also important
for successful virtual screening results.
3.2.2 Intermediate Accuracy Modeling
The results of the GPCR Dock assessments demonstrated that it was only possible
to achieve intermediate accuracy for the A2AAR-ZM241385 and CXCR4-IT1t
complexes [45, 46]. The use of homology models with this accuracy will be more
limited in the area of ligand discovery. Nevertheless, docking screens against such
structural models, which can be further improved by access to extensive experi-
mental data, have aided identification of novel ligands.
• A direct comparison between the performance of homology models and crystal
structures in ligand discovery was made by Mysinger et al. in connection to the
GPCR Dock 2010 assessment [79]. Analogous to Carlsson et al. [76], two
docking screens were carried out: one against a homology model of the
CXCR4, and the other against the subsequently released crystallographic coor-
dinates for that receptor. The hit-rates obtained were, in this case, significantly
lower for the homology model compared to the crystal structure (4% and 17%,
respectively) [79].
• The studies by Zhukov et al. and Langmead et al. utilized mutagenesis data to
refine homology models of GPCR–ligand complexes and to discover ligands by
molecular docking [74, 143]. Zhukov et al. built homology models of the A2AAR
using the turkey β1ADR crystal structure (PDB code 2VT4, 25% sequence
identity) as template. Both in-house and publicly available site-directed muta-
genesis data were used to validate and optimize structural models bound to
ligands of different classes. Homology models generated with a similar approach
were used by Langmead et al. to identify novel ligands for the A2AAR using
molecular docking of 545,000 compounds against the predicted orthosteric site
[74]. From this screen, 230 molecules were experimentally evaluated, resulting
in the identification of 20 ligands (IC50 < 55 μM), which corresponds to a
hit-rate of 9%. Although this hit-rate was somewhat lower than that obtained
with crystal structures of the same receptor (see Sect. 2.2), novel ligands that
could be optimized to potent leads were identified.
• To summarize, homology models of medium accuracy have generally resulted in

docking screens with lower hit-rates compared to those using crystal structures.
Such models could be used to identify potential residues for mutagenesis studies,
which can guide further refinement of the receptor models and ligand binding
modes, until reaching a regime where they can successfully guide ligand dis-
covery and optimization.
3.2.3 Low Accuracy Modeling
For the CXCR4-CVX15 and SMO receptors, low accuracy models were achieved
for the complexes with ligands. The best submitted solutions only predicted a few
of the residues involved in ligand binding [46, 47]. At this level of accuracy, the
models may be of limited value in molecular docking screening studies. For the
CXCR4 case, the highly flexible nature of the cyclic peptide ligand was very
difficult to model, whereas the SMO complexes faced both a lack of suitable
structural templates (sequence identity < 15%) and ligand data to support
prediction of the binding mode(s). For these more challenging scenarios, develop-
ment of new methods for GPCR modeling or structure determination for more
closely related GPCRs will likely be required for successful structure-based ligand
design. In challenging cases, model building and optimization would likely have to
rely on structure prediction methods that sample wider spectra of receptor confor-
mations than homology modeling, preferably guided by restraints derived from
sequence analysis, mutagenesis studies, and ligand binding data.
4 Conclusions and Outlook
The extraordinary growth in GPCR structural information in recent years has

enabled the use of in silico methods to discover ligands for many important drug
targets. After the first molecular docking screens against the β2ADR, which pro-
vided the first glimpse of the potential of structure-based drug design for GPCRs,
ligands for an additional nine targets have been identified using the same technique.
The crystal structures have guided the discovery of both orthosteric ligands (inverse
agonists, antagonists, and agonists) and allosteric modulators. Access to atomic-
resolution information for several subtypes has also enabled the identification of
subtype-selective lead candidates. For each crystallized GPCR, it becomes possible
to use homology modeling with these structures as templates to access an even
larger fraction of the GPCRome. Given the rapid increase in structural information
for GPCRs and its impact on our understanding of signaling at the molecular level,
structure-based modeling techniques can be expected to play a key role in future
drug discovery.
Acknowledgements This work was supported by grants from the Swedish Foundation for
Strategic Research (ICA10-0098), Swedish Research Council (2013–05708), and the Science for
Life Laboratory to J.C. The Sven och Lilly Lawski Foundation supported D.R. with a postdoctoral
fellowship. A.R., D.R., and J.C. participate in the European COST Action CM1207 (GLISTEN).
References
1. Alexander SP, Benson HE, Faccenda E, Pawson AJ, Sharman JL, Spedding M, Peters JA,
Harmar AJ, CGTP Collaborators (2013) Br J Pharmacol 170:1459
2. Lagerstrom MC, Schioth HB (2008) Nat Rev Drug Discov 7:339
3. Rosenbaum DM, Rasmussen SG, Kobilka BK (2009) Nature 459:356
4. Liu W, Chun E, Thompson AA, Chubukov P, Xu F, Katritch V, Han GW, Roth CB, Heitman
LH, IJzerman AP, Cherezov V, Stevens RC (2012) Science 337:232
5. Overington JP, Al-Lazikani B, Hopkins AL (2006) Nat Rev Drug Discov 5:993
6. Macarron R, Banks MN, Bojanic D, Burns DJ, Cirovic DA, Garyantes T, Green DV,
Hertzberg RP, Janzen WP, Paslay JW, Schopfer U, Sittampalam GS (2011) Nat Rev Drug
Discov 10:188
7. Eckert H, Bajorath J (2007) Drug Discov Today 12:225
8. Shoichet BK (2004) Nature 432:862
9. Kitchen DB, Decornez H, Furr JR, Bajorath J (2004) Nat Rev Drug Discov 3:935
10. Katritch V, Cherezov V, Stevens RC (2013) Annu Rev Pharmacol Toxicol 53:531
11. Cooke RM, Brown AJ, Marshall FH, Mason JS (2015) Drug Discov Today 20:1355
12. Rodriguez D, Ranganathan A, Carlsson J (2015) Curr Top Med Chem 15:2484
13. Deupi X, Kobilka BK (2010) Physiology (Bethesda) 25:293
14. Piscitelli CL, Kean J, de Graaf C, Deupi X (2015) Mol Pharmacol 88:536
15. Rasmussen SGF, Choi H-J, Rosenbaum DM, Kobilka TS, Thian FS, Edwards PC, Burghammer M,
Ratnala VRP, Sanishvili R, Fischetti RF, Schertler GFX, Weis WI, Kobilka BK (2007) Nature
450:383
16. Cherezov V, Rosenbaum DM, Hanson MA, Rasmussen SGF, Thian FS, Kobilka TS, Choi
H-J, Kuhn P, Weis WI, Kobilka BK, Stevens RC (2007) Science 318:1258
17. Lebon G, Warne T, Tate CG (2012) Curr Opin Struct Biol 22:482
18. Rasmussen SG, DeVree BT, Zou Y, Kruse AC, Chung KY, Kobilka TS, Thian FS, Chae PS,
Pardon E, Calinski D, Mathiesen JM, Shah ST, Lyons JA, Caffrey M, Gellman SH, Steyaert J,
Skiniotis G, Weis WI, Sunahara RK, Kobilka BK (2011) Nature 477:549
19. Kooistra AJ, de Graaf C, Timmerman H (2014) Neurochem Res 39:1850
20. Vaidehi N, Floriano WB, Trabanino R, Hall SE, Freddolino P, Choi EJ, Zamanakos G,
Goddard WA (2002) Proc Natl Acad Sci U S A 99:12622
21. Michino M, Chen J, Stevens RC, Brooks 3rd CL (2010) Proteins 78:2189
22. Shacham S, Topf M, Avisar N, Glaser F, Marantz Y, Bar-Haim S, Noiman S, Naor Z, Becker
OM (2001) Med Res Rev 21:472
23. Cavasotto CN, Palomba D (2015) Chem Commun 51:13576
24. Martı́-Renom MA, Stuart AC, Fiser A, Sánchez R, Melo F, Sali A (2000) Annu Rev Biophys
Biomol Struct 29:291
25. Sali A, Blundell TL (1993) J Mol Biol 234:779
26. Goldfeld DA, Zhu K, Beuming T, Friesner RA (2011) Proc Natl Acad Sci U S A 108:8275
27. Kmiecik S, Jamroz M, Kolinski M (2014) Biophys J 106:2408
28. de Graaf C, Foata N, Engkvist O, Rognan D (2008) Proteins 71:599
29. Chen KY, Sun J, Salvo JS, Baker D, Barth P (2014) PLoS Comput Biol 10:e1003636
30. Ray A, Lindahl E, Wallner B (2010) Bioinformatics 26:3067
31. Shen MY, Sali A (2006) Protein Sci 15:2507
32. Katritch V, Rueda M, Abagyan R (2012) Methods Mol Biol 857:189
33. Rodrı́guez D, Bello X, Gutiérrez-de-Terán H (2012) Mol Inf 31:334
34. Worth CL, Kreuchwig A, Kleinau G, Krause G (2011) BMC Bioinformatics 12:185
35. Sandal M, Duy TP, Cona M, Zung H, Carloni P, Musiani F, Giorgetti A (2013) PLoS One 8:
e74092
36. Latek D, Pasznik P, Carlomagno T, Filipek S (2013) PLoS One 8:e56742
37. Isberg V, Vroling B, van der Kant R, Li K, Vriend G, Gloriam D (2014) Nucleic Acids Res
42:D422
38. Costanzi S, Tikhonova IG, Harden TK, Jacobson KA (2009) J Comput Aided Mol Des 23:747
39. Kooistra AJ, Roumen L, Leurs R, de Esch IJ, de Graaf C (2013) Methods Enzymol 522:279
40. Rodrı́guez D, Gutiérrez-de-Teran H (2013) Curr Pharm Des 19:2216
41. Engel S, Skoumbourdis AP, Childress J, Neumann S, Deschamps JR, Thomas CJ, Colson
AO, Costanzi S, Gershengorn MC (2008) J Am Chem Soc 130:5115
42. Kellenberger E, Springael JY, Parmentier M, Hachet-Haas M, Galzi JL, Rognan D (2007)
J Med Chem 50:1294
43. Becker OM, Marantz Y, Shacham S, Inbal B, Heifetz A, Kalid O, Bar-Haim S,
Warshaviak D, Fichman M, Noiman S (2004) Proc Natl Acad Sci U S A 101:11304
44. Evers A, Klabunde T (2005) J Med Chem 48:1088
45. Michino M, Abola E, GPCR Dock 2008 Participants, Brooks CL, Dixon JS, Moult J, Stevens
RC (2009) Nat Rev Drug Discov 8:455
46. Kufareva I, Rueda M, Katritch V, GPCR Dock 2010 Participants, Stevens RC, Abagyan R
(2011) Structure 19:1108
47. Kufareva I, Katritch V, Participants of GPCR Dock 2013, Stevens RC, Abagyan R (2014)
Structure 22:1120
48. Carlsson J, Yoo L, Gao Z-G, Irwin JJ, Shoichet BK, Jacobson KA (2010) J Med Chem
53:3748
49. Lenselink EB, Beuming T, Sherman W, van Vlijmen HW, IJzerman AP (2014) J Chem Inf
Model 54:1737
50. de Beer SB, Vermeulen NP, Oostenbrink C (2010) Curr Top Med Chem 10:55
51. Lorber DM, Shoichet BK (2005) Curr Top Med Chem 5:739
52. Neves MA, Totrov M, Abagyan R (2012) J Comput Aided Mol Des 26:675
53. Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, Olson AJ (2009)
J Comput Chem 30:2785
54. Halgren TA, Murphy RB, Friesner RA, Beard HS, Frye LL, Pollard WT, Banks JL (2004)
J Med Chem 47:1750
55. Korb O, Stutzle T, Exner TE (2009) J Chem Inf Model 49:84
56. Verdonk ML, Cole JC, Hartshorn MJ, Murray CW, Taylor RD (2003) Proteins 52:609
57. Leach AR, Shoichet BK, Peishoff CE (2006) J Med Chem 49:5851
58. Sousa SF, Fernandes PA, Ramos MJ (2006) Proteins 65:15
59. Mysinger MM, Carchia M, Irwin JJ, Shoichet BK (2012) J Med Chem 55:6582
60. Phatak SS, Gatica EA, Cavasotto CN (2010) J Chem Inf Model 50:2119
61. Evers A, Klebe G (2004) Angew Chem Int Ed Engl 43:248
62. de Graaf C, Rognan D (2009) Curr Pharm Des 15:4026
63. Irwin JJ, Sterling T, Mysinger MM, Bolstad ES, Coleman RG (2012) J Chem Inf Model
52:1757
64. eMolecules. http://www.emolecules.com
65. Lipinski CA, Lombardo F, Dominy BW, Feeney PJ (2001) Adv Drug Deliv Rev 46:3
66. Baell JB, Holloway GA (2010) J Med Chem 53:2719
67. Sabio M, Jones K, Topiol S (2008) Bioorg Med Chem Lett 18:5391
68. Kolb P, Rosenbaum DM, Irwin JJ, Fung JJ, Kobilka BK, Shoichet BK (2009) Proc Natl Acad
Sci U S A 106:6843
69. Weiss DR, Ahn S, Sassano MF, Kleist A, Zhu X, Strachan R, Roth BL, Lefkowitz RJ,
Shoichet BK (2013) ACS Chem Biol 8:1018
70. Katritch V, Jaakola VP, Lane JR, Lin J, Ijzerman AP, Yeager M, Kufareva I, Stevens RC,
Abagyan R (2010) J Med Chem 53:1799
71. Chen D, Ranganathan A, IJzerman AP, Siegal G, Carlsson J (2013) J Chem Inf Model
53:2701
72. Rodrı́guez D, Gao ZG, Moss SM, Jacobson KA, Carlsson J (2015) J Chem Inf Model 55:550
73. Kolb P, Phan K, Gao ZG, Marko AC, Sali A, Jacobson KA (2012) PLoS One 7:e49910
74. Langmead CJ, Andrews SP, Congreve M, Errey JC, Hurrell E, Marshall FH, Mason JS,
Richardson CM, Robertson N, Zhukov A, Weir M (2011) J Med Chem 55:1904
75. Ranganathan A, Stoddart LA, Hill SJ, Carlsson J (2015) J Med Chem 58:9578
76. Carlsson J, Coleman RG, Setola V, Irwin JJ, Fan H, Schlessinger A, Sali A, Roth BL,
Shoichet BK (2011) Nat Chem Biol 7:769
77. Vass M, Schmidt E, Horti F, Keseru GM (2014) Eur J Med Chem 77:38
78. Lane JR, Chubukov P, Liu W, Canals M, Cherezov V, Abagyan R, Stevens RC, Katritch V
(2013) Mol Pharmacol 84:794
79. Mysinger MM, Weiss DR, Ziarek JJ, Gravel S, Doak AK, Karpiak J, Heveker N, Shoichet
BK, Volkman BF (2012) Proc Natl Acad Sci U S A 109:5517
80. Schmidt D, Bernat V, Brox R, Tschammer N, Kolb P (2015) ACS Chem Biol 10:715
81. de Graaf C, Kooistra AJ, Vischer HF, Katritch V, Kuijer M, Shiroishi M, Iwata S,
Shimamura T, Stevens RC, de Esch IJ, Leurs R (2011) J Med Chem 54:8195
82. Sirci F, Istyastono EP, Vischer HF, Kooistra AJ, Nijmeijer S, Kuijer M, Wijtmans M,
Mannhold R, Leurs R, de Esch IJP, de Graaf C (2012) J Chem Inf Model 52:3308
83. Istyastono EP, Kooistra AJ, Vischer HF, Kuijer M, Roumen L, Nijmeijer S, Smits RA, de
Esch IJP, Leurs R, de Graaf C (2015) MedChemComm 6:1003
84. Negri A, Rives ML, Caspers MJ, Prisinzano TE, Javitch JA, Filizola M (2013) J Chem Inf
Model 53:512
85. Kruse AC, Weiss DR, Rossi M, Hu J, Hu K, Eitel K, Gmeiner P, Wess J, Kobilka BK,
Shoichet BK (2013) Mol Pharmacol 84:528
86. Rodrı́guez D, Brea J, Loza MI, Carlsson J (2014) Structure 22:1140
87. Lam V, Rodriguez D, Zhang T, Koh E, Carlsson J, Salahpour A (2015) MedChemComm
6:2216
88. Rosenbaum DM, Cherezov V, Hanson MA, Rasmussen SGF, Thian FS, Kobilka TS, Choi
H-J, Yao X-J, Weis WI, Stevens RC, Kobilka BK (2007) Science 318:1266
89. Taylor MR (2007) Pharmacogenomics J 7:29
90. Wacker D, Fenalti G, Brown MA, Katritch V, Abagyan R, Cherezov V, Stevens RC (2010)
J Am Chem Soc 132:11443
91. Rasmussen SG, Choi HJ, Fung JJ, Pardon E, Casarosa P, Chae PS, Devree BT, Rosenbaum
DM, Thian FS, Kobilka TS, Schnapp A, Konetzki I, Sunahara RK, Gellman SH, Pautsch A,
Steyaert J, Weis WI, Kobilka BK (2011) Nature 469:175
92. Rosenbaum DM, Zhang C, Lyons JA, Holl R, Aragao D, Arlow DH, Rasmussen SG, Choi HJ,
Devree BT, Sunahara RK, Chae PS, Gellman SH, Dror RO, Shaw DE, Weis WI, Caffrey M,
Gmeiner P, Kobilka BK (2011) Nature 469:236
93. Mysinger MM, Shoichet BK (2010) J Chem Inf Model 50:1561
94. Ballesteros JA, Weinstein H (1995) Methods Neurosci 25:366
95. Muller CE, Jacobson KA (2011) Biochim Biophys Acta 1808:1290
96. Fredholm BB, IJzerman AP, Jacobson KA, Linden J, Muller CE (2011) Pharmacol Rev 63:1
97. Jaakola V-P, Griffith MT, Hanson MA, Cherezov V, Chien EYT, Lane JR, IJzerman AP,
Stevens RC (2008) Science 322:1211
98. Xu F, Wu H, Katritch V, Han GW, Jacobson KA, Gao ZG, Cherezov V, Stevens RC (2011)
Science 332:322
99. Lebon G, Warne T, Edwards PC, Bennett K, Langmead CJ, Leslie AG, Tate CG (2011)
Nature 474:521
100. Tosh DK, Phan K, Gao ZG, Gakh AA, Xu F, Deflorian F, Abagyan R, Stevens RC, Jacobson
KA, Katritch V (2012) J Med Chem 55:4297
101. Beaulieu JM, Espinoza S, Gainetdinov RR (2015) Br J Pharmacol 172:1
102. Chien EYT, Liu W, Zhao Q, Katritch V, Han GW, Hanson MA, Shi L, Newman AH, Javitch
JA, Cherezov V, Stevens RC (2010) Science 330:1091
103. Scholten DJ, Canals M, Maussang D, Roumen L, Smit MJ, Wijtmans M, de Graaf C, Vischer
HF, Leurs R (2012) Br J Pharmacol 165:1617
104. Wu B, Chien EY, Mol CD, Fenalti G, Liu W, Katritch V, Abagyan R, Brooun A, Wells P, Bi
FC, Hamel DJ, Kuhn P, Handel TM, Cherezov V, Stevens RC (2010) Science 330:1066
105. Panula P, Chazot PL, Cowart M, Gutzmer R, Leurs R, Liu WL, Stark H, Thurmond RL, Haas
HL (2015) Pharmacol Rev 67:601
106. Shimamura T, Shiroishi M, Weyand S, Tsujimoto H, Winter G, Katritch V, Abagyan R,
Cherezov V, Liu W, Han GW, Kobayashi T, Stevens RC, Iwata S (2011) Nature 475:65
107. Dhawan BN, Cesselin F, Raghubir R, Reisine T, Bradley PB, Portoghese PS, Hamon M
(1996) Pharmacol Rev 48:567
108. Vanderah TW (2010) Clin J Pain 26:5
109. Granier S, Manglik A, Kruse AC, Kobilka TS, Thian FS, Weis WI, Kobilka BK (2012)
Nature 485:400
110. Manglik A, Kruse AC, Kobilka TS, Thian FS, Mathiesen JM, Sunahara RK, Pardo L, Weis
WI, Kobilka BK, Granier S (2012) Nature 485:321
111. Wu H, Wacker D, Mileni M, Katritch V, Han GW, Vardy E, Liu W, Thompson AA, Huang
XP, Carroll FI, Mascarella SW, Westkaemper RB, Mosier PD, Roth BL, Cherezov V, Stevens
RC (2012) Nature 485:327
112. Kruse AC, Kobilka BK, Gautam D, Sexton PM, Christopoulos A, Wess J (2014) Nat Rev
Drug Discov 13:549
113. Kruse AC, Hu J, Pan AC, Arlow DH, Rosenbaum DM, Rosemond E, Green HF, Liu T, Chae
PS, Dror RO, Shaw DE, Weis WI, Wess J, Kobilka BK (2012) Nature 482:552
114. Haga K, Kruse AC, Asada H, Yurugi-Kobayashi T, Shiroishi M, Zhang C, Weis WI,
Okada T, Kobilka BK, Haga T, Kobayashi T (2012) Nature 482:547
115. Wess J, Eglen RM, Gautam D (2007) Nat Rev Drug Discov 6:721
116. Berger M, Gray JA, Roth BL (2009) Annu Rev Med 60:355
117. McCorvy JD, Roth BL (2015) Pharmacol Ther 150:129
118. Saxena PR, Peer T-H (2001) J Headache Pain 2:8
119. Wacker D, Wang C, Katritch V, Han GW, Huang XP, Vardy E, McCorvy JD, Jiang Y,
Chu M, Siu FY, Liu W, Xu HE, Cherezov V, Roth BL, Stevens RC (2013) Science 340:615
120. Wang C, Jiang Y, Ma J, Wu H, Wacker D, Katritch V, Han GW, Liu W, Huang XP, Vardy E,
McCorvy JD, Gao X, Zhou XE, Melcher K, Zhang C, Bai F, Yang H, Yang L, Jiang H, Roth
BL, Cherezov V, Stevens RC, Xu HE (2013) Science 340:610
121. Langmead CJ, Christopoulos A (2014) Curr Opin Cell Biol 27:94
122. Kruse AC, Ring AM, Manglik A, Hu J, Hu K, Eitel K, Hubner H, Pardon E, Valant C, Sexton
PM, Christopoulos A, Felder CC, Gmeiner P, Steyaert J, Weis WI, Garcia KC, Wess J,
Kobilka BK (2013) Nature 504:101
123. Dore AS, Okrasa K, Patel JC, Serrano-Vega M, Bennett K, Cooke RM, Errey JC, Jazayeri A,
Khan S, Tehan B, Weir M, Wiggin GR, Marshall FH (2014) Nature 511:557
124. Wu H, Wang C, Gregory KJ, Han GW, Cho HP, Xia Y, Niswender CM, Katritch V, Meiler J,
Cherezov V, Conn PJ, Stevens RC (2014) Science 344:58
125. Kooistra AJ, Leurs R, de Esch IJ, de Graaf C (2015) J Chem Inf Model 55:1045
126. Hert J, Irwin JJ, Laggner C, Keiser MJ, Shoichet BK (2009) Nat Chem Biol 5:479
127. Moult J (2005) Curr Opin Struct Biol 15:285
128. Lensink MF, Méndez R, Wodak SJ (2007) Proteins 69:704
129. Damm-Ganamet KL, Smith RD, Dunbar Jr JB, Stuckey JA, Carlson HA (2013) J Chem Inf
Model 53:1853
130. Geballe MT, Skillman AG, Nicholls A, Guthrie JP, Taylor PJ (2010) J Comput Aided Mol
Des 24:259
131. Okada T, Sugihara M, Bondar AN, Elstner M, Entel P, Buss V (2004) J Mol Biol 342:571
132. Murakami M, Kouyama T (2008) Nature 453:363
133. Warne T, Serrano-Vega MJ, Baker JG, Moukhametzianov R, Edwards PC, Henderson R,
Leslie AGW, Tate CG, Schertler GFX (2008) Nature 454:486
134. Katritch V, Rueda M, Lam PC, Yeager M, Abagyan R (2010) Proteins 78:197
135. Dore AS, Robertson N, Errey JC, Ng I, Hollenstein K, Tehan B, Hurrell E, Bennett K,
Congreve M, Magnani F, Tate CG, Weir M, Marshall FH (2011) Structure 19:1283
136. Roumen L, Sanders MPA, Vroling B, De Esch IJP, De Vlieg J, Leurs R, Klomp JPG, Nabuurs
SB, De Graaf C (2011) Pharmaceuticals 4:1196
137. Wang C, Wu H, Evron T, Vardy E, Han GW, Huang XP, Hufeisen SJ, Mangano TJ, Urban
DJ, Katritch V, Cherezov V, Caron MG, Roth BL, Stevens RC (2014) Nat Commun 5:4355
138. Wang C, Wu H, Katritch V, Han GW, Huang XP, Liu W, Siu FY, Roth BL, Cherezov V,
Stevens RC (2013) Nature 497:338
139. Rodrı́guez D, Ranganathan A, Carlsson J (2014) J Chem Inf Model 54:2004
140. Leo D, Mus L, Espinoza S, Hoener MC, Sotnikova TD, Gainetdinov RR (2014) Neurophar-
macology 81:283
141. Anighoro A, Bajorath J, Rastelli G (2014) J Med Chem 57:7874
142. Vass M, Agai-Csongor E, Horti F, Keseru GM (2014) ACS Med Chem Lett 5:1010
143. Zhukov A, Andrews SP, Errey JC, Robertson N, Tehan B, Mason JS, Marshall FH, Weir M,
Congreve M (2011) J Med Chem 54:4312
Top Med Chem (2019) 30: 101–162
DOI: 10.1007/7355_2016_24
Published online: 14 March 2017
Integration on Ligand and Structure

Based Approaches in GPCRs
Anil K. Saxena, Shome S. Bhunia, and Mridula Saxena
Abstract The GPCRs are involved in wide range of physiological functions and pa-
thological conditions and hence they are considered drug targets of immense pharma-
ceutical importance. Modification of the cell-specific signaling and functioning of the
GPCRs provides an excellent opportunity for drug design activities. In view of the
diverse and complex nature of GPCR signaling, the proper understanding of receptor
structure is essential to illustrate their elusive structure–function relationships for ra-
tional drug design by the application of efficient and inexpensive molecular modeling
techniques. Despite advances in GPCR protein crystallization, the structural informa-
tion of most GPCRs is still unclear that necessitates the application of homology mod-
eling techniques to derive protein models for structure based drug design. However low
sequence similarity in GPCRs coupled with high structural plasticity may hinder the
development of appropriate protein models. In order to circumvent the problems, the
development of ligand based models integrated with structure based models may con-
verge to an integrated model duly validated on internal and/or external test set models
which may be useful in virtual screening for identifying the hits and in generating the
focused libraries for prioritizing the molecules for synthesis. The integrated models
may also be helpful in increasing our understanding of drug–receptor interactions.
A.K. Saxena (*)

Division of Medicinal and Process Chemistry, Central Drug Research Institute, Lucknow 226
031, India
e-mail: anilsak@gmail.com
S.S. Bhunia
Global Institute of Pharmaceutical Education and Research, Kashipur, Uttarakhand, India
e-mail: shome.sankar@gmail.com
M. Saxena
Department of Chemistry, Amity University, Lucknow, India
e-mail: drmridula.saxena@gmail.com
102 A.K. Saxena et al.
Keywords α1 adrenergic, Docking, Dopamine, Histamine, GPCR molecular

modeling, Pharmacophore
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
2 Advantages and Disadvantages of Ligand and Structure Based Approaches . . . . . . . . . . . . . 105
3 Integration of Ligand and Structure Based Approaches for Understanding GPCR
Structure and Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
4 Case Studies on the Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
4.1 α1a Adrenergic Receptor Antagonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
4.2 Dopamine D2 Receptor Agonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
4.3 Histamine H1 Receptor Antagonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
Abbreviations
(S)-6-OH-DPAT (S)-6-hydroxy-2-(dipropylamino)tetralin
(S)-7-OH-DPAT (S)-7-hydroxy-2-(dipropylamino)tetralin
(S)-DPAT (S)-Dipropylamino tetralin
2-AMC 2-(Aminomethyl)chromans
2D QSAR Two-dimensional quantitative structure–activity relationships
2-OHNPA 2-Hydroxy-N-n-propylnorapomorphine
3D QSAR Three-dimensional quantitative structure–activity relationship
3-PPP 3-(3-Hydroxyphenyl)-N-n-propylpiperidine
5-OH-DPAT 5-Hydroxy-2-(dipropylamino)tetralin
7-OH-OHBQ 7-Hydroxy-1,2,3,4,4a,5,6,10b-octahydrobenzo[f]quinoline
9-OH-OHBQ 9-Hydroxy-1,2,3,4,4a,5,6,10b-octahydrobenzo[f]quinoline
α1a Adr α1a adrenergic receptor
α1b Adr α1b adrenergic receptor
α1c Adr α1c adrenergic receptor
α2 Adr α2 adrenergic receptor
β1 Adr β1 adrenergic receptor
β2 Adr β2 adrenergic receptor
AAA Active analog based approaches
ADTN 6-Amino-5,6,7,8-tetrahydronaphthalene-2,3-diol
APO Apomorphine
Ar Aromatic
BPH Benign prostate hyperplasia
CADD Computer aided drug design
CFP Common feature pharmacophore
CHARMm Chemistry at HARvard Macromolecular mechanics
CNS Central nervous system stimulant
CoMFA Comparative molecular field analysis
Integration on Ligand and Structure Based Approaches in GPCRs 103
CoMSIA Comparative molecular similarity index analysis

DA Dopamine
DHX Dihydrexidine
ECL2 Extracellular loop 2
GIP GPCR-interacting protein
Glide XP Glide extra precision
GRK G protein-coupled receptor kinases
HAl Hydrophobic aliphatic
HAr Hydrophobic aromatic
HASL Hypothetical active site lattice
HBA Hydrogen bond acceptor
HBD Hydrogen bond donor
HOMO Highest occupied molecular orbital
HY Hydrophobic
LBDD Ligand based drug design
LUMO Lowest unoccupied molecular orbital
LUTS Low urinary tract symptoms
MDDR MDL Drug Data Report
MR Molar refractivity
NCE Novel chemical entity
NPA N-n-propylnorapomorphine
PD Parkinson’s disease
PI Positive ionizable
RA Ring aromatic
RMSD Root mean square deviation
SBDD Structure based drug design
SSS Substituent spanned space
TM Transmembrane
VS Virtual screening
1 Introduction
The GPCRs are recognized as potential drug targets with multitude of successful
therapeutic interventions in human physiology [1]. This can be easily assessed as 50%
of marketed drugs including 20% best selling drugs target GPCRs [2]. However the
overall therapeutic modality for the GPCRs family is still huge as they are encoded by
more than 800 genes in the human genome [3] that provides an excellent opportunity
for drug development activities. Based on phylogenetic analysis of human repertoire,
the GPCRs are classified into five main families according to the GRAFS classifi-
cation that include Glutamate (Class C), Rhodopsin (Class A), Adhesion (Class B),
Frizzled (Class F), and Secretin (Class B) class of receptors [3, 4]. The GPCR family
share common receptor architecture of heptahelical transmembrane domains
connected by alternative extracellular and intracellular loops. With enormous sub-

types and heterogenous tissue distribution, the GPCRs are the targets for a wide array
of chemical entities ranging from photons, ions, amines, nucleotides, lipids, peptides,
and proteins [5, 6]. Activation of the GPCRs by these chemical entities induces
changes in the heptahelical conformation and signal-switching leading to activation
of G protein or binding of β-arrestin proteins or other GPCR-interacting proteins
(GIPs) resulting in signal transduction from cytosol to the interior of the cell [7–
11]. The highly intricate and integrated network of signal transduction by the GPCRs
has been strategically utilized for drug targeting [12–14], to modify or manipulate the
downstream signaling pathways that have clinical implications in cancer [15–17],
cardiac dysfunction [18–22], central nervous system disorders [23–26], diabetes [27–
29], inflammation [30, 31], pain [32–34], and several other diseases [35].
The diverse and superior pharmacological profile of the GPCRs has intensified
the research interest for understanding the receptor structure and its associated mol-
ecular mechanism at atomistic level. Unfortunately gaining a molecular picture at
atomistic level to gain insights about the GPCR structure and function is mainly
held back due to the challenges encountered in GPCR crystallization. The GPCRs
are membrane proteins hydrophobic in nature that require detergents for cell mem-
brane extraction. Further the flexible and unstable nature of the GPCRs also pre-
sents difficulties including expression, solubilization, and purification that provide
challenges to isolate them in a fully functional and biochemically stable form [36].
The first X-ray crystal structure of Bovine rhodopsin in the year 2000 was a step
forward in GPCR research that provided information about GPCR receptor orga-
nization and served as a universal template for GPCR homology modeling and drug
design. Advancement in receptor stabilization and protein crystallographic tech-
niques has been helpful in resolving the crystal structure of β2 adrenergic receptor
in the year 2007 and nine other GPCRs in the year 2012 [37]. The recent advance-
ment in protein crystallography techniques such as in meso in situ serial crystal-
lography has further eased the crystallization protocols of GPCRs [38]. Besides
protein crystallization techniques, solid state NMR techniques are also important
tools to gather the atomistic detail about GPCR proteins [39].
Computer aided drug design involving both ligand and structure based approaches
has been highly intended towards the identification and optimization of ligands for
their therapeutic use. Ligand based (indirect) drug design facilitates the identification
of important chemical features in a set of bioactive molecules sharing a common tar-
get for which protein structural information is naive. The objective of the ligand based
drug design (LBDD) is to develop predictive models that can differentiate the bio-
active molecules from the least active and/or inactive ones. Until the mid 1980s,
LBDD was the major choice in understanding the structure–function relationships
between the ligands and the receptors particularly GPCRs due to scarcity in target
protein structure information. Although the first homology model was reported in
1980s, success stories related to structure based (direct) drug design (SBDD) were
reported in the early 1990s [40–42]. Since then the rapid advancements in molecular
biology, human genetics, and functional genomics resulted in an increased number of
molecular targets for therapeutic intervention through SBDD approaches. This, coupled
with the concomitant application of protein modeling techniques like homology mod-
eling, has fueled the improvements in drug-screening output.
2 Advantages and Disadvantages of Ligand and Structure

Based Approaches
The LBDD is based on the concept of molecular similarity that states that struc-
turally similar molecules should provide similar biological response. The models
developed through LBDD have been useful for virtual screening in the absence of
protein structural information and require low computational cost [43, 44]. It iden-
tifies the important chemical finger prints necessary for biological activity and has
gone through several development phases starting from active analog based ap-
proaches (AAA) to classical 2DQSAR studies [45] with current emphasis being on
3D QSAR based pharmacophore models which provide the three-dimensional
arrangement of structural features necessary for a biological activity. The develop-
ment of predictive pharmacophore models started from approaches like hypothet-
ical active site lattice (HASL) [46], comparative molecular field analysis (CoMFA)
[47], comparative molecular similarity index analysis (CoMSIA) [48, 49], Apex 3D
[50] including the recent ones such as PHASE [51, 52] from Schr€odinger, Hiphop
and HypoGen from Biovia [53, 54], GASP [55], DISCO [56], and GALAHAD [57]
from Tripos.
Though they are time and cost effective, the major limitations are due to their in-
ability to consider the receptor flexibility and constraints in considering the diverse
chemical structures including large substituent spanned space (SSS). These limitations
lead to restricted pharmacophores as they contain the fingerprints of a particular struc-
tural class of ligands without having information about others and remain confined
within the structural space of the ligands used for generation of the models and thus do
not qualify to be universal pharmacophore models [58]. The SBDD on the other hand
present a more realistic picture of ligand interaction at the receptor site with better
understanding of molecular interactions at the binding site than LBDD. The techniques
used in SBDD like docking of ligands provide better understanding of interactions at
the binding site than ligand based approaches. The SBDD is not restricted to SSS
limitations like LBDD and has more wider applications in virtual screening of large
chemical libraries [59–63]. The docking models solely rely on the interactions of a
ligand at the receptor site and thus may lead to new chemical entities (NCEs) in terms
of novelty as compared to the ones obtained through LBDD.
Most of the docking algorithms [64–67] depend on scoring function [59, 68] that
has some drawbacks. It should be noted that docking studies are dependent on the
criteria of correct binding mode of the ligand at the receptor site as scores with false
docked conformation can be deceiving. Secondly not all the interactions at the
receptor site are meaningful, i.e., certain interactions at the receptor site are nec-
essary to elicit a response. In certain cases, it may be possible that a ligand may
show interactions with amino acids that may not be responsible for activity but as
docking algorithms do not discriminate interactions between important and non-
important residues rather they provide score depending on the number of interac-
tions that may also result in false positives. However current docking protocols had
constraint docking options to specify the important residue interactions for screen-
ing of ligands by virtual screening (VS) protocol [69–71]. Protein conformational
fluctuations may also alter the docking performances [72–75] and this is rather an
important factor to be considered while carrying docking studies on homology mod-
eled protein as they are highly dependent on the selection of template structures and
sequence alignment. It has been observed that multiple sequence alignment has
been the best alternatives to model proteins when single template alignment results
in low sequence similarity [76, 77]. Further validation of the homology models with
known ligands is a necessary criterion to find out if the model is suitable enough to
differentiate between known active and inactive ligands [78, 79]. In certain cases,
the role of water molecules in stabilizing a ligand–receptor complex is also impor-
tant and must be taken into consideration [80–82]. Thus both LBDD and SBDD
have their own merits and demerits (Table 1).
Table 1 The merits and demerits of LBDD and SBDD

Merits Demerits
Ligand based drug design
Very efficient virtual screening queries Limited to their domain of applicability
Use of generalized descriptors, features, and Requirement of bioactive conformation and
fingerprints molecular alignments
Has evolved well with the advancements in Efforts are required to develop universally
statistics and other machine learning applicable methods
algorithms
Structure based drug design
De novo design technique: aids in the identi- Quality of the docking and scoring results and
fication of diverse and novel structural technical feasibility of processing a large
prototypes database
Serves as a good filter to remove inactive Pose vs score calculation
compounds (decoys)
Can be used to discriminate between more and Adequate positional and conformational sam-
less active compounds pling in the active site
Accounting for protein flexibility
Treatment of water molecules during ligand
docking
3 Integration of Ligand and Structure Based Approaches

for Understanding GPCR Structure and Function
Unlike other receptors, the GPCR–ligand interactions are highly complex that can
trigger different receptor conformational switching with subtle differences in bind-
ing energy. The recent advances in the protein crystallization techniques in last
5 years have provided new insights about ligand mediated functional changes in the
GPCRs [36, 83]. This has highly enriched the knowledge about ligand–receptor
interactions and offered an opportunity for decoding the ligand–receptor relation-
ships for rational drug design. Structural investigation of these receptor switching
has enabled to understand the functional plasticity in GPCRs where agonists can
promote a conformational change in the receptor, transforming it from an inactive
to active state by stabilizing the basal state of the receptor. This dynamic transition
of the receptor from an inactive to active state by the agonist happens due to con-
formational rearrangement at the helix region that disrupts the ionic lock between
the positively charged arginine in transmembrane domain (TM) 3 (R3.50) and a
negatively charged residue in TM6 (D/E6.30) [84–86]. This disruption of the ionic
bond was first observed in the crystal structure of opsin in complex with the alpha
subunit of transducin (pdb ID: 3DBQ) [87] and later on the agonist bound crystal
structure of β2 Adr (pdb ID: 3P0G) [88]. In both these crystal structures, an outward
movement of TM6 was observed that facilitates a large binding site for the Gα
subunit that is more prominent in case of the β2 Adr crystal structure. The under-
lying mechanism of ionic lock disruption was determined by comparing the inac-
tive receptor with the active one. In the inactive receptor, the residues Leu3.43,
Phe6.44, and X6.40 where X is a bulky hydrophobic residue (e.g., valine, isoleu-
cine, leucine, or methionine) remain in close conjunction with one another mai-
ntaining the ionic lock between TMIII and TMVI. Binding of an agonist at the
receptor site promotes an upward movement of TMIII resulting in dislocation of
Leu3.43 and stabilization with Leu4.26. This stabilization compels Leu4.26 to bring
an upward movement for Asn7.49 of the NPxxY motif that enables Tyr7.53 to move
in the cytoplasmic region and make H-bond with Tyr5.58 through a water molecule.
The Tyr5.58 in turn gets H-bonded with the Arg3.50 of the (D/ERY) motif to stabilize
the activated receptor structure [89]. On the contrary, the inverse agonists [90] sta-
bilize the inactive conformation of the receptor by maintaining the ionic lock while the
neutral antagonists do not affect the equilibration between the active and inactive
states of the receptor.
Besides signal transduction by association with the heterotrimeric G proteins, the
7TM helices in their activated conformation also interact with two other protein
families: β-arrestins and G protein-coupled receptor kinases (GRKs). The β-arrestins
were originally identified to be involved in GPCR signal trafficking by promoting
desensitization, internalization, and recycling of activated receptors [91] until some
investigations underpinned their new role as G protein-independent signal transduc-
ers [92, 93]. This property of the 7-TM helices to bind β-arrestin in activated state
led to the conceptual development of the term “Biased ligand agonism” or “Biased
agonism.” Biased ligand agonism is a phenomenon where ligands can specifically and
precisely induce an activated receptor conformation able to bind either G proteins
or β-arrestin so as to create a balance between G protein-dependent and β-arrestin-
dependent signal transduction [94–97]. Due to specificity in signaling, biased li-
gands may be effective therapeutic agents [98, 99] with low side effects.
Majority of the drugs targeting GPCRs are confined to the class A rhodopsin like
receptors that constitute a group of receptors with significant pharmacological im-
portance [5, 100]. Structural investigations of the receptors in this family signify
prominent differences regarding the orthosteric binding architecture constituted by
the residues in the helices of TMIII, TMVI, and TMVIII and residues of the highly
variable ECL2 that has also contributory role in ligand binding [101, 102]. However
in certain cases the common heptahelical construct provides some similarities in the
receptor binding environment enabling receptor cross talks [103, 104]. This type of
receptor cross talks demand ligands that prefer selective receptor binding to avoid
unwanted receptor mediated side effects. The selectivity issues get more complex
when it converges to a single superfamily of GPCRs. The orthosteric binding site in
a single GPCR subfamily is highly conserved making it difficult to achieve high
selectivity for specific GPCR subtypes [105]. The problem of subtype selectivity in
GPCRs has been strategically resolved by targeting the allosteric site [106, 107] by
modulators capable of manipulating the potency and efficacy of orthosteric ligands
besides having self-agonistic or inverse agonistic behavior [24, 108–110]. In this
context, bitopic ligands [111–113] have been designed linking the allosteric and or-
thosteric pharmacophore components.
All the factors mentioned above are indicative of the complex and diverse behav-
ior of GPCR signaling which is influenced by ligand mediated subtle changes in the
receptor environment. These complexities can be handled by having proper under-
standing about the ligand properties capable of modifying the receptor environment in
eliciting a desired response. This necessitates the application of molecular modeling
techniques to understand the insights about ligand structural requirements and their
interaction at the receptor site. Drug design strategies targeting GPCRs started long
back due to their incredible role in therapeutics. However most of the studies prior to
2000 depend largely on ligand based information due to absence of protein 3D struc-
tures of GPCRs [114–117]. In the initial phase (till the year 2000), the drug design
projects targeting GPCRs started with the development of elemental speculative mod-
els derived from alignment of rigid bioactive ligands (low flexibility) and concomitant
prediction of putative receptor binding volume without any information about recep-
tor composition [115]. These elementary models unique in their concept provided ba-
sic ideas about the important functional groups required for ligand recognition and
their relative three-dimensional coordinates in space. However the models were li-
mited in their ability to account ligand flexibility as they were developed considering
structurally rigid molecules. The structure based drug design approaches during this
period were dependent on homology models developed on bacteriorhodopsin as tem-
plate and suffered with the limitation of low sequence similarity. The transition phase
(2000–2007) of GPCR in silico modeling utilized: (1) the advances in the LBDD by
developing pharmacophore models based on diverse chemical structures taking into
account ligand flexibility followed by the two-tier (internal and external test set) va-
lidation and (2) correlation of information from pharmacophore models with the avail-
able information about the 3D structure of the receptor derived through homology
models with bovine rhodopsin as template and were better than those models derived
from bacteriorhodopsin [118, 119]. Homology modeling is based on the concept of
resemblance in structure and function of proteins having the same evolutionary origin
with conserved motifs. The structural information gained from bovine rhodopsin high-
ly enriched the concepts about the constitutional arrangements of the 7TM helices and
was further utilized in structural prediction of other GPCRS by homology modeling
techniques to acquire improved understanding about receptor structure and function
[120]. However with no alternative in hand, modeling proteins having low sequence
similarity with bovine rhodopsin could only generate approximate models rather than
predictive models. For years, bovine rhodopsin was the only option to model GPCR
structures until the crystal structure of β2 adrenergic receptor was solved in the year
2007 [121, 122]. Since then technological advances in protein engineering and crys-
tallization have removed the bottlenecks in GPCR crystallization. So far, the most
successful application in protein engineering is the generation of GPCR constructs by
incorporating the fusion protein T-4 lysozyme in the third intracellular loop of GPCRs
that augmented protein stabilization and crystallization [88, 122–133]. The T4L was
also used to crystallize the β1 Adr structure by fusing it at the N-terminal domain [134].
Application of other protein stabilization techniques or methods such as receptor point
mutation [135, 136], stabilizing antibodies, and presence of covalent ligands has also
resulted in successful GPCR crystallizations [137]. The advance in GPCR crystalliza-
tion including proteomics and genomics highly enhanced the current knowledge of
GPCRs and has opened opportunities for computer assisted molecular modeling. With
the increasing number of GPCR crystal structures, it is now possible to construct ho-
mology models for other GPCRs by selecting the most close homolog using single
sequence alignment or by using a combination of multiple sequence alignment [121,
138, 139]. However successful structural models using homologs depend on several
factors such as selection of right templates [140], proper alignment of amino acid se-
quences for better identity/similarity matches, and proper conformational sampling of
the flexible and variable loop region [141]. Moreover, the reliability of these homology
models lies in their ability to predict the activity of the known ligands through docking
studies coupled with site-directed mutagenesis information [78, 142, 143]. Although
advances in SBDD improved the concepts of drug design and created lot of opportu-
nities, still LBDD has its unique capabilities that cannot be ignored. It should be noted
that LBDDs provide a strict view about the important functionalities required for a
certain response and by that way it demarcated the function to response selectivity.
This is extremely important for GPCRs where subtle changes in ligand functionality
can cause a drastic change in receptor response. Hence an integration of LBDD and
SBDD, that are mutually independent, provides better insights in terms of assumptions
and approximations about ligand features recognition and their interactions at the
receptor site. Further these integrated models provide additional filtering layers du-
ring the process of virtual screening reducing the number of false positives. The
applications of the integrated approaches have been exemplified in the following

case studies on GPCRs.
4 Case Studies on the Models
4.1 α1a Adrenergic Receptor Antagonists
The α1 adrenergic receptors (α1 Adr) involved in sympathetic response are distributed
mainly in the cardiovascular system and lower urinary tract. Benign prostate hyper-
plasia (BPH) is an excessive growth of the prostate gland leading to obstruction of the
bladder outlet resulting in low urinary tract symptoms (LUTS) manifested by de-
creased urine stream with increased frequency of urination and sensation of incom-
plete bladder emptying. The α1a Adr antagonists bring symptomatic relief in BPH by
relaxing the prostrate smooth muscle. Despite the proven role of α1a Adr in BPH, de-
signing subtype selective antagonists has been challenging due to high homology of
the binding site among the other α1 subtypes. Most of the nonselective α1 Adr an-
tagonists are associated with cardiovascular side effects such as orthostatic hypoten-
sion mediated by the α1b Adr subtype. Hence agents having specific antagonistic
affinity towards the α1a Adr subtypes having little affinity towards α1b Adr will be
safe agents for BPH. Some of the important aspects of α1a Adr subtype selectivity
derived from the preliminary ligand based models and integrated with SBDD have
been discussed here. Preliminary pharmacophore models to distinguish subtype selec-
tivity in α1a Adr subtype were reported by using the Apex 3D software [144].
The Apex 3D expert system is based on logico structural approach to drug design
developed by Goldener et al. and is used for classification and prediction of biological
activity. Apex 3D automatically identifies biophores (pharmacophores) which repre-
sent a certain structural and electronic pattern in a bioactive molecule which is res-
ponsible for its activity through interaction with the receptor and can be regarded as
the local arrays of descriptor centers (user defined atoms, pseudo atoms like ring
centers, hydrophobic region, or hydrogen binding sites) [50]. The Apex 3D phar-
macophore was developed to characterize the salient features for characterizing se-
lectivity among α1a and α1b subtypes. The α1a Adr antagonist biophore was developed
taking multiple conformations of silodosin (KMD-3213) (1) (pKi/α1a: α1b ¼ 10.4:7.7),
5-methyluropidil (2) (pKi/α1a: α1b ¼ 9.2:7.6), and (+) niguldipine (3) (pKi/α1a:
α1b ¼ 9.6:7.6) (Fig. 1a). The α1a Adr antagonist pharmacophore consisted of three
biophoric centers viz., aromatic groups, basic nitrogen atom, and aromatic or six-
membered ring bearing polar substituents. The distance of the aromatic ring center
from the basic nitrogen atom was stated to be 5.2–5.8 Å while an aromatic or six-
membered ring bearing polar substituents is 6–8 Å away from the basic nitrogen
(Fig. 1c). The α1b Adr antagonist biophore developed by using spiperone (4)
(pKi/α1a: α1b ¼ 8.1:9.3) and risperidone (5) (pKi/α1a: α1b ¼ 6.6:8.6) (Fig. 1b)
resulted in a similar biophore like the α1a Adr subtype regarding the chemical
Fig. 1 (a) The training set molecules silodosin (1), 5-methyluropidil (2), and (+)-niguldipine (3) and
their mapping with the α1a Adr antagonist pharmacophore model with blue spheres for aromatic ring,
violet spheres for PI, and peach spheres for aromatic moiety with polar groups. (b) The training set
molecules spiperone (4) and risperidone (5), and their mapping with the α1b Adr antagonist phar-
macophore model. (c) The α1a and α1b Adr antagonist pharmacophore model with distances in Å. (d)
The chemical structures of α1 Adr antagonists used to describe the selectivity among α1 Adr subtypes
by Bremner et al.
features but in case of α1b Adr subtype the distance between the basic nitrogen cen-
ter and the aromatic group was longer ranging between 6.2 and 7.8 Å (Fig. 1c) than in
α1a Adr. These biophores served as a preliminary model for differentiating between
the α1a and α1b Adr antagonists and specified the distance criteria between the pos-
itive nitrogen center and aromatic group in the α1a antagonists which is crucial for
activity. All the nonselective antagonists such as 5-methyluropidil (2), dicentrine (6),
and corynanthine (7) mapped both the α1a and α1b Adr pharmacophores according to
the nonselective property of these antagonists. As observed in Fig. 1c, the distances
between the aromatic ring with polar groups and the basic nitrogen in α1a Adr
pharmacophore were almost similar to the distance between the aromatic ring and
the basic nitrogen in the α1b Adr pharmacophore. It created the possibility for some of
the ligands to fit both the α1a and α1b Adr antagonist pharmacophores by just re-
versing their orientation. This is illustrated in case of nonselective antagonist WB4101
(8) in which the benzodioxane group mapped the aromatic feature of the α1a Adr
pharmacophore and the aromatic ring associated polar features in the α1b Adr while
the other aromatic ring containing two methoxy groups mapped the aromatic ring
associated polar features in α1a Adr and the aromatic feature in α1b Adr. Compounds
that did not fulfill these mapping criteria by reversing their orientation acted as se-
lective antagonists for α1a and α1b Adr subtypes. The α1a Adr selective silodosin (1)
has less chances of reverse orientation as the distance between the phenyl ring (con-
taining the trifluoro ethoxy substituent) and the basic nitrogen is optimum to fit the α1a
Adr pharmacophore only while the distance between the basic nitrogen and the phenyl
ring at the other end is not in the range to fit the distance criteria of 6–8 Å for α1b Adr.
For 5-methyluropidil (2), the presence of only one aromatic feature in its structure
makes it fit for only the α1a Adr pharmacophore while (+)-niguldipine (3) has only one
polar end. The α1b Adr selective antagonist risperidone (5) has five-membered het-
erocyclic ring to match the aromatic feature in the α1a Adr pharmacophore which is
less favorable than the usual six-membered ring mapping this feature. The other com-
pound spiperone (4) has a long distance between the aromatic feature and the basic
nitrogen that lessens its chances to fit the α1a Adr pharmacophore. However both
prazosin (9) and phentolamine (10) do not match the distance requirements of the
pharmacophore model. In prazosin (9), the distance calculated from both the aromatic
rings and the basic nitrogen atom varied from 2.7 to 7.6–8.8 Å, respectively, that did
not satisfy the required distance criteria for the α1a Adr antagonist pharmacophore.
The same was observed in case of phentolamine (10) where the distance between the
aromatic ring and the basic nitrogen center was 3 Å. This unsuccessful mapping of
prazosin (9) with the conventional α1a Adr pharmacophore indicated that prazosin
may have a different binding mode at the α1a Adr compared with other antagonists.
In order to attain more clarity about the features responsible for selectivity among
α1a, α1b, and α1d Adr subtypes, the catalyst based models were used to identify the
differences among the Adr subtypes [145]. All these pharmacophore models were
developed excluding the quinazolines and the niguldipines. In the development of the
α1a Adr pharmacophore, four compounds were used where two compounds silodos-
in (1) and methyluropidil (2) were used previously in the Apex 3D pharmacophore
model generation while two new compounds RS100975 (11) and GG818 (12) were
included (Fig. 2). The compound (+) niguldipine used previously was excluded as
it interacts at a different binding site at the α1 Adr receptor [146]. Although (+)-
niguldipine interacted with the same aspartic acid at the TMIII region, still its other
interactions were different from other antagonists at the α1 Adr [147]. The quinazoline
class of compounds (prazosin and related analogs) were also excluded from the series
as they may bind the receptor in a different manner [146]. The catalyst generated
pharmacophore model for α1a Adr has three pharmacophoric features viz., one RA
(ring aromatic), one HBA (hydrogen bond acceptor), and one PI (positive ionizable)
feature (Fig. 3a). In this pharmacophore model, the phenyl ring in silodosin having
the (2,2,2)-trifluoroethoxy group mapped the RA feature, while the basic nitrogen
Fig. 2 Structures of the compounds used for α1 subtype selective CATALYST model generation
by Bremner et al.
separated from this phenyl group by an ethoxy linker mapped the PI feature. The
HBA feature was mapped by the ketone oxygen atom of the amido fragment at-
tached to the other phenyl ring silodosin (Fig. 3a). This pharmacophore model also
failed to predict the α1a Adr activity of prazosin (9) similar to the previously de-
veloped model through Apex 3D. The arrangement of the features was the same as
observed in case of Apex 3D pharmacophore viz., the positive charge center in
between oppositely placed aromatic ring system (RA) and HBA feature. It also
agreed with the Apex 3D biophore regarding the distance criteria between the basic
nitrogen (PI) and aromatic group (RA) that is observed to be 5.5 Å in this case. This
three-featured pharmacophore model however failed to correctly predict the mol-
ecules having weak α1a Adr activity and predicted all of them to be active with
poor correlation (0.35) between the observed and the predicted values. The α1b
pharmacophore was developed considering spiperone, AH-11110A (13), and bro-
motopsentin (14) as structural inputs (Fig. 2). The α1b Adr pharmacophore is con-
stituted by four features namely one RA, one PI, one HY (hydrophobic), and one
HBD (hydrogen bond donor) features. The mapping of spiperone (4) with the
developed model is shown in Fig. 3b. The distance criteria between the features
were well defined and the distance between the basic nitrogen (PI) and aromatic
group (RA) was 6.2 Å while the distance between the PI and hydrophobic (H) fea-
ture was 7.8 Å. As reported previously for the Apex 3D model [144], the distance
between the aromatic feature and the PI feature is slightly less in the α1a Adr
pharmacophore as compared to the α1b Adr pharmacophore. The α1d pharmaco-
phore model developed from SKF-104856 (15), discretamine (16), and SNAP8719
(17) (Fig. 2) is similar to α1a Adr pharmacophore model in terms of three biophoric
centers except the RA feature in α1a Adr corresponds to the HY feature in α1d Adr.
The mapping of SNAP8719 (17) with the developed α1d Adr pharmacophore model
is shown in (Fig. 3c). In order to decode the important features responsible for α1 Adr
subtype selectivity, the pharmacophore models for α1a, α1b, and α1d Adr subtypes
Fig. 3 Mapping of: (a) silodosin on the α1a Adr, (b) spiperone on the α1b Adr, and (c) SNAP-
8719 on the α1c Adr antagonist pharmacophore models developed by Bremner et al. (d) Compar-
ison of pharmacophore features between the α1a, α1b, and α1d Adr antagonist pharmacophore
models (reproduced with permission from Bioorg. Med. Chem., 2000, 8, 201–214 Copyright 2000
Elsevier Science Ltd. for Figs. a–c)
were compared (Fig. 3d) where the HBD feature was present in α1b Adr phar-
macophore model only while the α1a and α1d Adr had the HBA feature. The distance
between the HBA and PI features in the α1a Adr pharmacophore was 7.1 Å while in
the α1d Adr pharmacophore model it was less (4.5 Å). From these comparisons, it was
deduced that with certain structural modifications such as converting the O-methyl
group corresponding to HBA feature in cyclazosin (18) to alkyl groups may render it
α1b Adr selective. Pharmacophore models to determine the selectivity among α1 and
α2 Adr receptor subtypes have been reported where the α1 Adr pharmacophore model
was developed taking 24 compounds by using the pyridazinone-arylpiperazine deriv-
atives with general structures 19, 20, 21, and 22 and compounds published in the
literature (23–32) (Fig. 4) [148]. The best pharmacophore model was constituted by
three hydrophobic features (HY1, HY2, and HY3), a HBA, and a positive ionizable
(PI) group (Fig. 5a). The most active compound 23 mapped the HY1 and HY2 feature
with the o-methoxyphenyl moiety, while the tricyclic system mapped the HY3 feature
(Fig. 5a). The HBA feature was mapped by one of the ketone oxygen atoms present in
the tricyclic ring system while the PI feature was mapped by the piperazine N atom.
This model also failed to predict the activity of prazosin similar to the previous models
(Fig. 5b). The essential features for α1 antagonists from the developed pharmacophore
model have been used to postulate the possible α1a ADR topography. It may be
summarized as: (1) A basic, positively ionizable nitrogen to interact with the aspartate
side chain in TMIII; (2) The HY1 and HY2 features mapped by the ortho and meta
substituted phenyl ring situated at a distance of 6.69 and 6.17 Å from the PI feature,
respectively, suggest that HY1 and HY2 together may from a large hydrophobic cavity
at the receptor site; (3) A polar group at the other end of the molecule relative to the
arylpiperazine fragment; (4) An additional pharmacophore element (HY3) that can
accommodate terminal hydrophobic residues. The pharmacophore model developed
by Barbaro et al. is similar in composition to a CATALYST based α1 pharmacophore
model developed in our group. This pharmacophore model also contains three HY,
one HBA, and one PI features (Fig. 5c) that was used for further screening and iden-
tification of 33 (Fig. 5d) as a potent α1 Adr antagonist for which patents have been
granted [149, 150]. The mapping of compound 33 with the α1 Adr pharmacophore is
shown in Fig. 5e. A more robust pharmacophore model for α1a Adr was developed
[151] by Li et al. using 30 antagonists (Figs. 6 and 7) considering both the active and
inactive molecules (activity range of 0.036–3,300 nM) among which silodosin (1),
spiperone (4), SNAP8719 (17), and BMY-7378 (51) were in the training set [151].
Fig. 4 Structure for the training set of molecules used for α1 and α2 Adr selectivity
Fig. 5 Pharmacophore model for α1 Adr antagonist mapped with (a) compound 23 and (b) prazosin
(9) by Barbaro et al. (reproduced with permission from J. Med. Chem., 2001, 44, 2118–2132
Copyright 2001 Am. Chem. Soc.). (c) Mapping of prazosin with the α1 Adr pharmacophore
model developed in our lab. (d) Structure of the compound 33 identified as α1 Adr antagonist.
(e) Mapping of 33 with the α1 Adr pharmacophore model. (f) Mapping of silodosin on the α1a Adr
pharmacophore model developed by Li et al. (g) Mapping of tamsulosin on the α1a Adr phar-
macophore model (reproduced with permission from Bioorg. Med. Chem. Lett., 2005, 15, 657–664
Copyright 2004 Elsevier Ltd)
This pharmacophore model had four features: one PI, one HBD, one RA, and one HY
features (Fig. 5f) that was different from the Bremner pharmacophore model as it
contained a HBD feature instead of an HBA feature. Silodosin mapped the RA feature
with the phenyl ring containing the (2,2,2)-trifluoroethoxy group, the basic nitrogen
atom mapped the PI feature, the pyrrolidine group mapped the HY feature while the
hydroxy group at the end of the propyl chain mapped the HBD feature (Fig. 5f). The
distances between the pharmacophoric features were compared with the pharma-
cophore model developed by Bremner et al. where the distance 5.5 Å between the
Ar-PI features was almost similar with the corresponding distance (5.82 Å) in the
model developed by Li et al. The distances and the pharmacophoric features for the all
these α1 Adr models are represented in Table 2. The tamsulosin in the test set mapped
the hydrophobic feature with its methoxy substituent; the phenyl ring mapped the RA
feature (Fig. 5g) while the HBD feature was mapped by the sulphonamide group and
Fig. 6 Structures of the α1a Adr antagonists used as training set by Li et al. Other structures also
considered are silodosin (1), niguldipine (3), spiperone (4), risperidone (5), and corynanthine (6)
the PI feature was mapped by the nitrogen atom of the aliphatic chain. This phar-
macophore model was further correlated with a structure based model derived through
homology modeling using the β2-Adr crystal structure as template [152].
The docking studies were in agreement with the mutational studies reported on
the α1a Adr receptor. The important mutations that had impact on ligand binding at
the α1a Adr receptor included the Asp106 in TMIII, Phe308 and Phe312 in TMVII,
Phe193 in TMV, and Leu290 in TMVI that provide the hydrophobic/aromatic
interaction points at the receptor site [147, 153]. The serine residues (Ser188 and
Ser192) in TMV are also important as they make hydrogen bond interaction with
the ligands [147]. The docked conformation of silodosin (1) at the α1a Adr receptor
site that showed interactions with residues proved important through mutational
studies (Fig. 8) [147, 153, 154]. The compound silodosin (1) has aromatic/hydro-
phobic interactions with Trp102, Ile178, Phe288, Phe308, Phe309, Phe312, and
Tyr316 (Hyd feature) with its indole moiety while the phenyl ring at the other
terminal is situated inside the hydrophobic pocket lined by Val107, Cys110, Tyr111,
Ser158, Ala189, Phe193, and Phe289 (RA feature). The polar interactions of silo-
dosin include interaction with Ser83 by the hydroxyl group (H-bond donor) attached
with the indole moiety and interactions of basic nitrogen with Asp106 (PI feature)
(Fig. 8a and b). All these interactions matched the features in the developed phar-
macophore model as shown in (Fig. 5f). However some additional polar interactions
Fig. 7 Training set of compounds used (48–56) by Li et al.
Table 2 The distance and angular criteria of the pharmacophore features reported for α1a Adr
antagonists
Ligand based
model Software Distance between features (Å)
Bremner α1a Apex 3D Ar-PI 5.2–5.8, PI-PG 5.2–6.7
[1996]
Bremner α1a Catalyst Ar-PI 5.5, PI-HBA 7.1, Ar-PI-HBA 100
[2000]
Barbaro α1 Catalyst HY1-PI 6.69, HY2-PI 6.17, PI-HBA 5.62
[2001] PI-HY3 9.78
Li α1 [2005] Catalyst PI-RA 5.82, PI-HBD 9.08, PI-HY 7.53, HBD-RA 13.27, HBD-HY
3.95, HY-RA 10.87, RA-PI-HBD 125
were noticed for the phenoxy group where the O-atom formed H-bond contact with
Ser188 and Ser192. The electronegative fluorine atom of the trifluoroethoxy group
also formed H-bond contact with the backbone N-atom of Phe193. The salient fea-
tures obtained from the docking studies were: (1) hydrophobic/aromatic interactions
with Phe193 (TMV), Phe308 (TMVII), and Phe312 (TMVII), (2) polar contacts either
H-bond acceptor on donor interactions with Ser188 (TMV) and Ser192 (TMV), and
(3) salt bridge interaction with Asp106 (TMIII). This suggested that integration of
these methods helped in the identification of crucial features necessary for α1a Adr
antagonistic activity. As it was not possible to construct a single pharmacophore for
all the structural class of α1a antagonists, separate common feature pharmacophore
Fig. 8 (a) Docking of silodosin (1) at the α1a Adr receptor and (b) the two-dimensional in-
teraction of the docked complex (reproduced with permission from J. Mol. Model., 2008, 14,
957–966 Copyright Springer-Verlag 2008)
models were built to classify the high affinity α1a antagonists. Structural analysis of
the α1a antagonists revealed that they can be classified into two pharmacophore clas-
ses corresponding to class I and class II pharmacophore models [103]. The phar-
macophore models were developed using the compounds as represented in Fig. 9. The
pharmacophore model for class I antagonists had the positively ionizable nitrogen
atom situated at a distance of 2–3 bond length from the first aromatic ring and 5–6
bond length (9.5 Å) from the second aromatic ring (Fig. 10a ) while in class II phar-
macophore model the corresponding distance is 2–4 bond lengths (7.2 Å) (Fig. 10b).
The class I pharmacophore consisted of five features with the PI group being mapped
by the N2 atom of the quinazoline ring of prazosin, the HBA group being mapped by
the amide group of prazosin, and three hydrophobic features (Fig. 10a). The class I
pharmacophore model reported here has similarities with the pharmacophore model
developed by Barbaro et al. (Fig. 5a). The class II pharmacophore model constructed
with small molecular size α1a Adr antagonists had four pharmacophoric features
represented by one hydrophobic, two ring aromatic, and one positively ionizable
features (Fig. 10b) and lacked the HBA feature of the class I pharmacophore. However
the class I and class II models were similar regarding the features at the right side of
the PI pharmacophoric feature (class I: hydrophobic, hydrophobic, class II: ring aro-
matic, hydrophobic) indicating a common binding site for interacting with these two
and the PI feature at the α1a Adr adrenergic receptor for both class I and class II
ligands. Compared with the model developed by Klabunde et al., the pharmacophore
model developed by Bremner et al. [145] for distinguishing the α1a Adr subtype
selectivity is quite generic rather than selective. The pharmacophore models were fur-
ther compared with binding site of the α1a Adr receptor homology modeled on bovine
rhodopsin as template to provide the interaction of each pharmacophoric feature at the
receptor site (Fig. 10c and d). The PI feature in the pharmacophore model is expected
to form a salt bridge with the aspartate residue in TMIII. The head portion of the
ligands having aromatic and hydrophobic features is located in the hydrophobic zone
formed by the aromatic and aliphatic side chain of the residues in TM4, TM5, and
Fig. 9 The chemical structures of the compounds used for model generation by Klabunde et al.
Other structures used are spiperone (4), WB4104 (8), prazosin (9), cyclazosin (18), NAN-190 (46),
and RS17053 (58)
TM6. The aspartate residue in TMIII divides the binding region into two hydrophobic
pockets. The residues in TM4–TM7 (Val5.39, Phe6.51, Phe6.52, Met6.55, Phe7.35,
and Phe7.39) constitute one hydrophobic pocket while the aromatic residues in TM1–3
and 7 (Phe2.60, Phe2.64, Trp3.28, Phe7.35, and Phe7.39) form another hydrophobic
pocket. The HBA feature in the class I pharmacophore was assumed to interact with
Lys7.36 at the binding site. Pharmacophore validation of both class I and class II
pharmacophore models was performed by their ability to screen α1a antagonists from
MDL Drug Data Report (MDDR) dataset of 1,000 compounds that was further ex-
panded by addition of 50 known α1a Adr antagonists [155].
The purpose of adding known active set of 50 α1a Adr antagonists was to judge the
retrieval ability of the pharmacophore models from the total dataset. Virtual screen-
ing of the remodeled dataset by class I pharmacophore retrieved a total of 82 α1a Adr
antagonists in which 26 (52%) known α1a antagonists were recognized, while the
class II pharmacophore model performed better by recognizing 42 (84%) known α1a
Adr antagonists from a total of 146 hits. The quality of the models was further analyzed
by judging the number of known α1a Adr antagonists in the top ranked compounds.
This further confirmed the good predictive ability of the class II pharmacophore model
as six out of the top ten compounds were known α1a antagonists. Among the top 5%
of retrieved hits, the class I pharmacophore has 44% while the class II pharmacophore
has 50% of known active α1a antagonists that further indicate the ability of the models
Fig. 10 (a) The class I pharmacophore model for α1a Adr antagonists. (b) The class II pharma-
cophore model for α1a Adr antagonists. (c) The class I pharmacophore model mapped at the α1a Adr
binding site. (d) The class II pharmacophore model mapped at the α1a Adr binding site (reproduced
with permission from ChemBioChem, 2005, 6, 876–889 Copyright 2005 Wiley-VCH Verlag GmbH
& Co.). (e and f) Structure of the α1a Adr antagonists screened by integration of pharmacophore and
docking studies. (g) Docking interactions of compound 75 at the α1a Adr. (h) Docking interactions of
compound 76 at the α1a Adr (reproduced with permission from J. Med. Chem., 2005, 48, 1088–1097
Copyright 2005 Am. Chem. Soc.)
in identification of α1a Adr antagonists. The models were also nine and ten times more
superior than random selection for identification of α1a Adr antagonists.
The pharmacophore models were further integrated with a structure based model of
α1a Adr receptor built on bovine rhodopsin as template. A sequential virtual screening
was performed where the retrieved hits (22,950 compounds) from the pharmacophore
models were further docked at the homology modeled receptor to result in 300 top
scoring hits. The 300 hits were further clustered on the basis of structural similarity by
UNITY fingerprint similarity and a set of 80 diverse compounds were evaluated for
α1a antagonist activity. Among the 80 compounds, 37 compounds have affinity below
10 μM, 24 compounds showed affinity in the submicromolar range with ten com-
pounds having affinity below 100 nM, and three compounds below 10 nM. The best
compound in the series 75 (Fig. 10f) showed an α1a ADR antagonistic activity of
1.4 nM while the second best analog of 5-methylurapidil 76 (Fig. 10e) has a Ki value
of 3.6 nM. From the docking studies, it was confirmed that interaction of these com-
pounds with Trp3.28 and Phe2.64 is responsible for α1a selectivity. The role of
Phe2.64 for α1 subtype selectivity has been discussed earlier by mutational studies
[156, 157]. Interestingly, the hydrophobic feature adjacent to HBA feature in class I
pharmacophore model interacts with Phe2.64 and thus makes class I pharmaco-
phore selective for α1a Adr antagonism. The 2,6-dichlorophenyl moiety in com-
pound 75 and the pyrimidine ring in compound 76 have aromatic interactions with
Trp3.28 and Phe2.64 (Fig. 10g and h). The sequence alignment of all the α1 ad-
renergic subtypes with important residues highlighted is shown in Fig. 11. Further
the most active compound has the N3 atom of the pyrimidine ring at H-bond distance
from the hydroxy group in Ser2.61 while another H-bond with Lys7.36 by the amide
group is possible. The latter H-bond interaction with Lys7.36 complements the HBA
feature of the class I pharmacophore as described previously. However the interaction
with Lys7.36 may not be responsible for binding affinity at the α1a receptor. Thus the
study provides a clear concept about α1a antagonist activity by undertaking an in-
tegrative approach. Further comparisons of different screening methods in identifying
α1a antagonists have also been reported [158]. The selectivity of the class I phar-
macophore for α1a Adr antagonists was supported in another study by MacDougall
et al. where integration of ligand and structure based design was utilized to study the
selectivity among α1 Adr subtypes with the clarifications on the hitherto unclarified
interactions of prazosin at the α1a and α1b sites [159]. This pharmacophore model
was developed for compounds where the α1a and α1d antagonists exhibited >100-
fold selectivity over α1b and >40-fold selectivity over α1a/α1d (Figs. 12 and 13).
The α1a Adr pharmacophore model was developed taking 27 compounds including
active (Ki value less than 1 nM) eight and two compounds from class I and class II,
respectively. Since the majority including two most active compounds of the training
set belong to class I, the generated model favored class I pharmacophore. This α1a
Adr pharmacophore model had four featured pharmacophores in which the isopropyl
fragment, the phenyl ring of the phthalimide moiety, the keto group, and the piper-
azine nitrogen mapped the HAl, HAr, HBA, and PI features, respectively, for a
substituted compound having 82 as core structure with R¼H, X¼4-CH3 (α1a
Ki ¼ 0.16 nM: α1a/α1b ¼ >12,500: α1a/α1d ¼ 231) (Figs. 14a and b). Among
the active ten compounds, all the eight class I antagonists mapped the PI feature
while the two class II compounds failed to map the PI feature. Among the eight
class I active compounds, the top three mapped all the features of the pharma-
cophore while the rest five failed to map the HBA feature. The α1b Adr phar-
macophore was developed by using prazosin analogs.
Interestingly after protonation of the quinazoline nitrogen atom N1, the pharma-
cophore model generated for α1b Adr subtype does not contain any PI feature that is
considered to be an important feature for binding at the biogenic amine receptor site.
A four-featured pharmacophore model consisting of two HBA, one HAl, and one
HAr feature was observed in case of the α1b. The mapping of cyclazosin (18) (α1b
Ki ¼ 0.13 nM: α1b/α1a ¼ 92: α1b/α1c ¼ 25) with the α1b pharmacophore is shown
in Fig. 14c, d. The α1d Adr pharmacophore model was developed with compounds
Fig. 11 The amino acid sequence alignment of the α1 adrenergic receptor (α1 Adr) subtypes with
important binding residues highlighted in black rectangles
having >100-fold selectivity over α1a and >40-fold selectivity over α1b. Two
compounds used for generation of the pharmacophore model belonged to class II
ligands while the rest of the compounds having a single phenyl group cannot be
classified to any class mentioned by Klabunde et al. Most of the compounds in the
training set were structurally related to BMY-7378 that is having good selectivity for
the α1d Adr subtype. The α1d Adr pharmacophore contained five features viz., HAr,
two HAl, HBA, and PI features that mapped the phenyl ring, chloro group, pentane
Fig. 12 Structure of representative class of α1a Adr antagonists considered for selectivity study
by MacDougall et al.
Fig. 13 Structure of representative class of α1b Adr (86–95) and α1d Adr (96–99) antagonists
considered for selectivity study by MacDougall et al.
ring, keto group, and the basic nitrogen groups, respectively, for compound having the
basic structure as compound 66 with R¼2,5-Cl2 (α1d Ki ¼ 0.11 nM: α1d/α1a ¼ 386:
α1d/α1b ¼ 72) (Fig. 14e and f). Besides the pharmacophore models, subtype selectivity
in the α1 receptor subtypes was also guided by the size of the antagonists as the average
molecular weight of the compounds used as training set is highest for α1a Adr
Fig. 14 (a) The pharmacophore model and (b) 2D representation of the selective α1a Adr
antagonist. (c) The pharmacophore model and (d) representation of the selective α1b Adr antagonist.
(e) The pharmacophore model and (f) representation of the selective α1d Adr antagonist. (g) Docked
conformation of prazosin at the (g) α1a Adr and (h) α1b Adr. Mapping of Prazosin with the (i) α1a
Adr pharmacophore model and (j) α1b Adr pharmacophore model (reproduced with permission
from J. Mol. Graph. Model., 2006, 25, 146–157 Copyright 2005 Elsevier Inc.)
(MW ¼ 485), lowest for α1d Adr (MW ¼ 443) while for α1b Adr (MW ¼ 400) it is
between α1a and α1d Adr antagonists. The molecular weight variability in these
antagonists suggests that the binding site of α1a Adr is large in size compared to α1b
and α1d Adr subtypes. This pharmacophore model contained two additional hydropho-
bic features: one mapping the five-membered spiro ring and the other adjacent to the
aromatic ring compared to the previous pharmacophore model on α1d Adr. The best part
of the modeling experiment was the regression of each pharmacophore models by the
training set of the other two subtypes that resulted in a bad correlation signifying the
models to be selective ones. The generated pharmacophore model for α1a Adr has more
similarity with the class I pharmacophore model reported earlier. Interestingly prazosin
(9) was not able to map the pharmacophore model of α1a ADR by unsatisfying the HBA
feature of the pharmacophore (Fig. 14i). This was also observed for prazosin in the
model reported by Bremner et al. that was also a class I pharmacophore model. This
suggests that the class I pharmacophore model may be useful for identification of
selective α1a Adr antagonists. The α1b Adr pharmacophore developed here differed
from the conventional α1 Adr models by not having the PI feature that interacts with
Asp106 in TMIII. Interestingly prazosin mapped all the features of the α1b Adr
pharmacophore (Fig. 14j) signifying a different binding site compared to the site
of the conventional α1 Adr antagonists. This was further confirmed by the docking
studies of prazosin performed at the α1 Adr subtypes. Prazosin was found to have
no H-bond contact with the aspartate residue of TMIII in both α1a and α1b Adr
and failed to even fit in the α1d binding site. For α1a Adr, the shortest distance
between the charged Asp and positively charged nitrogen was found to be 3.94 Å
(Fig. 14g) while for α1b Adr the distance of ASP-PI feature was found to be 8.00 Å
that supported the pharmacophore model of the α1b Adr subtype (Fig. 14h).
The interaction of prazosin with Asp 106 is controversial as mutation of D106A
on α1b Adr was reported to have no effect on the binding of prazosin in one study
while two other studies confirmed the importance of Asp106 in the binding of
prazosin. However in a recent study, the homologous and heterologous binding
experiments have demonstrated that the Asp106Ala had no effect on prazosin
affinity. The inability of prazosin to form salt bridge with Asp106 at the α1a
binding site has also been reported on a homology modeled α1a Adr receptor
developed on β2 Adr template [160]. In this model, the di-methoxy groups
attached to the quinazoline moiety of prazosin lie near S188 and S192 residue of
the TMV that is similar in interaction like the β2 inverse agonists (carazolol,
timolol, and ICI118551) while the furan ring made hydrophobic interaction with
F312 in TM7. The inevitable importance of the α1a/d antagonists in BHP/LUTS
has regenerated research interest with the aim of designing subtype selective
antagonists and this has been evidenced by recent reports in this area [161] and
several pharmacophore studies have been published [162–165]. However design-
ing α1 selective antagonists based on receptor models is challenging due to high
homology among the receptor subtypes. Hence integration of ligand based infor-
mation may be helpful in validation of structure based models.
4.2 Dopamine D2 Receptor Agonists
The dysregulation in the dopamine signaling pathway due to loss of dopaminergic

neurons in the basal ganglia results in the pathogenesis of Parkinson’s disease (PD).
Treatments of PD include L-dopa which is a precursor of dopamine and other orally
administered D2 agonists such as pergolide, bromocriptine, ropinirole, and prami-
pexole [166–168]. However the D2 agonists used in PD have psychotic side effects
due to D2 receptor activation. The earlier molecular modeling studies on dopamine
receptors have been highly prioritized on the nature and alignment of the N-alkyl
substituents at the dopamine receptor site to deduce the dopamine receptor topog-
raphy. Most of these models were developed preferring the constrained structural
analogs rather than the flexible analogs for easy determination of the N-alkyl sub-
stituents to characterize the topography of the D2 receptor. The first D2 agonist model
was reported by McDermed et al. [169, 170] taking into account the constrained
dopamine analogs such as S-5-OH-DPAT (100), apomorphine (APO) (101), and
(6-amino-5,6,7,8-tetrahydronaphthalene-2,3-diol) ADTN (102) (Fig. 15). The study
reported the active enantiomer of ADTN (102) to be opposite in conformation com-
pared to S-5-OH-DPAT (100) and apomorphine (101). The model also postulated the
location of the hydroxyl groups in one of the two possible meta positions that de-
termine the orientation of the amino group binding in the D2 receptor and in order to
fit at the receptor site, ADTN (102) must be rotated like APO to fit at the D2 receptor
site (Fig. 17a). The D2 agonistic properties are mainly due to the phenylethylamine
moiety. It also explains why iso-APO (iso-apomorphine) (103) is inactive at the
receptor site (Fig. 17a). It is also stipulated from the model that the distance between
the oxygen atom (meta hydroxyl group) and the basic nitrogen atom is 7.3 Å while
the nitrogen atom is nearly orthogonal to the plane of the phenyl ring. Later, this
distance of 7.3 Å has been found to be crucial for D2 agonism through a molecular
modeling study on 2-(aminomethyl)chromans (2-AMCs) reported by Mewshaw et al.
[171] where an increase in distance between N–O to 7.8 Å has been postulated to be
responsible for decrease in D2 agonism affinity by 24-folds between two analogs 104
(D2 High: Ki ¼ 0.9 0.2, D2Low: Ki ¼ 15.9 6.7) and 105 (Fig. 15). It was also
addressed by Seeman et al. [172] that potent D2 agonists have the hydroxyl group to
nitrogen atom distance of 7.3 Å or less. However the pharmacophore model of
Mewshaw et al. had the basic nitrogen atom situated 0.92 Å below the plane of the
phenyl ring and has a difference from the earlier models where the nitrogen atom is
coplanar to the plane of the phenyl ring and this may be due to the rigid structures
considered during generation of earlier models. The dopamine D2 models were
built on the basis of the interesting finding that stereochemical aspects in the 2-
aminotetralines determine the D2 receptor agonist activity. For example, the S-
enantiomer of 5-OH-DPAT (100) having 2-aminotetralin substructure was found
to be active as D2 agonist and compared to apomorphine it was ten times more po-
tent in eliciting stereotypy in the rat and emesis in the dog while the R-enantiomer
of 5-OH-DPAT was inactive as D2 agonist [173]. Further pharmacological evaluation
Fig. 15 Structure of different chemical class of D2 agonists
of compounds 5,6-dihydroxy-2-(di-n-propylamino)tetralin (106), 6,7-dihydroxy-2-

aminotetralin (117), and 7-hydroxy-2-(di-n-propylamino)-tetralin (108) as D2 ago-
nists revealed the S-enatiomers to be active in the compounds 100 and 106 while an
opposite trend was observed for compounds 107 and 108 where the R-enantiomer was
active as D2 agonist (Fig. 15) [174, 175]. Deriving the D2 receptor topography from
ligand based information got further complicated when 3-(3-hydroxyphenyl)-N-n-
propylpiperidine (3-PPP) (109) was found to be a selective presynaptic (autorecep-
tors) D2 agonist [176, 177]. For compound 109, the (+)R enantiomer behaved as
agonist at both presynaptic (low doses) and postsynaptic (high doses) DA (dopamine)
receptors while the ()S enantiomer acted as agonist in the presynaptic (low doses)
and as antagonist (high doses) at the postsynaptic DA receptors. This opposite be-
havior of the (+)R enantiomer and ()S enantiomer of 3-PPP (109) at the postsynaptic
DA receptor resulted the racemic compound to be devoid of activity making 3-PPP

(109) presynaptic DA selective compound. Encouraged by the behavior of 3-PPP
(109) it appeared that the stereochemistry around the piperidine ring plays an impor-
tant role in deciding the D2 agonist behavior. This was further supported by apomor-
phine where the (+)6aS enantiomer antagonizes the agonist behavior of the (+)6aR
enantiomer [178]. Based on this information, further studies were carried out to es-
tablish a relationship between the stereochemistry of the piperidine ring and D2
agonist activity. In a series of monophenolic octahydrobenzo[f]quinolines, the trans
isomer (111) was more active as D2 agonist than the cis isomer (110) [179]. In com-
pound 111, the trans (4aS, 10bS) was found to be most active enantiomer whereas
among the cis enatiomers, 110 (4aR, 10bS) showed weak D2 agonist activity sup-
porting that the central DA receptors (pre- and postsynaptic) are activated by planar
molecules [180]. In cis isomers, the piperidine ring of compound 110 (4aR, 10bS)
protrudes downward the aromatic plane whereas in the trans isomer 111 (4aS, 10bS)
the piperidine ring remains in the plane of the aromatic ring. This was further support-
ed by the totally inactive isomer of 110 (4aR, 10bS). This proves that S-configuration
at carbon 4a is necessary for activity and is further supported by the compounds 100,
103, and 105 all of which have S-configuration at this center with the stereochemistry
of the carbon atom attached to the nitrogen atom. These findings strongly rationalize
that the trans conformation with S-configuration at carbon 4a is required for D2
agonism. However interestingly for compound 112 the cis isomer was found to be
as active as 109 with the 4aR (4aR, 10bR) enantiomer more active than the 4aS
enantiomer thus differing with the previous hypothesis where the stereochemistry was
to be of S-configuration at the 4a carbon atom attached to the nitrogen atom. In-
terestingly in the analogue series of compound 109, the nature of N-alkyl substituent
largely determines the D2 agonistic activity rather than the configuration at the 4a
carbon that suggests lipophilicity and/or steric factors to have effect on the intrinsic
activity of these compounds [181]. With further investigation on a series of com-
pounds, it was deduced that the nature of the substituent attached to the basic nitrogen
is important for activity. Especially it was observed that certain compounds such as
113 and N-n-Bu-Apomorphine (115) where the N-alkyl substituents point downward
a maximum substitution up to n-propyl (114) are favorable towards activity whereas
compounds having n-butyl substitution are devoid of activity [179]. This has been the
cause of inactivity for ergoline derivatives that have n-butyl substitutions. An align-
ment of the active conformations of 100, 106, 111, 112, 116, 117, and apomorphine
(101) on the basis of interaction of the OH group at the receptor site resulted in two
important conclusions that may complement the DA-receptor structure volume:
(1) Aromatic functionality with m-OH group or instead a pyrrole or pyrazole ring
imitating the m-hydroxyaryl group. (2) The distance criteria between the aromatic
function and the basic nitrogen should be like the extended phenylethylamine struc-
ture. (3) The N-substituents may position them in two main directions viz., upward or
downward direction, where the downward direction is sterically restricted and can
accommodate maximum a piperidine ring or an n-propyl group, whereas the upward
direction has less sterical requirements. (4) The stereochemistry of the carbon at-
tached to the basic nitrogen atom is also important as it provides direction to the
carbon–nitrogen bond. For example, in compounds 100, 106, 111, 112, 116, 117
(Fig. 17b), and apomorphine, the active enantiomers have the carbon–nitrogen di-
rected downwards, towards the plane of the paper.
The model however failed to identify the structural requirement for D2 agonist
activity of the unnatural ergoline derivatives relative to the natural ergoline. The chi-
rality of the carbon atom next to the basic nitrogen atom in natural ergolines has 5R
configuration while the 5S configuration is the inactive one. In order to map the natural
ergolines, some of the unnatural ergolines (S-configuration) have to turn 180 down as
shown for compound 118 at the D2 receptor site. The S-configuration of compound
119 has the piperidine ring fitting exactly in the well-defined cleft and substitution at
position-8 will provide steric hindrance rendering compound 119 as inactive. So an
unsubstituted 119 is expected to be active as D2 agonist. Hence no substitution at
position-8 is preferable for compounds having S-configuration and having the pyrrol-
ethylamine moiety responsible for D2 agonist activity. The partial agonist 120 fulfills
these criteria of the ergoline pharmacophore like 119 when the pyrrole portion interacts
at the receptor site, alternatively it was also postulated that 120 may get hydroxylated
to 121 during metabolism and the active pharmacophore may be the phenyl ethylamine
moiety. In further investigations, the compound 122 having both the R (pyrrolyleth-
ylamine moiety) and S (phenyl ethylamine) isomers were active as D2 agonist. Hence
the structural requirements (pyrrolylethylamine or phenyl ethylamine) for D2 agonism
in the ergolines and their mode of interaction at the receptor site remained unclear
[180, 182, 183].
The nature of the substituents at the basic nitrogen and their orientation at the D2
receptor have been studied more elaborately with (S)-3-(3-hydroxyphenyl)-N-n-
propylpiperidine ((S)-3PPP) that can act both as agonist and antagonist in different
rotamaric forms [184]. As mentioned previously, the (+)R enantiomer of 3-PPP
(109) behaved as agonist at both presynaptic (low doses) and postsynaptic (high
doses) DA (dopamine) receptors while the ()S enantiomer acted as agonist in the
presynaptic (low doses) and as antagonist (high doses) at the postsynaptic DA re-
ceptors. The differences in response for 3-PPP have been attributed due to different
interactions of the N-alkyl substituent at these receptor sites (presynaptic and post-
synaptic). However as our main discussion is focused on D2 agonist pharmacophore
so the agonist interactions of 3-PPP at the D2 receptor model will be described here.
The study describes the existence of a propyl cleft in the downward direction and a
sterically “unrestricted upward” direction at the D2 receptor site that accommodate
the propyl chains attached to the basic nitrogen moiety (Fig. 17d). The alignments of
compounds 125 and 126 both of which are active further depicted the upward and
downward orientation of the propyl group (Fig. 17c). This unrestricted upward por-
tion of the receptor can accommodate larger N-substituents such as phenethyl groups
or the nonphenolic phenyl group in apomorphine (101). The receptor interaction
points of the D2 agonists and the relative orientation of the propyl cleft at the receptor
site have also been proposed keeping in view the McDermed receptor concept as
shown in Fig. 17d. The D2 agonist pharmacophore was further extended to elaborate
the D2 receptor topography near the basic nitrogen atom. The orientation of the
propyl cleft was proposed to be situated above the plane of the ligand at the receptor
site (Fig. 17e). The conclusion was made based on two compounds 123 and 124
having a cyclic structure embedded with the positively charged nitrogen. Although
both these compounds are similar in structures to 109, they were inactive in vivo. The
inactivity of compound 123 can be easily accounted due to its large ring size that is
not able to fit properly in the well-defined propyl cleft but the inactivity of compound
123 is not expected as it has a small ring size that may easily fit the propyl cleft at the
receptor site. The inactivity of compound 123 indicated that the basic nitrogen atom
does not tolerate any steric bulk just in front of it and as a result an anti-conformation
of the propyl group is more preferred as in the gauche conformation the propyl group
will be oriented just in front of the nitrogen atom (Fig. 17f and g). Hence the propyl
cleft was stated to lie orthogonal to the plane of the aromatic moiety [184]. A more
detailed description about the D2 agonist pharmacophore model was reported by
Chidester where molecular modeling studies have been carried out on a set of 11
tricyclic molecules (127–137) (Fig. 16) having X-ray crystal structures [185]. The D2
pharmacophore model generated had two H-bond requirements: (1) the one being
involving the basic nitrogen atom and (2) the second being H-bond groups (OH, NH)
as donors in the aromatic ring (secondary hydrogen bonding). The donor character of
the substituents was determined on the basis of preference of the hydroxy group over
O-methyl substituents on the aromatic ring. The model also describes the existence of
an aromatic group with donor substituents and the propyl cleft. An area located south
of the amine was proposed to confer antagonist or partial agonist character at the D2
receptor (Fig. 17h). However the 11 X-ray crystal structures used to develop the D2
pharmacophore model were having the propyl and allyl groups in gauche conforma-
tion rather than anti-conformation as proposed by Liljefors et al. Our study on the
alignment of the active apomorphine analogue ()6a-R-apomorphine (101) and the
active D2 antagonist ()12aS-centbutindole (138) showed similar stereochemical
superposition at the adjacent carbon next to the basic nitrogen [186] (Fig. 18a ) that
was further utilized to synthesize derivatives where the catechol portion of the apo-
morphine was replaced by the indole moiety to remove the emetic side effect of apo-
morphine. Two of the synthesized analogs 139 and 140 showed marked dopaminergic
activity [187–191]. Thus the proper location of the propyl cleft remained elusive.
A detailed study about these earlier classical D2 models has been studied and
compared through receptor models with subsequent development of pharmacophore
model [192]. It should be noted that most of the earlier models were developed by
superimposition of the NH containing scaffold of ergolines with the hydroxy group
attached to the aromatic ring but while doing so the large N-substituents will be guided
towards the confined n-propyl cleft that is unwanted as groups larger than propyl are
not accepted in this cleft. The unexplained binding of ergoline containing scaffolds as
well as certain compounds with large n-alkyl groups has been studied by docking these
molecules in a homology modeled D2 receptor developed on the co-crystallized
structure of 5HT1b with ergotamine as template (sequence identity 57%). The se-
lection of the template was made as the ergotamine scaffold co-crystallized with
5HT1b structure has a large C8 substituent. The compounds 125 ((4aS, 10bS) trans-7-
OH-OHBQ) and 126 ((4aR, 10bR) trans-9-OH-OHBQ) were docked at the D2
receptor to find the binding orientation of these two compounds at the D2 receptor
Fig. 16 The chemical structures of D2 agonists
(Fig. 18b). The protonated nitrogen atom in compounds 125 and 126 formed a salt
bridge with the Asp1143.32 and the hydroxy group in both the compounds formed
H-bond contact with Ser1935.42. The aromatic scaffold in these compounds formed
edge to face aromatic interactions with Phe1906.52 and cation–pi interactions with the
protonated His3936.55. The docked conformations of compounds 125 and 126 are in
agreement with conformations reported earlier viz., the propyl groups as nitrogen
substituents are placed in upward (compound 125) and downward (compound 126)
direction. The propyl cleft stated earlier was formed by the side chains of Cys1183.36,
Trp386 6.48, Phe3896.51, Thr 4127.39, and Tyr4167.43 and the propyl group in com-
pound 126 is resided in this cleft. The compounds 142 and 144 having the same
configuration but having large alkyl substituents however failed to fit in this cavity
and hence were inactive. For compound 125, the n-propyl substituent was directed
upwards in the large spacious cavity heading towards the extracellular surface of the
receptor demarcated by extracellular loop 2 (ECL2) that explains that compound 125
can have substituents larger than propyl groups at this position and this has been
confirmed by the D2 agonistic activity of compounds 141, 143, and bifeprunox (148)
(Fig. 18c). The docked conformation of ergotamine (146) and bromocriptine (145)
also had the large N-substituents located in this spacious cavity. However the earlier
presumption that the NH group in ergotamine and the OH group of the tetralin have
the same binding site at receptor is not true and it has been observed that the NH
Fig. 17 (a) The D2 receptor topography as proposed by McDermed et al. (b) The upward and
downward conformation of the N-alkyl substituents of D2 agonist molecules. (c) Superimposition
of 125 and 126 with the hydroxy groups in both the molecules being considered to interact at the
same receptor site. (d) The existence of propyl cleft at the D2 receptor that can accommodate alkyl
groups up to the length of propyl chain. (e) The proposed anti-conformation of the propyl group
indicating N-substituents is not tolerated just in front of the N-atom. (f and g) In the gauche
conformation, the N-substituent will lie in the plane of the N-atom while in anti-conformation it
will lie above the plane. (f) The D2 receptor model proposed by Chidester et al.
group of ergotamine interacts with Ser1975.46 while the OH group of tetralin interacts
with Ser1935.42. It was also interesting that the ergot derivatives have dual binding
mode at the D2 receptor site where some of them may form H-bond with Ser1935.42
and Ser1975.46 and this dual binding behavior was observed for the docked conforma-
tion of 147R and 147S that also explained how the ergoline derivatives having large
Fig. 18 (a) The alignment of ()6a-R-apomorphine (101) and ()12aS-centbutindole (138). (b)
The docked conformation of the compounds 125 and 126 at the D2 receptor. (c) The superposition of
the compounds 125, 126, ergotamine (146), and bifeprunox (148) and the revised D2 agonist phar-
macophore model. (d) Alignment of the compounds 125, 126, 146, and 147. (e) The docked
conformation of R-147 (light pink) and S-147 (deep purple) at the D2 receptor binding site showing
reversed binding mode of the enantiomers (reproduced with permission from Neurochem. Res.,
2014, 39, 1997–2007 Copyright Springer Science+Business Media New York 2014)
C-8 substituents showed dopamine agonistic activity (Fig. 18e). The –NH group in the
docked conformation of 147S has H-bond interactions with Ser1935.42 and occupies a
similar position at the receptor site as observed for the hydroxy group in compounds
125 and 126. The –NH group of 147R however overlapped with ergotamine and
formed an H-bond with Ser1975.46. The docked conformation of 147S and 147R
showed partial structural overlap where the phenyl moiety in the indole group and
the basic nitrogen group occupied the same position at the receptor site. From these
docking studies, a refined pharmacophore model was defined that contains two donor
sites, one aromatic region, a basic protonated nitrogen, the propyl cleft, and the large
spacious cavity that can accommodate large substituents. This is different from the
classical D2 pharmacophore in having one extra donor site (Fig. 18c). The dopamine
D2 agonist pharmacophore was developed considering a set of full agonists and
inactive compounds by Malo et al. [193] (Fig. 19). The study was performed to deduce
the features responsible for selectivity between D1 and D2 agonist activity. A set of
diverse structures active at the D1 and D2 receptors were selected and compared with
structurally similar analogs that were inactive at both these receptors. However our
discussion will focus on the D2 agonist pharmacophore model. The pharmacophore
model was developed in two steps by using the Molecular Operating Environment
(MOE) software package [194]. The development of pharmacophore model on the

basis of projected features provided a more realistic view about the interactions of the
ligand with the receptor models and at the same time did not force the important
features in the active compounds to occupy the same position, instead ensured that
the active ligands interact with the receptor residues in different orientations. The
preliminary pharmacophore model was developed using the PCHD annotation
scheme in MOE and further optimized using the unified annotation scheme. The
preliminary D2 pharmacophore model was developed using two structurally different
full agonists (R)-NPA (149) and talipexole (156) by selecting conformations for both
the compounds where the n-propyl group was in an extended conformation. The
alignment of these compounds was performed using the aromatic ring having the
HBD group in (R)-NPA (149) with the amino group containing thiazole ring of
talipexole (156) (Fig. 20a). The preliminary pharmacophore model consisted of two
HBD and two HBA features representing the serine residues in TM5 (Ser193,
Ser194) illustrated as SerTM-5 in Fig. 20a, one projected HBD feature representing
the Asp114 in TM3 of the receptor. Besides this, the models consisted of aromatic
features represented by inclusion volumes for the aromatic ring centroid and for two
ring normals. The aromatic pharmacophore features represented the aromatic interac-
tions with the aromatic residues in TM6 (mainly F390 but also W386 and F389). The
Hyd feature of the model represented the N-substituents that are present in most of the
potent D2 agonists and represented the propyl cleft. Among all the pharmacophore
features, the aromatic ring system and the HBD feature around the cationic nitrogen
were stated to be important for differentiating the active compound from the inactives.
The hydroxy features were stated to be less important as D2 agonist (S)-DPAT (155) is
a full agonist (intrinsic activity is lower than compounds having hydroxyl group)
despite lacking the HBA or donor group features. The PCHD annotation scheme was
next refined to fit all the active D2 agonists in the pharmacophore model (Fig. 20b).
The only D2 active agonist sumanirole (151) was unable to fit the developed model for
which slight outward adjustment of the top Ser-TM5 feature (A, increasing the
distance to Aro) was done for fitting the sumanirole (151) in the model. In this
optimized model, one HBA and HBD features were kept instead of two donor and
acceptor features to fit the hydrogen bond accepting carbonyl function or a hydrogen
bond donating –NH function as present in sumanirole (151). The retention of only one
hydrogen bonding feature instead of two is acceptable as many monohydroxy-
substituted full D2 agonists, such as (S)-5-OH-DPAT (100) and (R,R)-PHNO (150),
contain only one H-bond feature. The existence of hydrophobic feature representing the
propyl pocket was ignored in the final model so that it may not be a strong determinant
for D1 and D2 selectivity. The final model for D2 selective agonist was developed by
the unified annotation scheme implemented in MOE. The modifications in the phar-
macophore included an increase: (1) in the distance of the hydrogen bonding feature
from the heavy atoms in the unified model to 2.8 from 2.1 Å present in the PCHD
model and (2) in the angle between the two possible projected features from an HBD or
HBA (Ser-TM5). In the unified annotation scheme, the imidazole ring of sumanirole
was identified as an aromatic feature while the cationic nitrogen was identified as N+
Fig. 19 Structure of the D2 agonists considered for model generation by Malo et al.
feature which was HBD in the PCHD model (Fig. 20c). This annotation of the cationic
nitrogen as N+ group provided a more specific pharmacophore feature that is important
in the GPCRs. The tolerance feature of the pharmacophore model was also set to 1.5 Å
for introducing flexibility in the model. The refined model was able to identify all
12 active derivatives (12/12 hits), two of four partial agonists (2/4 hits), and in dis-
criminating six of 14 inactives (8/14 hits). Further refinement of the pharmacophore
model was performed by introducing excluded volumes to separate the active D2 ag-
onists from the inactive ones. For this, nine excluded volumes (V1–V9) were incor-
porated and they included V1 (r ¼ 2.1) to exclude ()-DHX (177), V2,V4,V5,V6
Fig. 20 (a) Mapping of full agonists (R)-NPA (149) (green) and talipexole (156) (purple) with the D2
agonist model using the PCHD annotation scheme. (b) Mapping of (R)-NPA with the refined PCHD-
generated D2 agonist model having excluded volume (Excl: grey). (c) Mapping of the full agonist
sumanirole (151) on the pharmacophore models by PCHD (gold carbons) and unified annotation
(green carbons) schemes. (d) The distance between the pharmacophore features of the D2 agonist
models. (e and f) The pharmacophore features (unified annotation scheme) with excluded volumes
(V1–V9 and Excl O) (reproduced with permission from ChemMedChem, 2010, 5, 232–246 Copyright
2010 Wiley-VCH Verlag GmbH & Co.)
(r ¼ 1.8 Å) and V3 (r ¼ 1.6 Å) to exclude A77636 (170) and A70108 (160). The
excluded volume V7 (r ¼ 1.8 Å) was included to exclude the enantiomer A70360 (175)
of compound 160 along with the compounds 173 and A77641 (174), V8 (r ¼ 1.8 Å)
was added to exclude SKF38393 (171), and ()-sumanirole (166) was excluded by
adding V9 (r ¼ 1.3 Å) (Fig. 20e and f). Another volume excluding oxygen (O) was
added near the hydrophilic ether functionality that may be responsible for making the
DHX analogue doxanthrine (172) less active at the D2 site. Finally the refined pharma-
cophore model with excluded volumes was able to recognize all full agonists (12/12),
two out of four partial agonists (2/4) and all the inactives (14/14). Interestingly the
mapped conformation of (R)-3-ppp (154) on the refined pharmacophore model had its
aromatic ring in the same plane as piperidine ring and was in agreement with the ag-
onist model reported by Liljefors and Wikstrom [65] where the crucial features es-
sential for D2 agonism at the receptor site were: (1) the salt bridge between Asp114-
TM3 and the amino group of the ligand, (2) the hydrogen bond(s) with the serine
residues in TMV by the phenolic groups, and (3) the aromatic interactions with the
hydrophobic residues in TMVI. The distances between the important features for this
model are shown in Fig. 20d. This developed D2 agonist pharmacophore model was
further integrated with a structure based model developed by the docking of the agonist
()-(R)-2-OHNPA (161) on the homology modeled D2 receptor. In the docked
conformation the C10 hydroxy group of ()-(R)-2-OHNPA (161) interacted with
Ser193 (H-bond distance ¼ 3.0 Å, O-HO (Ser1935.42) angle is 164 ) while the C11
hydroxy group interacted with the imidazole nitrogen atom of His393 (H-bond dis-
tance ¼ 2.9 Å, O-H-N (His3936.55) angle is 157 ). No hydrogen bond contact with the
oxygen atom attached with C11 and Ser197 was observed due to unfavorable H-bond
distance between them (4.9 Å). This was in support towards the mutational studies
where the efficacy and affinity of (R)-NPA (149) was less hampered due to Ser1975.46-
Ala mutation. The positively ionizable nitrogen atom formed salt bridge with the side
chain of Asp1143.32 while the hydroxy group attached to 2-position of ()-(R)-2-OH-
NPA made two H-bond interactions with the NH (H-bond distance ¼ 2.9 Å, O-H-N
(Asp186) angle is 142 ) and carbonyl (H-bond distance ¼ 2.9 Å, O-H-O (Asp186)
angle is 143 ) atom of Asn186 located at ECL2. The phenyl moiety of the catechol-
amine moiety formed face to edge pi–pi interactions with Phe3906.52 and hydrophobic
contact with the side chain of Val111. The propyl chain of ()-(R)-2-OH-NPA (161)
occupied the characteristic N-alkyl/propyl pocket that provided a hydrophobic envi-
ronment formed by the residues Val832.53, Cys1183.36, Trp3866.48, Thr4127.39, and
Tyr4167.43. The D2 agonist pharmacophore model developed earlier was superimposed
in the structural model of D2 agonist to compare the features of the pharmacophore
with important interacting binding site residues and for analysis of the correct location
of excluded volumes (Fig. 21a and b). It was observed that location of all the features in
the developed pharmacophore model was in agreement with the important interacting
residues except for the Ser–TMV interaction that was shifted towards Val190. The
location of the excluded volumes was also not correct regarding the shape of the bind-
ing cavity. All this mismatches were further rectified by the development of a refined
pharmacophore model where the Ser-TM5 feature was placed near Ser1935.42 and the
excluded volumes were adjusted according to the agonist-binding cavity (Fig. 21c and
d). All the features of the generated pharmacophore model were treated as essential
features except the modified Ser-TM5 feature as the full agonist (S)-DPAT (155) did
not interact with this residue. Hence the Ser-TM5 feature was treated as an optional
feature. The modified excluded volumes were constructed considering the H-atom of
the amino acid residues that are within 3 Å of the docked conformation of ()-(R)-2-
OH-NPA (161) (1.2 Å for aliphatic and 1.0 Å for aromatic hydrogen atoms) with fur-
ther radius optimizations until the model differentiated between the active and inac-
tives. The excluded oxygen feature (exclO) was also retained to identify doxanthrine as
an inactive. The decreased efficacy of DHX may be attributed due to lack of N-propyl
substituent. Analysis was performed to compare the conformation of the hits fitting the
pharmacophore model with their receptor interactions. The hits that mapped the pharma-
cophore model were suggested to have: (1) a distance of 2.4–3.8 Å from the Ser1935.42
and His3936.55, (2) the angle between the heavy atom and the hydrogen atom of the
ligand to oxygen atom of the interacting residue (N/O-H-O (Ser1935.42) and N/O-H-N
(His3936.55)) should ideally be 180 40, respectively. The refined pharmacophore mod-
el was then utilized to screen similar set of ligands as used previously. Among the
13 full agonists and five partial agonists, the 11 agonists except (R)-3-PPP (154) and
A70108 (160) and four partial agonists except (S)-3-PPP (164) fitted the pharmaco-
phore model. The model also failed to exclude one of the inactive compound ((S)-
Fig. 21 (a and b) The alignment of the pharmacophore models (Fig. 20e and f) at the D2 receptor
site. (c and d) The refined D2 agonist models with modified Ser–TMV and refined excluded
volumes (reproduced with permission from ChemMedChem, 2012, 7, 471–482 Copyright 2012
Wiley-VCH Verlag GmbH & Co.)
7-OH-DPAT) (169) from the 12 inactive compounds. The 3-PPP failed to map the
pharmacophore model due to perpendicular orientation of the piperidine and the
phenyl ring instead of being in the same plane. When the pharmacophore confor-
mation of these compounds was compared with the receptor interactions, it was
observed that the agonists and the partial agonists did not fulfill the angular criteria
((N/O-H-O (Ser1935.42) and N/O-H-N (His3936.55)) that should ideally be 180 40).
All the full agonists which fitted the pharmacophore model had one proper H-bond
interaction at the receptor site except (S)-DPAT (155) that did not have H-bond
functionalities. All the partial agonists also had one H-bond except (S)-6-OH-DPAT
(163) that may interact differently at the binding site. Among the inactives, (S)-7-
OHDPAT (169) fits into the pharmacophore model while in the receptor model it
failed to maintain H-bond contact with His3936.55 and Ser1935.42. The pharmacophore
model also explained the interactions of the full agonist quinpirole (157). The best
pharmacophore hit of quinpirole did not interact with the serine residues of TMV that
was in full agreement with the mutational studies where Ser193-Ala mutation had no
effect on the binding of quinpirole (157). However from the mutational studies it was
observed that quinpirole is affected by His3936.55-Ala mutation. The pharmacophore
mapped conformation of quinpirole (157) did satisfy the H-bond criteria with His3936.55.
It was assumed that the interaction between the pyrazole nitrogen atom in quinpirole and
His3936.55 may be water mediated or it may be possible that Asp186 and His3936.55 may
arrange in a conformation to interact with quinpirole. The study presented here integrates
both ligand and structure based methods for identification of the crucial features for
dopamine D2 agonism.
4.3 Histamine H1 Receptor Antagonists
The histamine mediates important physiological responses such as inflammation,

gastric acid secretion, and neurotransmission by acting on four histamine receptors
subtypes (H1–H4). The histamine H1 receptor is an attractive drug target due to its
potential role in allergic reactions. Increased histamine levels are associated with
several clinical conditions such as multiple sclerosis, rheumatoid arthritis, allergic
asthma, and psoriatic arthritis. The first generations antihistaminics mepyramine
(178), chlorpheniramine (179), diphenhydramine (180), and piperoxan (181) had
sedation as the main CNS side effect while the second-generation drugs such as
cetirizine (182), fexofenadine (183), terfenadine (184), and loratadine (185) have low
BBB penetration with low sedation potential (Fig. 22). The classical H1 antagonists in
general contain two aromatic rings and basic nitrogen atom separated by three to four
carbon atoms. Pharmacophore models on classical antagonists taking the semirigid
cyproheptadine (186) have been reported by several groups among which the
pharmacophore model developed by Van Drooge et al. from triprolidine (187), phe-
nindamine (188), and cyproheptadine (186) reported a boat conformation for the
piperidylene ring of cyproheptadine (186). However the model was not satisfactory
regarding the fitting of semirigid H1 antagonists that prompted Terlaack et al. to de-
velop another pharmacophore model taking semirigid conformations of classical H1
receptor antagonists (cyproheptadine (186), phenindamine (188), triprolidine (187),
epinastine (189), mequitazine (190), IBF28145 (191), and mianserine (192)). The mod-
el was developed by selecting the aromatic moieties as template and allowing freedom
to the basic nitrogen atom that interacted with Asp116 of TM 3. The Asp116 was also
incorporated in the model that helped in the development of a stereoselective models
that can identify the absolute bioactive configuration of antihistamines such as phe-
nindamine (S), epinastine (S), and IBF28145 (R). The nonclassical H1 antihistaminics
generally have more than one aromatic ring and a basic nitrogen atom. However some
of them have a single aromatic moiety such as the benzimidazole derivatives.
The 2-substituted 1,2,3,4,5,6,7,12,12a-octahydropyrazino[20 ,10 :6,1]pyrido[3,4-b]
indoles having a combination of tryptamine and piperazine moieties in rigid confor-
mation have shown wide spectrum of biological activities such as antihistaminics,
neuroleptics, and anti-inflammatory. The nature of the substituent at position 2 of the
1,2,3,4,5,6,7,12,12a-octahydropyrazino[20 ,10 :6,1]pyrido[3,4-b]indoles governed the
biological activity in this class of compounds. The antihistamine compounds were
designed by the introduction of aroylaminoethyl side chain at position 2 of octahydro-
pyrazinopyridoindole core because a number of piperazine derivatives incorporating
aroylaminoethyl substructure have shown antihistaminic H1 activity. In the QSAR
studies on 2-β-aroylaminoethyl-1,2,3,4,6,7,12,1a-octahydropyrazino(20 , 10 :6,1) pyrido
(3,4-b)indoles ((1) in Table 5), it was observed that hydrophobicity of the substituents
at the ortho and para position and bulk at the ortho position of the aromatic ring of
aroyl aminoethyl side chain increased the activity. Among the synthesized 34 com-
pounds (Table 3), 28 compounds were used for model generation and the rest six
compounds (217–222) were found to be outliers, the activities of which were not
suitably predicted by the model [199]. In view of the similarity in terms of positive
steric effect of substitution at the phenyl ring of these molecules and diphenhydra-
mines, it was suggested that these molecules bind to H1-receptor in a folded confor-
mation in which the phenyl and indole rings of these molecules occupy similar
positions as the two phenyl rings of diphenhydramine (Fig. 25a). Based on these
studies and SAR in semirigid analogs of diphenhydramine, benzylhydrylamine, and
phenbenzamine, a model for H1 receptor was proposed. In order to gain more insights
into the discussed topography of H1 receptor, it was considered of interest to study
some semirigid analogs of known antihistaminics such as diphenylhydramine (180,
R¼C6H5, X¼O), benzhydrylamine (193, R¼C6H5, X¼NH), and phenbenzamine
(193, R¼H, X¼NC6H5) (Table 4). The locking of α and β carbons atoms of the
ethyl side chain adjacent to the dimethylamino groups with one of the aromatic rings
of 193 can produce two semirigid structures 194 and 195. Each of these compounds
194 and 195 having two asymmetric centers may produce two distereoisomers. Some
of these compounds (194a–c, 195a–c) were synthesized and evaluated for
H1-antagonistic activity in isolated guinea pig ileum. The five-membered semirigid
analog 194 was equipotent to the parent open chain diphenylhydramine 193a while
the corresponding six-membered semirigid analog 195b of benzhydrylamine (193a)
and five-membered semirigid analog (194b) of phenbenzamine (193b) were less
active than the parent compounds 193b and 193c, respectively. Further the analogs
Fig. 22 The structure of the antihistamine H1 compounds

194c and 195c of 194a and 195a in which the free phenyl ring is replaced by methyl
group were less active than the 194a and 195a, respectively. The SAR study of these
compounds based on their Dreiding models also revealed that π rich hydrophobic
subsites A and D and electron density subsites C and anionic site B are essential for
binding with the H1 receptor. All the above requirements were met by diphenylhy-
dramine (180), and also by 194a. In the case of the corresponding six-membered
analog (195a) though the distance of the basic nitrogen (subsite B) from the aromatic
ring (subsite A) is almost the same, both the aromatic rings, being coplanar, inhibit the
interactions at the subsite A and this may be the reason for the decreased activity. The
same would be true for 195c. However in the case of semirigid analog of phenben-
zamine (193b) though the noncoplanarity of the aromatic rings is maintained yet the
distant between subsites A and B is reduced causing 1,000-fold decrease in activity.
Further support for the noncoplanarity of the aromatic rings is evident from the ob-
served 100-fold decrease in activity of fluorene analog (196) of diphenylhydramine
where both rings are coplanar. These studies showed that: (1) pyrazinopyridoindoles
(197), semirigid analogs of 193, and diphenylhydramine type of antihistaminics act on
common receptor, (2) the o-substitution in the phenyl ring of the side chain of 197 has
conformational effect causing noncoplanarity to the phenyl ring of the side chain,
(3) hydrophobic interactions are more important in A region than electronic and steric
interactions, (4) the distance of the anionic site B from A is of prime importance for
the activity while the high electron density in C region also contributes to the activity.
With a view to further explore this model, four different prototype molecules
(Fig. 23) including the ones with the change in the side chain from aroylaminoethyl
to arylaminocarbonylethyl were synthesized and evaluated for antihistaminic H1
activity. All these molecules incorporate the above suggested essential structural
requirements to interact at proposed sites A, B, C, and D and the phenyl ring attached
to the aroylaminoethyl side chain should experience the same biomolecular interac-
tions being at the same site A which is also occupied by the phenyl ring of diphen-
hydramine. The QSARs were developed for each class in terms of antihistaminic
activity as dependent and physicochemical parameters particularly hydrophobicity as
independent variables. The almost identical slope values (0.375 0.045) with
hydrophobicity parameters in all the four prototypes including pyrazinopyridoindoles
clearly indicated that there is a similar change in activity for the same substructural
variation in the side chain phenyl ring of all the prototypes. Equation (2) (Table 5)
reported for only six compounds of the prototype pyrazinopyridoindoles with aroyl
amino ethyl side chain in terms of all the three physicochemical effects parameterized
as hydrophobic (π), electronic (σ), and steric (MR) compared well with the Eqn.
(3) (Table 5) in terms of their slope values π (0.325 0.021), σ (0.154 0.01), and
MR (0.0115 0.004) for all new 27 compounds belonging to other prototypes thus
indicating that the side chain phenyl ring in all these prototypes occupies the same
receptor site and at subsite A all these molecules experience the same kind of
biomolecular interactions. The overall Eqn. (4) for 33 compounds is provided in
Table 5. In order to further integrate the results of 2D-QSAR with the newly
developing 3DQSAR techniques which had no limitations on the inclusion of similar
congenic series, the HASL approach was used. In the HASL approach, a molecular
representation lattice is constructed using the energy minimized Cartesian coordinates
Table 3 The structure of the compounds synthesized with antihistamine H1 activity
Comp R H1-receptor blocking activity (IC50 μg/mL)a

198 H –
199 CH2CN –
200 (CH2)2CH2NH2 –
201 (CH2)2NHC(¼O)C6H4-2-NO2 0.157 0.06
202 (CH2)2NHC(¼O)C6H4-3-NO2 0.19 0.06
203 (CH2)2NHC(¼O)C6H4-4-NO2 0.41 0.02
204 (CH2)2NHC(¼O)C6H4-2-NH2 0.36 0.19
205 (CH2)2NHC(¼O)C6H4-3-NH2 0.24 0.048
206 (CH2)2NHC(¼O)C6H4-4-NH2 0.51 0.05
207 (CH2)2NHC(¼O)C6H4-2-CH3 0.11 0.08
208 (CH2)2NHC(¼O)C6H4-4-CH3 0.15 0.02
209 (CH2)2NHC(¼O)C6H3-2,4-(CH3)2 0.068 0.034
210 (CH2)2NHC(¼O)C6H4-2-OCH3 0.147 0.06
211 (CH2)2NHC(¼O)C6H2-3,4,5-(OCH3)3 0.172 0.05
212 (CH2)2NHC(¼O)C6H4-2-Cl 0.07 0.009
213 (CH2)2NHC(¼O)C6H4-4-Cl 0.14 0.03
214 (CH2)2NHC(¼O)C6H4-3,4-Cl2 0.095 0.03
215 (CH2)2NHC(¼O)C6H4-4-CN 0.177 0.04
216 (CH2)2NHC(¼O)C6H4-4-SO2CH3 0.48 0.2
197 (CH2)2NHC(¼O)C6H5 0.2 0.02
217 (CH2)2NHC(¼O)C6H4-3-CH3 0.54 0.2
218 (CH2)2NHC(¼O)C6H4-4-OCH3 0.07 0.0
219 (CH2)2NHC(¼O)C6H4-4F 0.56 0.06
220 (CH2)2NHC(¼O)C6H4-4CO2C2H5 0.345 0.02
221 (CH2)2NHC(¼O)C6H4-CONH2 0.08 0.03
222 (CH2)2NHC(¼O)C5H4-4-N 2.2 0.12
223 (CH2)2CN –
224 CH2CH2CH2-NH2 –
225 (CH2)3NHC(¼O)C6H4-2-CH3 0.32 0.1
226 (CH2)3NHC(¼O)C6H3-2,4-(CH3)2 0.32 0.1
227 (CH2)3NHC(¼O)C6H4-4-OCH3 0.22 0.08
228 (CH2)2OCH(C6H5)2 1.0 0.1
229 (CH2)2OCH(C6H5)-C6H4-4-F 1.0 0.1
230 (CH2)OCH-C6H4-4-F C6H4-4-Cl 1.35 0.2
231 (CH2)2NH-CH(C6H5)-C6H4-4-F 0.75 0.08
232 (CH2)2N¼CHC6H4-4-NO2 5.25 1.02
233 (CH2)3(CH3)OH-C6H4-4F 0.75 0.10
234 (CH2)2NH-CH2C6H4-4-NO2 2.20 0.22
235 (CH2)2NHC(¼S)NHC6H5 0.47 0.11
a
IC50 against histamine induced contraction in isolated guinea pig ileum: (–) described not done
[199]
Table 4 The structure of the constrained analogs of diphenhydramine
Comp Substituents IC50 (mg/mL)a Structure Substituents IC50 (mg/mL)a

180 R Diphenhydramine 0.001 195b R¼C6H5, X¼NH 3.5
194a R¼C6H5, X¼O 0.001 193b R¼H, X¼N-C6H5 0.01
195a R¼C6H5, X¼O 0.5 194c R¼CH3, X¼0 0.75
193a R¼C6H5, X¼NH 0.03 195c R¼CH3, X¼0 1.0
194b R¼H,X¼N-C6H5 10
a
In guinea pig ileum
of the molecule. A molecular volume is drawn enclosing the space within Van der
Waals radii of all the atoms lying within this volume, and a set of equidistant lattice
points are generated orthogonally to each other and separated by a distance called
resolution. Additional information like electro density is incorporated as a fourth
dimensional at the occupied lattice points and thus 4D lattice of a reference molecule
is generated. Next the 4D lattices of other molecules are compared with this reference
lattice by stepped progression of translational and rotational movements to find the
best common points among the lattices (maximum FIT). The first application of the
HASL approach not only included the β-benzoylaminoethyl and 2-(anilinocarbonyl)-
ethylpiperazines,-piperidines, -pyrazinopyrioindoles, and -pyrazinoiso-quinolines but
also diphenhydramine and its semirigid analogs reinforced the importance of major
sites for the interaction of the tertiary nitrogen and aromatic rings and showed that the
β-aroylamino/arylaminocarbonyl ethylamine substructure is the possible antihistaminic
H1 pharmacophore (Fig. 25b) for compounds 197, 245, 249, 254, 259, and 263 (Fig. 24).
These studies were further improved by advanced pharmacophore models using the
Apex 3D and CATALYST molecular modeling programs. The HipHop module in
CATALYST was used for the generation of pharmacophore models where diphenhy-
dramine was selected as a template and the rest 43 molecules were superimposed on
it. The 3D structures of all the compounds considered were generated within a 20 Kcal
cutoff by applying the poling algorithm by the application of CHARMm force field
[195]. The common feature pharmacophore was generated to find the common
chemical features present in all the training set molecules. The best alignments
obtained from the common feature pharmacophore generation were used in the
MOPAC for the calculation of different physicochemical and quantum chemical
parameters such as π-population, atomic charge, H-bond acceptor and donor index,
hydrophobicity, LUMO, HOMO, and molar refractivity on the basis of atom proper-
ties that were used by the Apex 3D program in the generation of 3D-QSAR and
pharmacophore models. The common feature pharmacophore protocol in CATA-
LYST generated eight hypotheses with the ranking score ranging from 80.3518 to
25.2954 units among which six hypotheses had similar combination of
Fig. 23 Chemical structure of the compounds used for 2D-QSAR generation
Table 5 The 2D-QSAR equations for the generated models

No Equation na rb sc Fd
1 log1/C ¼ 0.324 πo + 0.268 πp + 0.150 Io + 0.702 17 0.93 0.11 27.57
2 log1/I50 ¼ 0.304π(0.014) + 0.164σ(0.046) + 0.019MR 6 0.998 0.021 188.4
(0.005) + 0.262(0.024)
3 log1/I50 ¼ 0.347π(0.049) + 0.144σ(0.072) + 0.011MR 27 0.915 0.090 39.3
(0.006) 0.224(0.038)
4 log1/I50 ¼ 0.328π(0.034) + 0.143σ(0.059) + 0.013MR 33 0.953 0.083 68.7
(0.005) + 0.53IA(0.041) 0.229(0.003)
a
Number of dataset
b
Multiple correlation coefficient
c
Standard deviation
d
Statistical significance
pharmacophore features: two ring aromatic and one PI features (Fig. 25d). The
features of the developed pharmacophore model were in agreement to the earlier
models and the model proposed by Ter Laack et al. However the interfeature distance
in the model differed significantly from the model developed by Ter Laack et al.
[196]. The differences in interfeature distance (Å) are summarized in Table 6. These
differences in interfeature distances in these models may arise due to different
chemical class of compounds used for model generation. The features of the catalyst
pharmacophore complement with the homology model of H1 receptor developed by
Wieland where one of the aromatic rings interacted with Phe 433 and Phe 436 while
the other interacted with Trp167. The positively ionizable nitrogen atom made contact
with Asp116 of TMIII. The Apex 3D-biophoric models were developed taking the
alignment of training set molecules for hypothesis 1 of the CFP in CATALYST.
logðIC50 Þ ¼ 0 : 5640 ð0:140Þ ½Hydrophobicity at ss1

þ 10:097ð1:287Þ ðHydrophobicity at ss2Þ 1 : 638ð0:752Þ
½Hydrophobicity at ss3 þ 2 : 209ð0:738Þ
½Hydrophobicity at ss4 0:270ð0:44Þ ½Refractivity at ss5
þ 0:117ð0:034Þ ½Refractivity at ss6
þ 0:12 ðn ¼ 42; R ¼ 0:855; F6; 36 ¼ 16:376; Q ¼ 0:794; S ¼ 0:335Þ
ð5Þ
None of the generated biophoric models mapped all the molecules in the training
set, hence a model that can map maximum number of compounds (42 out of 45)
with good statistical parameters (correlation coefficient r2 > 0.7, the difference of
RMSA and RMSP <0.03 (a measure of cross-validation), chance 0.1, no. of
variables <7, and compounds >41) was selected for further analysis. The generated
Apex 3D model had three biophoric features ABD with A and D being the aromatic
features while site B corresponds to the positive ionizable feature that interacts with
Asp116. The mean interatomic distances between the three biophoric features are as
follows: A–D (5.585 0.398), A–B (6.2181 0.421), and B–D (5.5013 0.488).
Six secondary sites (ss1–ss6) parameters related to hydrophobicity were also gen-
erated in the Apex 3D model where the secondary sites ss1, ss3, and ss5 that signify
refractivity and hydrophobicity contributed negatively towards biological activity.
The secondary site ss2 that represents hydrophobicity at the phenyl moiety con-
tributes positively towards biological activity along with secondary sites ss4 and ss6
(Fig. 25c). The final Eqn. (5) showed low SD (S ¼ 0.335), good correlation co-
efficient (R ¼ 0.86) with cross validated Q ¼ 0.794. The 3D QSAR model also
predicted well an external set of four molecules (triprolidine, mepyramine, doxepin,
and ()-trans-1-phenyl-3-(dimethylamino)-1,2,3,4-tetrahydronaphthalene) with a
correlation coefficient of R2 ¼ 0.8904.
Fig. 24 Structure of the compounds used in HASL study

Fig. 25 (a) Alignment of compound 197 with diphenhydramine (180). (b) The alignment of
compound 197 with diphenhydramine (180) in HASL. (c) The mapping of compound 243 with
the Apex 3D model. (d) The mapping of diphenhydramine (180) with the CATALYST model con-
taining two RA (orange) and one PI (red) features (reproduced with permission from Bioorg. Med.
Chem., 2006, 14, 8249–8258 Copyright 2006 Elsevier Ltd.)
From the above studies, it is obvious that application of the 3D-QSAR ligand based
approaches identified the classical antihistaminic pharmacophore comprised by two
ring aromatic/hydrophobes and a PI feature that is highly expected as all the ligand
based models discussed here were developed to identify the common chemical func-
tionalities present in the diverse set of ligands. Although these models are predictive and
have the strength to identify lead molecules with antihistaminic activity, still they lack
the property to discriminate the leads having additional features that may have contrib-
utory role towards activity. For example, the methyl and chloro substituted phenyl rings
in compounds 209 and 214 indicate hydrophobic features to have contributory role
towards antihistaminic activity. This assumption was however opposed by two com-
pounds having 4-amido and 4-methoxy substitution at the phenyl ring with different
electronic and hydrophobic environments so these compounds are not predicted prop-
erly in the [36] earlier classical QSAR studies. To address this issue and with the crystal
structure of bovine rhodopsin in hand, protein homology models were constructed for
the histamine H1 receptor to gain information about the receptor environment that can
Table 6 The differences in features between the antihistamine H1 pharmacophore models

R1-Pa R1-R2a R2-Pa
Saxena et al. 5.247 4.971 6.88
Ter Laack et al. 8.73 4.79 9.10
R1 ¼ Ring Aromatic1, R2 ¼ Ring Aromatic2, P ¼ Positive ionizable [195, 196]
a
facilitate the binding of all these ligands [197]. The homology modeled H1 receptor
having 18.2% identical residues with bovine rhodopsin (RMSD ¼ 1.329 Å) was further
optimized by ligand assisted methods using mepyramine and diphenylhydramine in two
consecutive steps. The volume of the binding site cavity with the docked conformation
of mepyramine was 210.516 Å3 that increased to 270.4219 Å3 when diphenhydramine
was docked. Both mepyramine and diphenhydramine showed the important residue
interactions at the receptor site, the most crucial being the salt bridge formation with
Asp107 of TMVII. However the generated receptor model of H1 was unable to accom-
modate the folded conformation of the octahydropyrazinopyridoindoles as superimposed
with diphenhydramine. Few years later after the discovery of the crystal structure of
human H1 receptor, attempts were made to explain the SAR of the octahydropyra-
zinopyridoindoles using docking studies [198]. The crystal structure of human HR1 was
able to explain the SAR and outlier behavior of the compounds observed in the classical
2D-QSAR studies. Comparison of the homology modeled H1 and the crystal structure of
H1 revealed substantial differences between the protein structures. The RMSD between
these protein structures was 3.1 Å. The major difference observed between the protein
structures was in the ECL2 region. The ECL2 region in the homology modeled protein
similar to bovine rhodopsin has a β-hairpin structure and is located deep inside the
binding cavity preventing solvent access whereas the ECL2 region of the crystal structure
is more extended compared to the homology modeled receptor. This extended confor-
mation of the ECL2 region in the crystal structure induced changes in the helical region
increasing the volume of the binding cavity by moving the TMIII and TMV extracellular
regions apart (Fig. 26a). For this, the receptor bound crystal structure of doxepin was used
and the receptor interactions were translated into pharmacophore information. The gen-
erated receptor based pharmacophore consisted four features viz., two hydrophobes, one
PI, and one HBA features (Fig. 26a and c). This pharmacophore was further used to map
the folded conformation of compound 1 excluding the HBA feature (Fig. 26d). Changes
were also observed regarding the aromatic residues in TMVI for which greater bending
away from the binding region was observed for W4286.48 (~4 Å from the centroid of the
phenyl rings of the tryptophan moiety) in the crystal structure compared to the homology
modeled protein. The other aromatic residue F4246.44 shifted in the same plane by 3.7 Å
while F4326.52 is located 4.2 Å towards the binding cavity as compared to the homology
modeled H1 receptor. Minor shifts were also observed for F4356.55 (1.9 Å) and Y4316.51
(1.8 Å). The docking of the compounds 197, 209, 212, 214, 218, and 221 that was
performed on the crystal structure of human H1 showed a longitudinal orientation of the
molecular structures with the aroylethylamine portion being situated deep inside the
binding cavity while the octahydropyrazinopyridoindole moiety resided at the extracel-

lular region. In the docked conformation, the 2-chloro phenyl moiety of compound 1 had
hydrophobic interactions with the aromatic residues of TMVI (W4286.48, Y4316.51, and
F4326.52). The crucial salt bridge with Asp1073.32 was formed by the pyrazine N1 atom.
This is in agreement with both 2DQSAR and 3DQSAR studies where the first pyrazine
N1 atom was described to have important interaction at the receptor site. It also com-
plemented with the earlier finding in the 2DQSAR study where an optimum chain length
of two carbons is essential between the first pyrazine nitrogen and the NHCOAr moiety
to maintain the required distance between the hydrophobic/aromatic (phenyl ring of
NHCOAr fragment) and the PI features. The other interactions of compound 212 include
hydrophobic interactions of the indole moiety with M4517.36 and I4547.39 and H-bond
contact of the indole nitrogen with H4507.35 (Fig. 27a). The compounds 197 and 214
showed similar orientation at the receptor site like compound 212. However notably the
phenyl moiety in compound 1 was observed to be located at a distance of ~4 Å from the
tertiary carbon of I1153.40 when heavy atoms are considered that reduced to 2.2 Å when
hydrogen atoms were added. The combination of I1153.40 and F4246.44 creates a barrier
and defines a boundary for the binding site beyond which lies the ligand inaccessible site.
For compound 214, a shift of 0.9 Å backwards for the phenyl ring of the 3,4 dichloro-
phenyl moiety was observed relative to the phenyl ring in compound 212. The backward
shift of the phenyl ring in compound 3 resulted in order to accommodate the more bulky
chloro atom at the limited space provided and suggests that the space between D1073.32
and I1153.40/F4246.44 can tolerate ligands with small aryl substituents. This was further
supported by compounds 209, 218, and 221 having 2,4-dimethyl, 4-methoxy and 4-
amido substituents, respectively. The large aryl substituents in these compounds com-
pelled the NHCOAr moiety to move towards TMV and accommodate in the extra space
between TMIII and TMV due to an elongated ECL2 structure (Fig. 27b). The compar-
ison of docked conformation of diphenhydramine (180), 212, and 221 is shown in
Fig. 27c. The –NH2 fragment of 4-amido moiety maintained H-bond contact with the
backbone carbonyl oxygen atom and occupied a similar position as observed for the
acetic acid side chain in olopatadine. The aryl moiety of compound 221 maintained
hydrophobic contacts with Y1083.33, Y4316.51, F4326.52, F4356.55, and W4286.48 while
the receptor based interaction of the octahydropyrazinopyridoindole core is similar to
compound 212. The interactions of the octahydropyrazinopyridoindole core are also the
same for compound 218 while the 4-methoxyphenyl substitution in it has hydrophobic
contacts with Y1083.33, S1113.36, Y4316.51, F4326.52, and W4286.48. This suggests that the
octahydropyrazino pyridoindole class of compounds have different orientations at the
receptor site depending on the substitution at the aryl ring. Further studies were per-
formed to find the feasibility of compound 212 to acquire a folded conformation at the
receptor site (Fig. 27d). The HBA feature corresponding to the ring oxygen atom was
excluded as it is absent in many antihistaminics. A folded conformation for compound
212 was achieved when the three-featured pharmacophore was used for mapping. This
folded conformation when rescored at the H1 receptor site through docking studies (Glide
XP) showed good docking score (10.24) with the phenyl moiety of the indole
nucleus and the 2-chlorophenyl moiety being aligned on the two phenyl rings of
Fig. 26 (a) The comparison between the crystal structure of the homology modeled H1 receptor and
the crystal structure showing the gap between TMIII and TMV. (b) The mapping of doxepin in the
structure based pharmacophore aligned at the H1 receptor site. (c) The structure based phar-
macophore including two hydrophobic (HY) (blue), one hydrogen bond acceptor (HBA) (green),
and one positive ionizable (PI) (red) features with distance constraints. (d) Folded conformation of
compound 212 mapping the pharmacophore (reproduced with permission from SAR and QSAR in
Env. Res., 2012, 23, 311–325 Copyright 2012 Taylor & Francis)
diphenhydramine (Fig. 27d). The pyrazine nitrogen N1 formed the necessary salt
bridge with D1073.32. However flexible docking using Glide XP module resulted in
extended conformation for compound 1 that suggests that although the extended con-
formation is more favored, the folded conformation is energetically feasible. These
studies on the octahydropyrazinopyridoindoles showed that the association of
ligand and structure based methods supplement each other to recognize the important
receptor–ligand interactions.
5 Conclusion
The recent advances in the understanding of structure and function of GPCRs have
provided great enrichment in the development of small molecules modulating GPCR
function. Apart from structural information, many GPCR targets have a wealth of in-
formation about the chemical structure of small molecules modulating their function.
Despite these advances in structure based approaches (SBDD) and 3D-QSAR and phar-
macophore modeling (LBDD) in GPCRs, an integration of both approaches provides
an efficient protocol for ligand screening and optimization by reducing the number of
false positives and false negatives during the virtual screening (VS). In addition, the
integrated approach also provides an excellent platform for the ligand design process in
GPCRs as exemplified in three case studies reported in this chapter. In the first case of
α1 Adr, the integrated approach provided understanding about the important pharma-
cophore features and receptor residues important for determining the subtype selec-
tivity between α1a, α1b, and α1d Adrs. The studies have led to the identification of
Fig. 27 (a) Docked conformation of: (a) compound 212 at the active site of H1 receptor; (b) the
compound 221 at the active site of H1 receptor; (c) a comparison of docked conformation of 212
(blue) and 221 (green) with diphenhydramine (180) (yellow). Residues I1153.40 and F4246.44 act as
a barrier for bulky substitution at para position of the phenyl ring. (d) Folded conformation of
compound 212 at the active site of the histamine receptor (reproduced with permission from SAR
and QSAR in Env. Res., 2012, 23, 311–325 Copyright 2012 Taylor & Francis)
important leads. In the second case, the dopamine receptor topography has been de-
termined using the D2 ligands as agonists where the nature of the N-alkyl substituent
played an important role in modulating receptor function. Its integration with structure
based models led to the identification of important residues necessary for binding of the
ligands as well as in designing of potent D2 receptor agonists. In the third case, the
binding interactions and relative orientation of different class of antihistaminics H1
including the nonclassical 2-aroylaminoethyloctahydro-pyrazinopyridoindoles. The 2D
and 3D QSAR models (LBDD) integrated with the docking studies on modeled re-
ceptors (SBDD) have not only served as a powerful tool for the prediction of activity in
diverse type of small molecules but also explained the outlier behaviors of the mol-
ecules. With the current progress in computer aided drug design, the integrated ap-
proach may serve as a powerful cost and time effective technique not only in the
identification of new lead and candidate molecules but also in improving the under-
standing of structure and functions in GPCR family.
References
1. Lundstrom K (2006) Latest development in drug discovery on G protein-coupled receptors.

Curr Protein Pept Sci 7:465–470
2. Ma P, Zemmel R (2002) Value of novelty? Nat Rev Drug Discov 1:571–572
3. Fredriksson R, Lagerstrom MC, Lundin LG, Schioth HB (2003) The G-protein-coupled recep-
tors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and
fingerprints. Mol Pharmacol 63:1256–1272
4. Schioth HB, Fredriksson R (2005) The GRAFS classification system of G-protein coupled
receptors in comparative perspective. Gen Comp Endocrinol 142:94–101
5. Lagerstrom MC, Schioth HB (2008) Structural diversity of G protein-coupled receptors and
significance for drug discovery. Nat Rev Drug Discov 7:339–357
6. Kobilka B (2013) The structural basis of G-protein-coupled receptor signaling (nobel lec-
ture). Angew Chem Int Ed Engl 52:6380–6388
7. Lefkowitz RJ, Shenoy SK (2005) Transduction of receptor signals by beta-arrestins. Science
308:512–517
8. Luttrell LM, Lefkowitz RJ (2002) The role of beta-arrestins in the termination and transduc-
tion of G-protein-coupled receptor signals. J Cell Sci 115:455–465
9. Azzi M, Charest PG, Angers S, Rousseau G, Kohout T, Bouvier M et al (2003) Beta-arrestin-
mediated activation of MAPK by inverse agonists reveals distinct active conformations for G
protein-coupled receptors. Proc Natl Acad Sci U S A 100:11406–11411
10. Bock A, Kostenis E, Tränkle C, Lohse MJ, Mohr K (2014) Pilot the pulse: controlling the
multiplicity of receptor dynamics. Trends Pharmacol Sci 35:630–638
11. Smith JS, Rajagopal S (2016) The beta-arrestins: multifunctional regulators of G protein-
coupled receptors. J Biol Chem 291(17):8969–8977
12. Jacoby E, Bouhelal R, Gerspacher M, Seuwen K (2006) The 7 TM G-protein-coupled receptor
target family. ChemMedChem 1:761–782
13. Klabunde T, Hessler G (2002) Drug design strategies for targeting G-protein-coupled recep-
tors. Chembiochem 3:928–944
14. Jimonet P, Jager R (2004) Strategies for designing GPCR-focused libraries and screening
sets. Curr Opin Drug Discov Devel 7:325–333
15. Dorsam RT, Gutkind JS (2007) G-protein-coupled receptors and cancer. Nat Rev Cancer
7:79–94
16. Lappano R, Maggiolini M (2012) GPCRs and cancer. Acta Pharmacol Sin 33:351–362
17. Singh A, Nunes JJ, Ateeq B (2015) Role and therapeutic potential of G-protein coupled
receptors in breast cancer progression and metastases. Eur J Pharmacol 763(Part B):178–183
18. Salazar NC, Chen J, Rockman HA (2007) Cardiac GPCRs: GPCR signaling in healthy and
failing hearts. Biochim Biophys Acta 1768:1006–1018
19. Tang CM, Insel PA (2004) GPCR expression in the heart; “new” receptors in myocytes and
fibroblasts. Trends Cardiovasc Med 14:94–99
20. Hunt SA, Abraham WT, Chin MH, Feldman AM, Francis GS, Ganiats TG et al (2005)
ACC/AHA 2005 guideline update for the diagnosis and management of chronic heart failure in
the adult: a report of the American College of Cardiology/American Heart Association Task
Force on practice guidelines (writing committee to update the 2001 guidelines for the evaluation
and management of heart failure): developed in collaboration with the American College of
Chest Physicians and the International Society for Heart and Lung Transplantation: endorsed by
the Heart Rhythm Society. Circulation 112:e154–e235
21. Fernandez-Patron C, Filep JG (2012) GPCRs in cardiovascular pathologies. Drug Discov
Today Dis Mech 9:e75–e78
22. Belmonte SL, Blaxall BC (2011) G protein coupled receptor kinases as therapeutic targets in
cardiovascular disease. Circ Res 109:309–319
23. Dalet F-GE, Guadalupe T-FJ, Marı́a del Carmen C-H, Humberto G-AC, Antonio S-UM
(2013) Insights into the structural biology of G-protein coupled receptors impacts drug design
for central nervous system neurodegenerative processes. Neural Regen Res 8:2290–2302
24. Nickols HH, Conn PJ (2014) Development of allosteric modulators of GPCRs for treatment
of CNS disorders. Neurobiol Dis 61:55–71
25. Catapano LA, Manji HK (2007) G protein-coupled receptors in major psychiatric disorders.
Biochim Biophys Acta 1768:976–993
26. Thathiah A, De Strooper B (2011) The role of G protein-coupled receptors in the pathology of
Alzheimer’s disease. Nat Rev Neurosci 12:73–87
27. Ahren B (2009) Islet G protein-coupled receptors as potential targets for treatment of type
2 diabetes. Nat Rev Drug Discov 8:369–385
28. Rayasam GV, Tulasi VK, Davis JA, Bansal VS (2007) Fatty acid receptors as new therapeutic
targets for diabetes. Expert Opin Ther Targets 11:661–671
29. Swaminath G (2008) Fatty acid binding receptors and their physiological role in type 2 diabetes.
Arch Pharm 341:753–761
30. Sun L, Ye RD (2012) Role of G protein-coupled receptors in inflammation. Acta Pharmacol
Sin 33:342–350
31. Cash JL, Norling LV, Perretti M (2014) Resolution of inflammation: targeting GPCRs that
interact with lipids and peptides. Drug Discov Today 19:1186–1192
32. Stone LS, Molliver DC (2009) In search of analgesia: emerging poles of GPCRs in pain. Mol
Interv 9:234–251
33. Harrison C (2013) G protein-coupled receptors: a double attack on pain. Nat Rev Drug
Discov 12:665–665
34. Geppetti P, Veldhuis NA, Lieu T, Bunnett NW (2015) G protein-coupled receptors: dynamic
machines for signaling pain and itch. Neuron 88:635–649
35. Schoneberg T, Schulz A, Biebermann H, Hermsdorf T, Rompler H, Sangkuhl K (2004) Mutant
G-protein-coupled receptors as a cause of human diseases. Pharmacol Ther 104:173–206
36. Ghosh E, Kumari P, Jaiman D, Shukla AK (2015) Methodological advances: the unsung
heroes of the GPCR structural revolution. Nat Rev Mol Cell Biol 16:69–81
37. Katritch V, Cherezov V, Stevens RC (2013) Structure-function of the G protein-coupled
receptor superfamily. Annu Rev Pharmacol Toxicol 53:531–556
38. Huang CY, Olieric V, Ma P, Howe N, Vogeley L, Liu X et al (2016) In meso in situ serial
X-ray crystallography of soluble and membrane proteins at cryogenic temperatures. Acta
Crystallogr D Struct Biol 72:93–112
39. Tikhonova IG, Costanzi S (2009) Unraveling the structure and function of G protein-coupled
receptors through NMR spectroscopy. Curr Pharm Des 15:4003–4016
40. Roberts NA, Martin JA, Kinchington D, Broadhurst AV, Craig JC, Duncan IB et al (1990)
Rational design of peptide-based HIV proteinase inhibitors. Science 248:358–361
41. Erickson J, Neidhart DJ, VanDrie J, Kempf DJ, Wang XC, Norbeck DW et al (1990) Design,
activity, and 2.8 A crystal structure of a C2 symmetric inhibitor complexed to HIV-1 protease.
Science 249:527–533
42. Dorsey BD, Levin RB, McDaniel SL, Vacca JP, Guare JP, Darke PL et al (1994) L-735,524: the
design of a potent and orally bioavailable HIV protease inhibitor. J Med Chem 37:3443–3451
43. Geppert H, Vogt M, Bajorath J (2010) Current trends in ligand-based virtual screening: molecu-
lar representations, data mining methods, new application areas, and performance evaluation.
J Chem Inf Model 50:205–216
44. Willett P (2006) Similarity-based virtual screening using 2D fingerprints. Drug Discov Today
11:1046–1053
45. Helguera AM, Combes RD, Gonzalez MP, Cordeiro MNDS (2008) Applications of 2D
descriptors in drug design: a DRAGON tale. Curr Top Med Chem 8:1628–1655
46. Doweyko AM (1988) The hypothetical active site lattice. An approach to modelling active
sites from data on inhibitor molecules. J Med Chem 31:1396–1406
47. Cramer RD, Patterson DE, Bunce JD (1988) Comparative molecular field analysis (CoMFA).
1. Effect of shape on binding of steroids to carrier proteins. J Am Chem Soc 110:5959–5967
48. Klebe G, Abraham U, Mietzner T (1994) Molecular similarity indices in a comparative
analysis (CoMSIA) of drug molecules to correlate and predict their biological activity. J Med
Chem 37:4130–4146
49. Bhunia SS, Roy KK, Saxena AK (2011) Profiling the structural determinants for the selec-
tivity of representative factor-Xa and thrombin inhibitors using combined ligand-based and
structure-based approaches. J Chem Inf Model 51:1966–1985
50. Apex-3D (1993) InsightII, version2.3.0. BIOSYM Technologies, San Diego
51. Dixon SL, Smondyrev AM, Rao SN (2006) PHASE: a novel approach to pharmacophore
modeling and 3D database searching. Chem Biol Drug Des 67:370–372
52. Dixon SL, Smondyrev AM, Knoll EH, Rao SN, Shaw DE, Friesner RA (2006) PHASE: a new
engine for pharmacophore perception, 3D QSAR model development, and 3D database
screening: 1. Methodology and preliminary results. J Comput Aided Mol Des 20:647–671
53. Dassault Systèmes BIOVIA (2015) Discovery studio modeling environment, release 4.5.
Dassault Systèmes, San Diego, CA
54. Bhunia SS, Singh S, Saxena S, Saxena AK (2015) Pharmacophore modeling, docking and
molecular dynamics studies on caspase-3 activators binding at beta-tubulin site. Curr Comput
Aided Drug Des 11:72–83
55. Jones G, Willett P, Glen R (2000) GASP: genetic algorithm superimposition program. In:
Guner OF (ed) Pharmacophore perception, development & use in drug design, vol 2. Inter-
national University Line, La Jolla, CA, pp 85–106
56. Martin YC, Bures MG, Danaher EA, DeLazzer J, Lico I, Pavlik PA (1993) A fast new approach
to pharmacophore mapping and its application to dopaminergic and benzodiazepine agonists.
J Comput Aided Mol Des 7:83–102
57. Richmond NJ, Abrams CA, Wolohan PR, Abrahamian E, Willett P, Clark RD (2006) GALAHAD:
1. Pharmacophore identification by hypermolecular alignment of ligands in 3D. J Comput Aided
Mol Des 20:567–587
58. Prathipati P, Dixit A, Saxena AK (2007) Computer-aided drug design: integration of structure-
based and ligand-based approaches in drug design. Curr Comput Aided Drug Des 3:133–148
59. Kitchen DB, Decornez H, Furr JR, Bajorath J (2004) Docking and scoring in virtual screening
for drug discovery: methods and applications. Nat Rev Drug Discov 3:935–949
60. Hajduk PJ, Greer J (2007) A decade of fragment-based drug design: strategic advances and
lessons learned. Nat Rev Drug Discov 6:211–219
61. Cavasotto CN, Orry AJ (2007) Ligand docking and structure-based virtual screening in drug
discovery. Curr Top Med Chem 7:1006–1014
62. Saxena M, Bhunia SS, Saxena AK (2015) Molecular modelling studies on 2-substituted
octahydropyrazinopyridoindoles for histamine H2 receptor antagonism. SAR QSAR Environ
Res 26:739–755
63. Pitta E, Tsolaki E, Geronikaki A, Petrović J, Glamočlija J, Soković M et al (2015)
4-Thiazolidinone derivatives as potent antimicrobial agents: microwave-assisted synthe-
sis, biological evaluation and docking studies. MedChemComm 6:319–326
64. Trott O, Olson AJ (2010) AutoDock Vina: improving the speed and accuracy of docking
with a new scoring function, efficient optimization, and multithreading. J Comput Chem
31:455–461
65. Verdonk ML, Cole JC, Hartshorn MJ, Murray CW, Taylor RD (2003) Improved protein–
ligand docking using GOLD. Proteins 52:609–623
66. Halgren TA, Murphy RB, Friesner RA, Beard HS, Frye LL, Pollard WT et al (2004) Glide: a
new approach for rapid, accurate docking and scoring. 2. Enrichment factors in database screen-
ing. J Med Chem 47:1750–1759
67. Clark RD, Strizhev A, Leonard JM, Blake JF, Matthew JB (2002) Consensus scoring for
ligand/protein interactions. J Mol Graph Model 20:281–295
68. Halperin I, Ma B, Wolfson H, Nussinov R (2002) Principles of docking: an overview of
search algorithms and a guide to scoring functions. Proteins 47:409–443
69. Krippahl L, Barahona P (2015) Protein docking with predicted constraints. Algorithms Mol
Biol 10:9
70. Azad CS, Bhunia SS, Krishna A, Shukla PK, Saxena AK (2014) Novel glycoconjugate of
8-fluoro norfloxacin derivatives as gentamicin-resistant Staphylococcus aureus inhibitors:
synthesis and molecular modelling studies. Chem Biol Drug Des 86(4):440–446
71. Saxena AK, Devillers J, Bhunia SS, Bro E (2015) Modelling inhibition of avian aromatase by
azole pesticides. SAR QSAR Environ Res 26:757–782
72. Fischer M, Coleman RG, Fraser JS, Shoichet BK (2014) The incorporation of protein
flexibility and conformational energy penalties in docking screens to improve ligand discov-
ery. Nat Chem 6:575–583
73. Jain AN (2009) Effects of protein conformation in docking: improved pose prediction through
protein pocket adaptation. J Comput Aided Mol Des 23:355–374
74. Boehr DD, Nussinov R, Wright PE (2009) The role of dynamic conformational ensembles in
biomolecular recognition. Nat Chem Biol 5:789–796
75. McGovern SL, Shoichet BK (2003) Information decay in molecular docking screens against
holo, apo, and modeled conformations of enzymes. J Med Chem 46:2895–2907
76. Larsson P, Wallner B, Lindahl E, Elofsson A (2008) Using multiple templates to improve
quality of homology models in automated homology modeling. Protein Sci 17:990–1002
77. Rataj K, Witek J, Mordalski S, Kościółek T, Bojarski AJ (2013) The importance of template
choice in homology modeling. A 5-HT(6)R case study. J Cheminform 5:P8
78. Evers A, Klebe G (2004) Ligand-supported homology modeling of G-protein-coupled receptor
sites: models sufficient for successful virtual screening. Angew Chem Int Ed Engl 43:248–251
79. Kumari P, Ghosh E, Shukla AK (2015) Emerging approaches to GPCR ligand screening for
drug discovery. Trends Mol Med 21:687–701
80. Santos R, Hritz J, Oostenbrink C (2010) Role of water in molecular docking simulations of
cytochrome P450 2D6. J Chem Inf Model 50:146–154
81. Kumar A, Zhang KY (2013) Investigation on the effect of key water molecules on docking
performance in CSARdock exercise. J Chem Inf Model 53:1880–1892
82. Wang L, Berne BJ, Friesner RA (2011) Ligand binding to protein-binding pockets with wet
and dry regions. Proc Natl Acad Sci 108:1326–1330
83. Salom D, Padayatti PS, Palczewski K (2013) Crystallization of G protein-coupled receptors.
Methods Cell Biol 117:451–468
84. Xie XQ, Chowdhury A (2013) Advances in methods to characterize ligand-induced ionic
lock and rotamer toggle molecular switch in G protein-coupled receptors. Methods Enzymol
520:153–174
85. Trzaskowski B, Latek D, Yuan S, Ghoshdastider U, Debinski A, Filipek S (2012) Action of
molecular switches in GPCRs – theoretical and experimental studies. Curr Med Chem
19:1090–1109
86. Kobilka BK, Deupi X (2007) Conformational complexity of G-protein-coupled receptors.
87. Scheerer P, Park JH, Hildebrand PW, Kim YJ, Krausz N, Choe H-W et al (2008) Crystal
structure of opsin in its G-protein-interacting conformation. Nature 455:497–502
88. Rasmussen SG, Choi HJ, Fung JJ, Pardon E, Casarosa P, Chae PS et al (2011) Structure of a
nanobody-stabilized active state of the beta(2) adrenoceptor. Nature 469:175–180
89. Tehan BG, Bortolato A, Blaney FE, Weir MP, Mason JS (2014) Unifying family A GPCR
theories of activation. Pharmacol Ther 143:51–60
90. Sato J, Makita N, Iiri T (2016) Inverse agonism: the classic concept of GPCRs revisited.
Endocr J 63(6):507–514
91. Goodman OB Jr, Krupnick JG, Santini F, Gurevich VV, Penn RB, Gagnon AW et al (1996)
Beta-arrestin acts as a clathrin adaptor in endocytosis of the beta2-adrenergic receptor. Nature
383:447–450
92. Kang DS, Tian X, Benovic JL (2014) Role of beta-arrestins and arrestin domain-containing
proteins in G protein-coupled receptor trafficking. Curr Opin Cell Biol 27:63–71
93. Gurevich VV, Gurevich EV (2006) The structural basis of arrestin-mediated regulation of G-
protein-coupled receptors. Pharmacol Ther 110:465–502
94. Reiter E, Ahn S, Shukla AK, Lefkowitz RJ (2012) Molecular mechanism of beta-arrestin-
biased agonism at seven-transmembrane receptors. Annu Rev Pharmacol Toxicol 52:179–197
95. Luttrell LM, Miller WE (2013) Arrestins as regulators of kinases and phosphatases. Prog Mol
Biol Transl Sci 118:115–147
96. Beaulieu JM, Sotnikova TD, Marion S, Lefkowitz RJ, Gainetdinov RR, Caron MG (2005) An
Akt/beta-arrestin 2/PP2A signaling complex mediates dopaminergic neurotransmission and
behavior. Cell 122:261–273
97. Shukla AK, Singh G, Ghosh E (2014) Emerging structural insights into biased GPCR
signaling. Trends Biochem Sci 39:594–602
98. DeWire SM, Violin JD (2011) Biased ligands for better cardiovascular drugs: dissecting G-
protein-coupled receptor pharmacology. Circ Res 109:205–216
99. Chang SD, Bruchas MR (2014) Functional selectivity at GPCRs: new opportunities in
psychiatric drug discovery. Neuropsychopharmacology 39:248–249
100. Heilker R, Wolff M, Tautermann CS, Bieler M (2009) G-protein-coupled receptor-focused
drug discovery using a target class platform approach. Drug Discov Today 14:231–240
101. Peeters MC, van Westen GJ, Li Q, IJzerman AP (2011) Importance of the extracellular loops in
G protein-coupled receptors for ligand recognition and receptor activation. Trends Pharmacol
Sci 32:35–42
102. Wheatley M, Wootten D, Conner MT, Simms J, Kendrick R, Logan RT et al (2012) Lifting
the lid on GPCRs: the role of extracellular loops. Br J Pharmacol 165:1688–1703
103. Klabunde T, Evers A (2005) GPCR antitarget modeling: pharmacophore models for biogenic
amine binding GPCRs to avoid GPCR-mediated side effects. Chembiochem 6:876–889
104. Lee SM, Booe JM, Pioszak AA (2015) Structural insights into ligand recognition and
selectivity for classes A, B, and C GPCRs. Eur J Pharmacol 763:196–205
105. Magnani F, Pappas CG, Crook T, Magafa V, Cordopatis P, Ishiguro S et al (2014) Electronic
sculpting of ligand-GPCR subtype selectivity: the case of angiotensin II. ACS Chem Biol
9:1420–1425
106. Kruse AC, Kobilka BK, Gautam D, Sexton PM, Christopoulos A, Wess J (2014) Muscarinic
acetylcholine receptors: novel opportunities for drug development. Nat Rev Drug Discov
13:549–560
107. Katritch V, Cherezov V, Stevens RC (2012) Diversity and modularity of G protein-coupled

receptor structures. Trends Pharmacol Sci 33:17–27
108. Conn PJ, Christopoulos A, Lindsley CW (2009) Allosteric modulators of GPCRs: a novel
approach for the treatment of CNS disorders. Nat Rev Drug Discov 8:41–54
109. Conn PJ, Lindsley CW, Meiler J, Niswender CM (2014) Opportunities and challenges in the
discovery of allosteric modulators of GPCRs for treating CNS disorders. Nat Rev Drug
Discov 13:692–708
110. Urwyler S (2011) Allosteric modulation of family C G-protein-coupled receptors: from
molecular insights to therapeutic perspectives. Pharmacol Rev 63:59–126
111. Lane JR, Sexton PM, Christopoulos A (2013) Bridging the gap: bitopic ligands of G-protein-
coupled receptors. Trends Pharmacol Sci 34:59–66
112. Kamal M, Jockers R (2009) Bitopic ligands: all-in-one orthosteric and allosteric. F1000 Biol
Rep 1:77
113. Valant C, Robert Lane J, Sexton PM, Christopoulos A (2012) The best of both worlds? Bitopic
orthosteric/allosteric ligands of G protein-coupled receptors. Annu Rev Pharmacol Toxicol
52:153–178
114. Charifson PS, Bowen JP, Wyrick SD, Hoffman AJ, Cory M, McPhail AT et al (1989)
Conformational analysis and molecular modeling of 1-phenyl-, 4-phenyl-, and 1-benzyl-
1,2,3,4-tetrahydroisoquinolines as D1 dopamine receptor ligands. J Med Chem 32:2050–2058
115. Mottola DM, Laiter S, Watts VJ, Tropsha A, Wyrick SD, Nichols DE et al (1996) Confor-
mational analysis of D1 dopamine receptor agonists: pharmacophore assessment and receptor
mapping. J Med Chem 39:285–296
116. Wilcox RE, Tseng T, Brusniak MY, Ginsburg B, Pearlman RS, Teeter M et al (1998)
CoMFA-based prediction of agonist affinities at recombinant D1 vs D2 dopamine receptors.
J Med Chem 41:4385–4399
117. Tonani R, Dunbar J Jr, Edmonston B, Marshall GR (1987) Computer-aided molecular
modeling of a D2-agonist dopamine pharmacophore. J Comput Aided Mol Des 1:121–132
118. Palczewski K, Kumasaka T, Hori T, Behnke CA, Motoshima H, Fox BA et al (2000) Crystal
structure of rhodopsin: a G protein-coupled receptor. Science 289:739–745
119. Archer E, Maigret B, Escrieut C, Pradayrol L, Fourmy D (2003) Rhodopsin crystal: new
template yielding realistic models of G-protein-coupled receptors? Trends Pharmacol Sci
24:36–40
120. Bissantz C, Bernard P, Hibert M, Rognan D (2003) Protein-based virtual screening of chemical
databases. II. Are homology models of G-protein coupled receptors suitable targets? Proteins
50:5–25
121. Mobarec JC, Sanchez R, Filizola M (2009) Modern homology modeling of G-protein coupled
receptors: which structural template to use? J Med Chem 52:5207–5216
122. Cherezov V, Rosenbaum DM, Hanson MA, Rasmussen SG, Thian FS, Kobilka TS et al
(2007) High-resolution crystal structure of an engineered human beta2-adrenergic G protein-
coupled receptor. Science 318:1258–1265
123. Wacker D, Fenalti G, Brown MA, Katritch V, Abagyan R, Cherezov V et al (2010) Con-
served binding mode of human beta2 adrenergic receptor inverse agonists and antagonist
revealed by X-ray crystallography. J Am Chem Soc 132:11443–11445
124. Jaakola VP, Griffith MT, Hanson MA, Cherezov V, Chien EY, Lane JR et al (2008) The 2.6
angstrom crystal structure of a human A2A adenosine receptor bound to an antagonist.
Science 322:1211–1217
125. Xu F, Wu H, Katritch V, Han GW, Jacobson KA, Gao ZG et al (2011) Structure of an agonist-
bound human A2A adenosine receptor. Science 332:322–327
126. Wu B, Chien EY, Mol CD, Fenalti G, Liu W, Katritch V et al (2010) Structures of the CXCR4
chemokine GPCR with small-molecule and cyclic peptide antagonists. Science 330:1066–1071
127. Chien EY, Liu W, Zhao Q, Katritch V, Han GW, Hanson MA et al (2010) Structure of the
human dopamine D3 receptor in complex with a D2/D3 selective antagonist. Science
330:1091–1095
128. Shimamura T, Shiroishi M, Weyand S, Tsujimoto H, Winter G, Katritch V et al (2011)

Structure of the human histamine H1 receptor complex with doxepin. Nature 475:65–70
129. Hanson MA, Roth CB, Jo E, Griffith MT, Scott FL, Reinhart G et al (2012) Crystal structure
of a lipid G protein-coupled receptor. Science 335:851–855
130. Haga K, Kruse AC, Asada H, Yurugi-Kobayashi T, Shiroishi M, Zhang C et al (2012)
Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist. Nature
482:547–551
131. Kruse AC, Hu J, Pan AC, Arlow DH, Rosenbaum DM, Rosemond E et al (2012) Structure
and dynamics of the M3 muscarinic acetylcholine receptor. Nature 482:552–556
132. Manglik A, Kruse AC, Kobilka TS, Thian FS, Mathiesen JM, Sunahara RK et al (2012) Crystal
structure of the micro-opioid receptor bound to a morphinan antagonist. Nature 485:321–326
133. Wu H, Wacker D, Mileni M, Katritch V, Han GW, Vardy E et al (2012) Structure of the
human kappa-opioid receptor in complex with JDTic. Nature 485:327–332
134. Rasmussen SG, DeVree BT, Zou Y, Kruse AC, Chung KY, Kobilka TS et al (2011) Crystal
structure of the beta2 adrenergic receptor-Gs protein complex. Nature 477:549–555
135. Warne T, Serrano-Vega MJ, Baker JG, Moukhametzianov R, Edwards PC, Henderson R et al
(2008) Structure of a beta1-adrenergic G-protein-coupled receptor. Nature 454:486–491
136. Dore AS, Robertson N, Errey JC, Ng I, Hollenstein K, Tehan B et al (2011) Structure of the
adenosine A(2A) receptor in complex with ZM241385 and the xanthines XAC and caffeine.
Structure 19:1283–1293
137. Chun E, Thompson AA, Liu W, Roth CB, Griffith MT, Katritch V et al (2012) Fusion partner
toolchest for the stabilization and crystallization of G protein-coupled receptors. Structure
20:967–976
138. Chaudhari R, Heim AJ, Li Z (2015) Improving homology modeling of G-protein coupled
receptors through multiple-template derived conserved inter-residue interactions. J Comput
Aided Mol Des 29:413–420
139. Latek D, Pasznik P, Carlomagno T, Filipek S (2013) Towards improved quality of GPCR
models by usage of multiple templates and profile-profile comparison. PLoS One 8:e56742
140. Perry SR, Xu W, Wirija A, Lim J, Yau MK, Stoermer MJ et al (2015) Three homology models
of PAR2 derived from different templates: application to antagonist discovery. J Chem Inf
Model 55:1181–1191
141. Levit A, Barak D, Behrens M, Meyerhof W, Niv MY (2012) Homology model-assisted
elucidation of binding sites in GPCRs. Methods Mol Biol 914:179–205
142. Rodriguez D, Ranganathan A, Carlsson J (2014) Strategies for improved modeling of GPCR-
drug complexes: blind predictions of serotonin receptors bound to ergotamine. J Chem Inf
Model 54:2004–2021
143. Cavasotto CN, Palomba D (2015) Expanding the horizons of G protein-coupled receptor
structure-based ligand discovery and optimization using homology models. Chem Commun
(Camb) 51:13576–13594
144. Bremner JB, Coban B, Griffith R (1996) Pharmacophore development for antagonists at
alpha 1 adrenergic receptor subtypes. J Comput Aided Mol Des 10:545–557
145. Bremner JB, Coban B, Griffith R, Groenewoud KM, Yates BF (2000) Ligand design for
alpha1 adrenoceptor subtype selective antagonists. Bioorg Med Chem 8:201–214
146. Sleight AJ, Koek W, Bigg DCH (1993) Binding of antipsychotic drugs at α1A- and α1B-
adrenoceptors: risperidone is selective for the α1B-adrenoceptors. Eur J Pharmacol 238:407–410
147. Wetzel JM, Salon SA, Tamm JA, Forray C, Craig D, Nakanishi H et al (1996) Modeling and
mutagenesis of the human alpha 1a-adrenoceptor: orientation and function of transmembrane
helix V sidechains. Receptors Channels 4:165–177
148. Barbaro R, Betti L, Botta M, Corelli F, Giannaccini G, Maccari L et al (2001) Synthesis,
biological evaluation, and pharmacophore generation of new pyridazinone derivatives with
affinity toward α1- and α2-adrenoceptors1. J Med Chem 44:2118–2132
149. Sinha N, Jain S, Saxena AK, Anand N, Saxena RM, Dubey MP et al (2000). Methods for
preparing 1-[4-arylpiperazin-1-yl]-3-[2-oxopyrrolidin/piperidin-1-yl] propanes. Google Patents
150. Sinha N, Jain S, Saxena AK, Anand N, Saxena RM, Dubey MP et al (2000). 1-[4-arylpiperazin-
1-yl]-3-[2-oxopyrrolidin/piperidin-1-yl]propanes and their use in medical treatments. Google
Patents
151. Li M-Y, Tsai K-C, Xia L (2005) Pharmacophore identification of α1A-adrenoceptor antag-
onists. Bioorg Med Chem Lett 15:657–664
152. Li M, Fang H, Du L, Xia L, Wang B (2008) Computational studies of the binding site of
alpha1A-adrenoceptor antagonists. J Mol Model 14:957–966
153. Waugh DJ, Gaivin RJ, Zuscik MJ, Gonzalez-Cabrera P, Ross SA, Yun J et al (2001) Phe-308
and Phe-312 in transmembrane domain 7 are major sites of alpha 1-adrenergic receptor
antagonist binding. Imidazoline agonists bind like antagonists. J Biol Chem 276:25366–25371
154. Ahmed M, Hossain M, Bhuiyan MA, Ishiguro M, Tanaka T, Muramatsu I et al (2008) Mu-
tational analysis of the alpha 1a-adrenergic receptor binding pocket of antagonists by radioligand
binding assay. Biol Pharm Bull 31:598–601
155. Evers A, Klabunde T (2005) Structure-based drug discovery using GPCR homology modeling:
successful virtual screening for antagonists of the alpha1A adrenergic receptor. J Med Chem
48:1088–1097
156. Hamaguchi N, True TA, Saussy DL Jr, Jeffs PW (1996) Phenylalanine in the second membrane-
spanning domain of alpha 1A-adrenergic receptor determines subtype selectivity of dihydro-
pyridine antagonists. Biochemistry 35:14312–14317
157. Hamaguchi N, True TA, Goetz AS, Stouffer MJ, Lybrand TP, Jeffs PW (1998) Alpha
1-adrenergic receptor subtype determinants for 4-piperidyl oxazole antagonists. Biochemis-
try 37:5730–5737
158. Evers A, Hessler G, Matter H, Klabunde T (2005) Virtual screening of biogenic amine-
binding G-protein coupled receptors: comparative evaluation of protein- and ligand-based
virtual screening protocols. J Med Chem 48:5448–5465
159. MacDougall IJ, Griffith R (2006) Selective pharmacophore design for alpha1-adrenoceptor
subtypes. J Mol Graph Model 25:146–157
160. Maı̈ga A, Dupont M, Blanchet G, Marcon E, Gilquin B, Servent D et al (2014) Molecular
exploration of the α1A-adrenoceptor orthosteric site: binding site definition for epinephrine,
HEAT and prazosin. FEBS Lett 588:4613–4619
161. Chen J, Campbell AP, Urmi KF, Wakelin LPG, Denny WA, Griffith R et al (2014) Human
α1-adrenoceptor subtype selectivity of substituted homobivalent 4-aminoquinolines. Bioorg
Med Chem 22:5910–5916
162. Pandey N, Yadav M, Nayarisseri A, Ojha M, Prajapati J, Gupta S (2013) Cross evaluation of
different classes of alpha-adrenergic receptor antagonists to identify overlapping pharmaco-
phoric requirements. J Pharm Res 6:173–178
163. Gupta AK, Saxena AK (2010) 3D-QSAR CoMFA and CoMSIA studies on a set of diverse
α1a-adrenergic receptor antagonists. Med Chem Res 20:1455–1464
164. Maciejewska D, Żołek T, Herold F (2006) CoMFA methodology in structure-activity anal-
ysis of hexahydro- and octahydropyrido[1,2-c]pyrimidine derivatives based on affinity
towards 5-HT1A, 5-HT2A and α1-adrenergic receptors. J Mol Graph Model 25:353–362
165. Li M, Xia L (2007) Rational design, synthesis, biologic evaluation, and structure–activity
relationship studies of novel 1-indanone α1-adrenoceptor antagonists. Chem Biol Drug Des
70:461–464
166. Thobois S (2006) Proposed dose equivalence for rapid switch between dopamine receptor
agonists in Parkinson’s disease: a review of the literature. Clin Ther 28:1–12
167. Taravini IR, Larramendy C, Gomez G, Saborido MD, Spaans F, Fresno C et al (2016) Con-
trasting gene expression patterns induced by levodopa and pramipexole treatments in the rat
model of Parkinson’s disease. Neuropharmacology 101:576–589
168. Ravenscroft P, Chalon S, Brotchie JM, Crossman AR (2004) Ropinirole versus L-DOPA
effects on striatal opioid peptide precursors in a rodent model of Parkinson’s disease: impli-
cations for dyskinesia. Exp Neurol 185:36–46
169. Freeman HS, McDermed JD (1982) Chemical regulation of biological mechanisms. Royal
Society of Chemistry, London, pp 154–165
170. McDermed JD, Freeman HS, Ferris RM (1979) Enantioselective binding of (+) and (-)
2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalenes and related agonists to dopamine
receptors. In: Usdin E (ed) Catacholamines: basic and clinical frontiers, vol 1. Pergamon
Press, New York, NY, p 568
171. Mewshaw RE, Kavanagh J, Stack G, Marquis KL, Shi X, Kagan MZ et al (1997) New
generation dopaminergic agents. 1. Discovery of a novel scaffold which embraces the D2 ago-
nist pharmacophore. Structure-activity relationships of a series of 2-(aminomethyl)chromans.
J Med Chem 40:4235–4256
172. Seeman P (1980) Brain dopamine receptors. Pharmacol Rev 32:229–313
173. McDermed JD, McKenzie GM, Freeman HS (1976) Synthesis and dopaminergic activity of
(+-)-, (+)-, and (-)-2-dipropylamino-5-hydroxy-1,2,3,4-tetrahydronaphthalene. J Med Chem
19:547–549
174. Freeman H, McDermed J (1982) Chemical regulation of biological mechanisms. In: Proceedings
of the 1st Medicinal Chemistry Symposium, Cambridge, England, Sept 1981 (special publica-
tion/Royal Society of Chemistry ISSN 0260-6291; No 42)
175. McDermed J, Freeman H, Ferris R (1979) In: Usdin E, Kopin I, Barchas J (eds) Chemical
regulation of biological mechanisms, vol 1. Pergamon Press, New York, NY, p 568
176. Hjorth S, Carlsson A, Wikstr€om H, Lindberg P, Sanchez D, Hacksell U et al (1981) 3-PPP,
a new centrally acting DA-receptor agonist with selectivity for autoreceptors. Life Sci
28:1225–1238
177. Hjorth S, Carlsson A, Clark D, Svensson K, Wikstr€om H, Sanchez D et al (1983) Central
dopamine receptor agonist and antagonist actions of the enantiomers of 3-PPP. Psychophar-
macology (Berl) 81:89–99
178. Riffee W, Wilcox R, Smith R, Davis P, Brubaker A (1981) Proceedings of a satellite sympo-
sium to the 8th International Congress of Pharmacology, Okayama, Japan. Pergamon Press,
New York, NY
179. Wikstroem H, Sanchez D, Lindberg P, Arvidsson L-E, Hacksell U, Johansson A et al (1982)
Monophenolic octahydrobenzo[f]quinolines: central dopamine- and serotonin-receptor stim-
ulating activity. J Med Chem 25:925–931
180. Wikstroem H, Andersson B, Sanchez D, Lindberg P, Arvidsson LE, Johansson AM et al
(1985) Resolved monophenolic 2-aminotetralins and 1,2,3,4,4a,5,6,10b-octahydrobenzo[f]
quinolines: structural and stereochemical considerations for centrally acting pre- and post-
synaptic dopamine-receptor agonists. J Med Chem 28:215–225
181. Wikstroem H, Sanchez D, Lindberg P, Hacksell U, Arvidsson LE, Johnsson AM et al (1984)
Resolved 3-(3-hydroxyphenyl)-N-n-propylpiperidine and its analogs: central dopamine re-
ceptor activity. J Med Chem 27:1030–1036
182. Wikstrom H, Andersson B, Sanchez D, Lindberg P, Arvidsson LE, Johansson AM et al
(1985) Resolved monophenolic 2-aminotetralins and 1,2,3,4,4a,5,6,10b-octahydrobenzo[f]
quinolines: structural and stereochemical considerations for centrally acting pre- and post-
synaptic dopamine-receptor agonists. J Med Chem 28:215–225
183. Liljefors T, Wikstrom H (1986) A molecular mechanics approach to the understanding of
presynaptic selectivity for centrally acting dopamine receptor agonists of the phenylpipe-
ridine series. J Med Chem 29:1896–1904
184. Liljefors T, Bogeso KP, Hyttel J, Wikstrom H, Svensson K, Carlsson A (1990) Pre- and
postsynaptic dopaminergic activities of indolizidine and quinolizidine derivatives of
3-(3-hydroxyphenyl)-N-(n-propyl)piperidine (3-PPP). Further developments of a dopamine
receptor model. J Med Chem 33:1015–1022
185. Chidester CG, Lin CH, Lahti RA, Haadsma-Svensson SR, Smith MW (1993) Comparison of
5-HT1A and dopamine D2 pharmacophores. X-ray structures and affinities of conforma-
tionally constrained ligands. J Med Chem 36:1301–1315
186. Seeman P, Westman K, Protiva M, Jilek J, Jain PC, Saxena AK et al (1979) Neuroleptic
receptors: stereoselectivity for neuroleptic enantiomers. Eur J Pharmacol 56:247–251
187. Saxena AK, Singh HK, Dhawan BN, Anand N (1986). A process for the synthesis of cis-4-
substituted 1,2,3,4,4a,5,6,11c-octahydro-7H-pyrido(2,3-c)carbazoles as potential dopaminer-
gic agents. Indian Patent 167494
188. Saxena AK, Singh HK, Dhawan BN, Anand N (1986). A process for the synthesis of 1-alkyl
substituted 1,2,3,4,4a,5,11,11a-octahydro-6H-pyrido(3,2-b)carbazoles. Indian Patent 167492
189. Saxena AK, Singh HK, Dhawan BN, Anand N (1986). A process for the synthesis of cis-1-
methyl 1,2,3,4,4a,5,11,11a-octahydro-6H-pyrido(3,2-b)carbazole. Indian Patent 165919
190. Saxena AK, Singh HK, Dhawan BN, Anand N (1986). A process for the synthesis of cis-4-alkyl
substituted 1,2,3,4,4a,5,6,11c-octahydro-7H-pyrido(2,3-c)carbazole. Indian Patent 167493
191. Mehta P, Kumar Y, Saxena AK, Gulati AK, Singh HK, Anand N (1991) Synthesis of cis &
trans 1-substituted 1,2,3,4,4a,5,11,11a-octahydro-6H-pyrido(3,2-b)carbazoles, 4-substituted-
1,2,3,4,4a,5,6,11c-octahydro-7H-pyrido(2,3-c)-carbazoles, cis-4-methyl-1,2,3,4,4a,5,6,12b-
octahydro-7H-pyrido(2,3-c)-acridine & cis-1-methyl-1,2,3,4,-4a,5,12,12a-octahydro-6H-pyrido
(3,2-b)acridine – a new class of potential antiparkinsonian agents. Indian J Chem 213–221
192. Krogsgaard-Larsen N, Harpsoe K, Kehler J, Christoffersen CT, Brosen P, Balle T (2014)
Revision of the classical dopamine D2 agonist pharmacophore based on an integrated
medicinal chemistry, homology modelling and computational docking approach. Neurochem
Res 39:1997–2007
193. Malo M, Brive L, Luthman K, Svensson P (2010) Selective pharmacophore models of dopa-
mine D(1) and D(2) full agonists based on extended pharmacophore features. ChemMedChem
5:232–246
194. Malo M, Brive L, Luthman K, Svensson P (2012) Investigation of D(2) receptor–agonist
interactions using a combination of pharmacophore and receptor homology modeling.
ChemMedChem 7:471–482
195. Saxena M, Gaur S, Prathipati P, Saxena AK (2006) Synthesis of some substituted pyrazino-
pyridoindoles and 3D QSAR studies along with related compounds: piperazines, piperidines,
pyrazinoisoquinolines, and diphenhydramine, and its semi-rigid analogs as antihistamines (H1).
Bioorg Med Chem 14:8249–8258
196. ter Laak AM, Venhorst J, Donne-Op den Kelder GM, Timmerman H (1995) The histamine
H1-receptor antagonist binding site. A stereoselective pharmacophoric model based upon
(semi-)rigid H1-antagonists and including a known interaction site on the receptor. J Med
Chem 38:3351–3360
197. Saxena AK, Alam I, Dixit A, Saxena M (2008) Internet resources in GPCR modelling. SAR
QSAR Environ Res 19:11–25
198. Saxena M, Bhunia SS, Saxena AK (2012) Docking studies of novel pyrazinopyridoindoles
class of antihistamines with the homology modelled H(1)-receptor. SAR QSAR Environ Res
23:311–325
199. Saxena AK, Dhaon MK, Ram S, Saxena M, Jain PC, Patnaik GK, Anand N (1983) Synthesis
& QSAR in 2-substituted 1,2,3,4,6,7,12,12a,-Octahydropyrazino[20 ,10 :6,1]pyrido[3,4-b]
indoles-a new class of H1-antagonists. Ind J Chem 22B:1224–1232
Top Med Chem (2019) 30: 163–180
DOI: 10.1007/7355_2017_14
Biased Agonist Pharmacochaperones:

Small Molecules in the Toolbox
for Selectively Modulating GPCR Activity
Bernard Mouillac and Christiane Mendre
Abstract In recent years, biased agonists as well as pharmacological chaperones

have demonstrated the potential to harness G protein-coupled receptor signaling
and trafficking and have collectively opened new possibilities in G protein-coupled
receptor drug discovery. Combining pharmacological chaperoning and biased
agonism properties into a unique given molecule would be of high therapeutic
interest in many human diseases resulting from G protein-coupled receptor muta-
tion and misfolding. This strategy perfectly applies to congenital nephrogenic
diabetes insipidus which is a typical conformational disease. In most of the cases,
it is associated with inactivating mutations of the renal arginine vasopressin V2
receptor leading to misfolding and intracellular retention of the receptor, causing
the inability of patients to concentrate their urine in response to the antidiuretic
hormone. Cell-permeable pharmacological chaperones have been successfully
challenged to restore plasma membrane localization of the receptor mutants and
to rescue their function. Interestingly, different classes of specific ligands such as
antagonists, agonists, as well as biased agonists of the V2 receptor have proven their
usefulness as efficient pharmacological chaperones. These compounds, and partic-
ularly small-molecule-biased agonists which only trigger the V2-induced Gs
protein-dependent signaling pathway, represent a potential therapeutic treatment
of this X-linked genetic pathology.
Keywords Antidiuretic hormone, Biased agonist, Congenital nephrogenic

diabetes insipidus, Pharmacological chaperone, Therapeutic rescue, Tolvaptan,
V2 vasopressin receptor, Vaptans
B. Mouillac (*) and C. Mendre

Institut de Génomique Fonctionnelle, CNRS, INSERM, Université de Montpellier, 34094
Montpellier, France
e-mail: bernard.mouillac@igf.cnrs.fr
164 B. Mouillac and C. Mendre
Contents
1 G Protein-Coupled Receptor Ligands with “Novel” Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
1.1 Biased Agonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
1.2 Pharmacological Chaperones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
2 The X-Linked Genetic Disease Congenital Nephrogenic Diabetes Insipidus (cNDI): The
V2R as a Target for PC Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
2.1 The Pathology of cNDI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
2.2 Pharmacological Chaperone Treatment: Antagonists First . . . . . . . . . . . . . . . . . . . . . . . . . . 170
3 Biased Agonist Pharmacochaperones: Ideal Therapeutics for Treating cNDI? . . . . . . . . . . . 172
3.1 Agonists Versus Antagonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
3.2 Biased Agonists Versus Agonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174
4 Perspectives: Insights from Structural Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
Abbreviations
3D Three-dimensional
AQP2 Aquaporin-2
AVP Arginine vasopressin
cAMP Cyclic adenosine monophosphate
cNDI Congenital nephrogenic diabetes insipidus
ER Endoplasmic reticulum
FDA US food and drug administration
GnRHR Gonadotropin-releasing hormone receptor
Gs G protein subunit αs
LSD Lysosomal storage disorder
OT Oxytocin
PC Pharmacological chaperone, pharmacochaperone, pharmacoperone
PCT Pharmacological chaperone therapy
TM Transmembrane
V2R Vasopressin type 2 receptor
1 G Protein-Coupled Receptor Ligands with “Novel”

Properties
1.1 Biased Agonists
The discovery and the characterization of ligand-biased signaling at G protein-

coupled receptors (GPCRs) from various classes has completely changed the
classical concepts of receptor pharmacology [1–4]. Classically, the activity of
Biased Agonist Pharmacochaperones: Small Molecules in the Toolbox for. . . 165
each GPCR was associated with only one major signaling pathway, in general a
specific coupling to a unique G protein and generation of a second messenger
(inositol phosphates, calcium, cAMP, for instance). If considering this single
downstream outcome, GPCR ligands were classified as agonists (able to generate
a maximal response), partial agonists (able to induce a submaximal response at
saturating concentration), antagonists (inhibitors of agonist response but without
intrinsic activity), or inverse agonists (ligands that can decrease or suppress basal
receptor activity). This view evolved rapidly, taking into account that GPCRs can
couple to several different G proteins, allowing a single receptor to engage multiple
signaling pathways simultaneously [5, 6], and that structurally distinct ligands can
activate the same GPCR in different ways [7–9]. In other words, a GPCR possesses
different active states, and ligand structure can “bias” downstream signaling.
Moreover, it also became evident that ligand-activated GPCRs can engage G
protein-independent signaling pathways, for instance, through the activation of
β-arrestins [10–12] whose primarily role was clearly defined in desensitization of
G protein-dependent signaling. Whereas second messengers generated via G
protein-dependent activation of enzymatic effectors account for most of the classi-
cal short-term consequences of GPCR activation, arrestin-mediated signals appear
to perform numerous functions on a much longer time scale (up to several hours),
among them protein synthesis, cell migration, cytoskeletal rearrangement, and cell
proliferation to apoptosis [13]. Thus, the discovery that GPCRs can elicit separable
G protein- and arrestin-dependent signaling pathways and that ligands can differ-
entially activate or inhibit one or the other process opened the way to a complete
pharmacological reassessment of known compounds and to the development of
novel ligands with unique properties, able to selectively modulate GPCR activity
and associated downstream cellular events. These properties have been referred to
as “biased agonism” or functional selectivity.
Either G protein-biased or β-arrestin-biased ligands have been identified. A full
G protein-biased agonist leads to robust coupling and activation of G proteins but
not interaction with β-arrestins, whereas a complete β-arrestin-biased agonist does
not promote G protein coupling but induces robust β-arrestin recruitment. These
notions uncovered a new paradigm in drug discovery that relies on the
pluridimensionality of GPCR signaling, with the aim to develop potential thera-
peutics with better efficacy and fewer adverse effects. Proof-of-concept studies
have demonstrated that both G protein and arrestin pathway-selective ligands can
promote beneficial effects in vivo while simultaneously antagonizing deleterious
ones. A few examples of drugs with biased properties toward β-adrenergic receptors
[14, 15], dopamine receptors [16], histamine receptors [17], the orphan GPR109a
receptor [18], opioid receptors [19], and angiotensin II receptors [20] are shown in
Table 1.
Table 1 Biased ligands used in vivo: their receptor target, in vitro activity, and beneficial
physiological advantages
Target
Receptor Biased ligand pathology In vitro activity Therapeutic advantages
β1-AR Carvedilol Cardiac arrhyth- β-Arrestin-biased Better cardioprotection
mia, heart agonist, no G pro-
failure tein signaling
β2-AR Salmeterol Respiratory dis- Gs-biased agonist, Long-acting
eases, asthma very low β-arrestin bronchodilatation
signaling
D2R Aripiprazole Psychiatric dis- β-Arrestin-biased Better antipsychotic
derivatives orders, agonist, no Gs pro- activity (mice)
schizophrenia tein signaling
H4R JNJ7777120, Allergies, G protein antago- Suppression of cough
VUF10214 asthma nist, nonselective through reduction airway
β-arrestin-biased inflammation (guinea
agonist pig)
GPR109a MK 0354 Lipid metabo- G protein-biased Reduced incidence of
lism, regulation agonist, no flushing
of FFA plasma β-arrestin signaling
levels
μ-OR Herkinorin, Pain Gi-biased agonist, Analgesic with less
TRV130 no β-arrestin adverse effects (consti-
signaling pation and respiratory
suppression)
AT1AR TRV027, SII Hypertension, Partial β-arrestin- Decreased blood pres-
acute heart biased agonist, full sure, improved
failure G protein cardiomyocyte
antagonist contraction
β1-AR β1-adrenergic receptor, β2-AR β2-adrenergic receptor, D2R dopamine type 2 receptor, H4R
histamine type 4 receptor, GPR109A orphan GPCR 109a, μ-OR μ-opioid receptor, AT1AR
angiotensin II type 1A receptor, FFA free fatty acid
1.2 Pharmacological Chaperones
Many genetic and neurodegenerative diseases in humans result from protein

misfolding and/or aggregation. These pathologies are classified as conformational
diseases [21, 22] in which misfolded proteins are rejected and misrouted by the
cellular quality control system, consequently being unable to play their physiolog-
ical roles. Many of the misfolded protein mutants responsible for these pathologies
are sequestered in an intracellular compartment (usually the endoplasmic reticu-
lum, ER), but can be rescued by chemical chaperones [23] or pharmacological
chaperones (PC). Contrary to chemical chaperones which are nonspecific small
organic compounds (glycerol, dimethyl sulfoxide, trimethyl-N-oxide), PC (also
termed pharmacochaperones or pharmacoperones) are specific small molecules
that bind to their protein targets to facilitate biogenesis and/or prevent/correct
misfolding [24–26]. These are usually hydrophobic structures that enter cells by
diffusion and rescue protein localization and function. The discovery and activity
of PC have also been extensively reviewed and discussed in recent years [27–
31]. Most of the PC target secretory pathway proteins include enzymes, trans-
porters, receptors (among them, many GPCRs), and ion channels. In addition, while
most PC have been used in vitro, the demonstration of their efficacy in animal
models and humans established that their use holds great promise as novel thera-
peutic strategy.
Lysosomal enzymes are best examples of protein targets that can be functionally
rescued in vivo by PC. Lysosomal storage disorders (LSDs) are metabolic diseases
caused by mutations in genes that encode proteins involved in different lysosomal
functions, in most cases enzymes, including acid-β-glucosidase (Gaucher disease),
α-galactosidase A (Fabry disease), and many other acidic hydrolases [32, 33]. The
biological and clinical interest of LSD is high, and different therapeutic approaches
have been developed to treat these disorders [34]. The therapeutic approach that has
been most successful is enzyme replacement therapy [35]. This strategy is based on
the periodic intravenous administration of a manufactured enzyme that is taken up
into cells and delivered to lysosomes and can reduce substrate storage. Alternative
strategies also exist and are directed toward reducing the synthesis of substrates by
enhancing clearance of substrates from cells and tissues [36]. Recently, PC therapy
(PCT) for a number of LSDs has been evaluated in the clinic [34]. This is an
emerging approach based on small-molecule ligands that selectively bind and
stabilize mutant enzymes, increase their cellular levels, and improve lysosomal
trafficking and activity. The PC migalastat for treating Fabry disease [37] and
afegostat or ambroxol for treating Gaucher disease [38] are very promising thera-
peutic avenues. Indeed very recently, migalastat has been approved in Europe by
EMA (the European Medicines Agency) as the first PC therapeutic molecule [39].
Membrane proteins like receptors, transporters, and ion channels for which
three-dimensional (3D) folding is tightly controlled by the cellular quality control
system and that are targeted to the plasma membrane through the secretory pathway
are major targets for PCT. PCT is of particular interest for GPCRs, since mutations
in GPCRs are responsible for many human pathologies and GPCRs constitute the
largest class of membrane targets for a majority of currently marketed drugs (more
that 30% of FDA-approved drugs). For instance, the human gonadotropin-releasing
hormone receptor (GnRHR) has been a central focus of drug development, and
many useful compounds (agonists and antagonists) have been characterized for the
treatment of reproduction disorders [40–42]. PC of the GnRHR have shown effi-
cacy in cell culture systems but also in a small animal model, a knock-in
mouse expressing the GnRHR E90K mutant which causes hypogonadotropic
hypogonadism in humans [43]. Indeed, pulsatile PCT rescued the E90K receptor
plasma membrane localization and responsiveness of the endogenous natural ligand
gonadotropin-releasing hormone. Spermatogenesis, proteins associated with steroid
transport and steroidogenesis, and androgen levels were restored in mutant male
mice following the PCT. A PC action can be generalized to many intracellular-
retained misfolded mutant receptors from many GPCR families. A few examples of
PC targeting GnRHR [43], vasopressin receptors [25, 28, 44], calcium-sensing
Table 2 Representative pharmacological chaperones for GPCRs: their specific receptor and
target pathology, their in vitro efficacy, and their in vivo effects
Receptor PC Target pathology In vitro efficacy In vivo effects
GnRHR IN3 Hypogonadotropic Plasma membrane Restoration of
(antagonist) hypogonadism rescue, restoration of testis function
GnRH responsiveness (mouse model)
a
V2R SR121463 cNDI Plasma membrane
(inverse rescue, restoration of
agonist) AVP responsiveness
SR49059 cNDI Equivalent to Decrease in urine
(antagonist) SR121463 volume and water
intake (humans)
MCF com- cDNI Plasma membrane Not tested
pounds rescue, direct activa-
(biased tion of V2R
agonists)
b
CaSR NPS R-568 Hypocalciuric Plasma membrane
(allosteric hypercalcemia and functional rescue
agonist)
LHR Org 42599c Reproductive dysfunc- Plasma membrane Not available
(allosteric tions, infertility due to and functional rescue
agonist) Leydig cell hypoplasia
FSHR Org 41841c Reproductive dysfunc- Plasma membrane Not available
(allosteric tions, infertility and functional rescue
agonist)
CaSR calcium-sensing receptor, cNDI congenital nephrogenic diabetes insipidus, LHR luteinizing
hormone receptor, FSHR follicle-stimulating hormone receptor
a
SR121463 is a compound of the vaptan family and is named satavaptan. The ligand is efficient in
patients with dilutional hyponatremia by increasing serum sodium concentrations (DILIPO study
[48]). The vaptans may also have therapeutic potential for heart failure
b
The calcimimetic cinacalcet which has been developed through optimization of ligands such as
NPS R-568 and NPS R-467 is widely used in clinic for treating hyperparathyroidism
c
Org 42599 and Org 41841 are thienopyrimidine compounds
receptor [45], and luteinizing and follicle-stimulating hormone receptors [46, 47]
are shown in Table 2. Most of these PC have been shown to be useful in cellular
systems and still have to confirm proof of concept for in vivo protein rescue.
2 The X-Linked Genetic Disease Congenital Nephrogenic

Diabetes Insipidus (cNDI): The V2R as a Target for PC
Therapy
Combining biased agonist properties with pharmacochaperone activity would be a

fantastic strategy to develop small-molecule compounds for treating diseases
related to protein misfolding and for which drug-induced beneficial versus delete-
rious effect ratio has to be improved. The kidney arginine vasopressin (AVP)
type 2 receptor (V2R), which regulates water homeostasis, constitutes a major

target for voiding disorders, and also possesses a large variety of pharmacological
ligands and therapeutic compounds, can be considered as a model GPCR system
for challenging this strategy.
2.1 The Pathology of cNDI
Regulated water excretion by the kidney is crucial to preserve water homeostasis of

our body. The adjustment of water renal reabsorption, to respond to the increase in
blood osmolality (hypernatremia) or decrease in blood volume (hypovolemia),
mainly depends on the release of the antidiuretic hormone AVP from the pituitary
[49–51]. Binding of AVP to the V2R, a Gs protein-coupled receptor localized at the
basolateral membrane of the principal cells of the kidney collecting duct, results in
an intracellular cyclic adenosine monophosphate (cAMP)-dependent signaling
cascade of events. Among them, phosphorylation of the water channel aquaporin-
2 (AQP2) and its translocation from storage compartments to the apical surface of
the cells are of primary importance. Water from pro-urine which enters the cells
exits via aquaporin-3 and aquaporin-4 at the basolateral side, leading to water
reabsorption and urine concentration. Upon restoration of water balance, the level
of plasma AVP drops and AQP2 is internalized, leaving the apical membrane
watertight again.
Disorders that interfere with proper urine concentration can be life-threatening,
especially in children. One such disease is cNDI [52]. Indeed, cNDI is a rare
inherited disease, characterized by insensitivity of the kidney to AVP and absence
of water reabsorption. It results in polyuria and compensatory polydipsia and may
lead to severe dehydration and electrolyte imbalance (hypernatremia) in the case of
inadequate water supply.
The X-linked form of cDNI is caused by mutations in the V2R gene [53]. To
date, over 200 different V2R mutations have been described. V2R mutations are
divided into five different classes. Class II mutations (the most prevalent, more than
50%) are most frequently missense mutations (amino acid substitutions). These
mutations result into misfolded, trafficking-deficient receptors that do not reach the
plasma membrane of the basolateral side of the principal cells of the kidney. Indeed,
most of the mutants are retained in the endoplasmic reticulum (ER) and in the
ER-Golgi intermediate compartment. Consequently, V2R mutants are unable to
interact with circulating AVP [54]. These V2R mutants, rather than resulting in
nonfunctional proteins (mutants from classes III and IV), are intrinsically func-
tional, as demonstrated by overexpression in heterologous cell expression
systems [55].
Untreated adult cNDI patients may have a daily output of 15–20 L of highly
dilute urine. Newborn infants often suffer from hypernatremic dehydration with
symptoms of irritability, poor feeding, and weight gain. In addition, repeated
periods of brain dehydration may result in permanent brain damage, and mental
retardation and seizures can occur. The main strategy for treating cNDI patients
consists of a sufficient water supply to replace the urinary water loss, but this can
seriously impact on the quality of life due to excessive drinking and urine voiding.
Some diuretics, like hydrochlorothiazide, amiloride, or the cyclooxygenase inhib-
itor indomethacin, have been proven effective to reduce urine output by up to 50%
[56]. However, diuretics may affect the sodium and potassium balance in patients,
and therefore these treatments require tight monitoring of serum electrolytes and
osmolality.
Although understanding of cNDI from molecular and cell biological point of
view has largely increased since the cloning of the V2R gene [57–59], developing
alternative strategies to manage water homeostasis and induce antidiuresis in cNDI
patients is still obvious. The V2R is a “natural” target for establishing new forms of
therapies, and PC rescue of its function is a very elegant and specific approach.
2.2 Pharmacological Chaperone Treatment: Antagonists

First
Chemical chaperones, like glycerol and DMSO, were shown, for instance, to
correct mutants of the AQP2 water channel, as assessed by protein maturation,
cellular targeting, and water permeability [60]. Taking the concept of chemical
chaperones further, artificial mutants of the multidrug resistance P-glycoprotein-1,
a cell surface transporter which interacts with a panel of cytotoxic agents, were
functionally rescued. Indeed, ER-retained mutants were targeted to the plasma
membrane, and their functional rescue was demonstrated using specific substrates
or inhibitors like vinblastine, cyclosporin, or verapamil [24]. These compounds
were proposed to stabilize a specific native-like conformation of the transporter,
allowing its release from the ER quality control cell system.
The concept was applied to the mutants of V2R responsible for cNDI, based on
the idea that pharmacological ligands act by binding to and stabilizing specific
conformations of their receptors. Selective cell-permeant nonpeptidic V2R antag-
onists (which block the V2R in an inactive conformation) were assessed to check
whether they could facilitate the folding of mutant receptors that are retained in ER
and unable to interact with AVP [25]. Given that these antagonists are specific to
the V2R and that they perform chaperone-like activity, these compounds were
named PC for the first time [26]. The first antagonist (or inverse agonist) to be
used was SR121463, a selective high-affinity V2R ligand [61]. An overnight
treatment of the cells retaining different V2R mutants into an intracellular com-
partment converted precursor forms into fully glycosylated mature receptors that
were targeted to the cell surface. Once correctly localized at the plasma membrane,
these mutants were able to differentially bind AVP and produce a correlated cAMP
intracellular signal [25]. Interestingly, V2R membrane-impermeable peptidic
antagonists were unable to mimic the SR121463 effect, indicating a PC intracellu-
lar effect. The PC-driven V2R mutant rescue was not limited to SR121463, because
another nonpeptidic antagonist, VPA985, reproduced equivalent results. Since the

publication of this study, SR121463 was used to rescue a larger panel of V2R
mutants [62–65]. Different other antagonists (or inverse agonists) were tested for
their PC properties, such as the V1AR-selective SR49059 [65, 66], the mixed
V1A/V2R YM087 [28], and the two V2R-selective OPC31260 and OPC41061
[67]. Very importantly in some cases, the PC effect was also reproducible in
polarized renal cells where V2R mutants were appropriately targeted to the
basolateral membrane, the natural localization of the wild-type V2R [68]. In addi-
tion, because of their target specificity, the PC compounds are active at nanomolar
concentrations on cultured cells [67]. This is a tremendous advantage compared to
Table 3 Different classes of V2R pharmacological chaperones: their efficiency in rescuing

mutant receptor plasma membrane localization and function
Intrinsic V2R mutants with plasma V2R mutants with
PCa activity Specificity membrane rescue functional rescue
SR121463 Inverse V2R L59P, Δ62-64, L83Q, All
(satavaptan, agonist Y128S, S167L, A294P,
Aquilda®) P322H, R337X
ΔV278, L292P, R337X L292P, R337X
S167T, V206D Both
Δ62-64, S167T, C319Y, Not tested
P322S, W323H
L44P, L59P, Y128S, A294P, L44P, A294P,
R337X R337X
V88M No affinity for
AVP
L83Q L83Q
SR49059 Antagonist V1AR L44P, I130F, S167T, Δ62- All
(relcovaptan) 64, R137H, W164S, C319Y
OPC41061 Antagonist V2R L44P, Δ62-64, R113W, L44P, I130F,
(tolvaptan, I130F, S167T, G201D, S167T
Samsca®) T204N, V206D
OPC31260 Inverse V2R L44P, Δ62-64, R113W, L44P, I130F,
(mozavaptan) agonist I130F, S167T, G201D, S167T
T204N, V206D
OPC51803 Agonist V2R None L44P, Y128S,
I130F, S167T,
Y280C, R337X
VA999088 and Agonists V2R None L44P, Y128S,
VA999089 I130F, S167T,
Y280C, R337X
MCF14, Biased V2R L44P, Y128S, A294P, L44P, A294P,
MCF18 and agonists R337X R337X
MCF57
a
Apart from SR49059 which is a selective antagonist for the AVP V1A receptor subtype, all other
ligands have been classified regarding their activity toward the V2R-induced Gs protein-dependent
signaling pathway. Interestingly, MCF compounds are Gs-biased agonists with no β-arrestin
signaling
chemical chaperones which are active at micromolar concentrations or even more.

Some prototypic PC for the V2R are presented in Table 3, as well as the different
cNDI mutants for which their beneficial effect was proven. Indeed, studies with
SR121463 [25, 26, 44, 62–65, 68]; SR49059 [66]; OPC41061 and OPC31260 [67];
OPC51803, VA999088, and VA999089 [69]; and MCF14, MCF18, and MCF57
[44] are listed.
Interestingly, the concept of PC developed for V2R mutants responsible for
cNDI was also studied with the V1A and V1B subtypes of the vasopressin receptor
family. SSR149415, which is a specific nonpeptidic antagonist for V1B, was
demonstrated to rescue plasma membrane localization and function of the V1B
mutant 341FN(X)2LL(X)3L350 [70]. In addition, the selective nonpeptidic antag-
onist SR49059 was shown to rescue the functional properties of surface-impaired
D148A/N/E mutants of the V1A receptor subtype [71]. To date, no more study has
been investigated using PC to rescue other artificial mutants of V1AR, V1BR, or the
vasopressin-related oxytocin receptor.
3 Biased Agonist Pharmacochaperones: Ideal

Therapeutics for Treating cNDI?
Using antagonists for rescuing function of the V2R and more generally GPCR
mutants responsible for inherited conformational diseases is somehow paradoxical.
First of all, because these antagonists specifically block (inhibit) their receptors,
they cannot directly stimulate receptor-associated signaling pathways. Regarding
patients who suffer from cNDI, the therapeutic beneficial effect would be
antidiuresis, through the activation of a cAMP-dependent signaling cascade and
particularly membrane translocation of AQP2. Indeed, using the PC antagonist
strategy, functional rescue of mutants of the V2R is a subtle balance between the
ability of the ligand to target cell surface expression of the mutants and its
possibility to be displaced by endogenous AVP for receptor activation [72]. In
this regard, considering the antagonist affinity is an important feature for this
challenge, and therefore low-affinity antagonists (those which are easily displaced
by AVP) may possess a higher clinical value [73]. However, the efficiency of such
low-affinity antagonist ligands in rescuing receptor function is lower than that of
high-affinity ligands (the higher the affinity, the better the rescue). Moreover, high
concentrations of low-affinity antagonists to be administered for clinical efficiency
might lead to unwanted side effects in patients (see paragraph below). In addition,
compound-intrinsic factors other than affinity may influence their capacity to confer
functional rescue and their extent to be displaced by AVP, like their localization in
the binding pocket of the V2R, their intrinsic activity, or their lipophilic value.
Overall, it seems that concerning cNDI patients, the high-affinity OPC31260
(mozavaptan) and OPC41061 (tolvaptan) nonpeptidic antagonists would combine

the best clinical potential, in terms of cell surface rescue, low concentration to be
used, and efficient displacement by AVP [67, 74].
Interestingly, a small-scale short-term clinical trial has been set up using
SR49059, a low-affinity antagonist for V2R (it is specific for the V1A receptor
subtype) [28]. Different patients with R137H, W164S, or Δ62-64 mutations were
treated with the nonpeptidic antagonist. Interestingly, SR49059 significantly
decreased the 24-h urine volume and water intake, demonstrating a successful PC
behavior in vivo. However, because of a potential interference with the cytochrome
P450 metabolic pathway (hepatic toxicity), the development of this molecule was
discontinued during clinical phase II. To date, no other clinical trial has been
developed for cNDI patients. Recently, the cell-permeable nonpeptidic antagonist
OPC41061 (tolvaptan, Samsca®) has been approved in the USA and Europe for the
treatment of hyponatremia, for instance, in the syndrome of inappropriate
antidiuretic hormone secretion and congestive heart failure [75]. This ligand may
be of high therapeutic value for novel clinical trials for treating cNDI in the future,
provided that it can be displaced by AVP in vivo.
3.1 Agonists Versus Antagonists
Other pharmacological approaches to treat cNDI patients may have a higher

potential than the antagonist PC strategy. Indeed, agonists and biased agonists of
the V2R may prove to be of higher clinical value. Theoretically, PC agonists
possess advantages over antagonists since they are able to directly stimulate
the V2R and induce receptor-associated signaling pathways. In this case,
V2R-selective high-affinity ligands are likely to be the most appropriate to effi-
ciently rescue plasma membrane targeting of the mutants and their direct activation.
However, agonists also promote V2R internalization, and consequently a decrease
in the cAMP signal, a phenomenon that could reduce the beneficial effects of these
compounds. Two agonist-based alternative approaches have been described and
constitute very promising therapeutic strategies.
Recent investigations indicated that ER-retained but intrinsically functional
V2R mutants can be activated intracellularly by different agonists [69]. The acti-
vation surprisingly leads to sufficient cAMP increase to induce AQP2 to be
translocated to the apical membrane of renal polarized cells. The recently devel-
oped nonpeptidic agonist OPC51803 and two novel agonists VA999088 and
VA999089 were used in this study to rescue the function of a panel of different
cNDI V2R mutants (see Table 3). In contrast to PC-assisted receptor folding and
rescue, the localization and maturation of the cNDI mutants did not change upon
ligand binding and receptor activation. Due to their structural features (small
lipophilic molecule compounds which have the ability to penetrate cell membranes
and reach the ER), it is surprising that these three ligands do not behave as PC
and likely induce plasma membrane targeting of the different V2R mutants.
Consequently, they cannot be classified as pharmacological chaperones but still

constitute promising future therapeutic candidates for cNDI clinical studies
[50]. Like for PC-based approaches, V2R rescue and intracellular receptor activa-
tion by OPC51803 and both VA compounds may only work for mutations that
affect folding or proper intracellular transport, but neither for highly truncated
receptors nor those that lost their Gs-dependent coupled signaling pathways.
These ligands may also be tested on a larger panel of cNDI mutants, in order to
check their PC potential properties.
3.2 Biased Agonists Versus Agonists
The discovery of biased agonist PC of the V2R is a novel therapeutic opportunity

for cNDI patients [44]. The V2R nonpeptidic agonists MCF14 (OPC23h), MCF18
(VNA932), and MCF57 possess only part of the AVP signaling properties. Indeed
(see Fig. 1), these molecules are full agonists of the Gs-dependent cAMP signal
(which is responsible for AQP2 translocation and water reabsorption) but do not
induce receptor internalization and arrestin-related signaling pathways (antagonists
for arrestin recruitment and associated events). On a therapeutic point of view, these
particular properties may lead to additional beneficial effects for cNDI patients, as
compared to common agonists. The ligands MCF14, MCF18, and MCF57 are high-
affinity agonists for V2R and are capable of inducing cNDI mutant receptor
maturation and translocation to the plasma membrane and can directly initiate a
cAMP response (they may act in synergy with circulating AVP in the case of in vivo
clinical trials). Functional rescue with MCF14, MCF18, and MCF57 was demon-
strated for different mutants of the V2R (see Table 3). These ligands are however
Agonists Antagonists / Biased Agonists

Inverse Agonists
OPC51803 SR121463 MCF
V2R V2R V2R
Gs arrestins Gs arrestins Gs arrestins
Fig. 1 The different classes of V2R pharmacological chaperones

not totally selective for the V2R, possessing a significant affinity for the V1AR and
the oxytocin (OT) receptor. Their pharmacological profile has thus to be improved
yet. They however constitute a novel class of PC. Indeed, the V2R-biased agonist
PC, able to generate a cAMP signal and acting as noninternalizing ligands, poten-
tially providing a long-lasting cellular response during drug administration, may
constitute ideal therapeutic compounds for treating cNDI [76].
4 Perspectives: Insights from Structural Studies
To develop new V2R-biased ligands with unique beneficial therapeutic effects and
no adverse effects (no activity on other V2R-induced signaling pathways but also
no activation of other AVP/OT receptors), rational drug development may be based
on biophysical and structural studies. In theory, to fully understand the structural
basis of biased signaling would be to crystallize a given receptor not only in its
inactive, active, and biased active conformations but also in complex with both
G protein and β-arrestin. In addition, crystallographic approaches have to be
complemented with dynamic studies of ligand-receptor-G protein/arrestin com-
plexes like NMR spectroscopy. Today, we are far from having such a complete
set of data for any given receptor, and no V2R 3D structure has been described to
date. Therefore, information on biased signaling molecular mechanisms can be
derived from other biophysical and structural approaches. Indeed, we used trypto-
phan fluorescence and lanthanide resonance energy transfer fluorescence to study
ligand-induced structural changes of the purified human V2R [77]. We compared
the effects of the reference unbiased agonist AVP to those of SR121463, a PC with
inverse agonist and partial agonist properties toward Gs and arrestin, respectively,
and of MC14, a PC with full Gs-biased properties. Decrease and increase in the
overall intrinsic tryptophan fluorescence were recorded using the SR121463 or
AVP/MCF14, respectively, indicating the existence of at least two different recep-
tor populations in response to inverse agonist or agonists of the V2R-dependent Gs
protein signaling pathway. Moreover, the introduction of lanthanide fluorescence
resonance energy transfer-based intramolecular sensors at transmembranes (TM) 6
and 7 and at the C-terminus of the V2R clearly demonstrated that β-arrestin- and
Gs-biased ligands differentially affected the average lifetime constant of the major
population of the receptor, indicating the existence of different conformational
states. Conformational movements of functional domains of the V2R relative to
each other are different depending on the biased ligands and consequently may
explain why different signaling pathways are activated or not. Indeed, movements
of the V2R TM 6 are involved in Gs signaling, whereas those of TM 7 and helix
8 are involved in β-arrestin recruitment.
Some other methodologies can also be used to study the structural basis of biased
signaling at GPCRs. For instance, bimane fluorescence in the ghrelin receptor
GHS-R1a was used to study ligand-induced conformational changes [78]. Indeed,
sensors were introduced in the second or the third intracellular loop, respectively, to
detect specific effects of biased and unbiased ligands on receptor conformations.

Interestingly, the JMV 3002 and JMV 3018 biased ligands for the G protein induced
smaller changes in bimane fluorescence compared to unbiased ligands, whereas the
SPA inverse agonist triggered a significant increase in fluorescence. These results
are in agreement with the presence of distinct conformations stabilized by func-
tionally different ligands. Chemical labeling coupled to 19F–NMR spectroscopy is
also a methodology that can help to decipher structural information in biased
signaling. It has been shown to demonstrate that the β-arrestin-biased ligand
carvedilol induces a unique conformational signature of the β2-AR [79]. Indeed,
introducing site-specific 19F-NMR labels in the structure of the β2-AR at cytoplas-
mic ends of TM 6 and 7 allowed to demonstrate that these regions adopt two major
conformational states and that carvedilol predominantly impact the conformation of
TM 7.
5 Conclusions
Over the last two decades, V2R-selective biased ligands and PC compounds have
been identified and pharmacologically characterized. Developing molecules which
combine PC properties and bias for Gs signaling pathway is a promising strategy for
treating cNDI and more generally of particular clinical interest in GPCR research.
In principle, V2R-specific PC have more desirable properties as therapeutics than
current nonspecific treatments like thiazides with indomethacin. Because the res-
cuing properties of these ligands have been analyzed to only a few misfolded
receptors, it would be important to investigate their PC properties on a larger
panel of V2R mutants (most of the mutants from class II are potential candidates
to be treated). Additionally, their Gs protein bias is also a major criteria to select
compounds that do not display β-arrestin recruitment, in order to favor beneficial
effects and abolish adverse effects. We anticipate that biased agonist PC are novel
small molecules in the toolbox that will become promising therapeutics and phar-
macological ligands useful for selectively modulating GPCR activity.
References
1. Galandrin S, Oligny-Longpré G, Bouvier M (2007) The evasive nature of drug efficacy:

implications for drug discovery. Trends Pharmacol Sci 28(8):423–430
2. Lutrell LM (2014) More than just a hammer: ligand “bias” and pharmaceutical discovery. Mol
Endocrinol 28(3):281–294
3. Luttrell LM, Maudsley S, Bohn LM (2015) Fulfilling the promise of “biased” G protein-
coupled receptor agonism. Mol Pharmacol 88(3):579–588
4. Reiter E, Ahn S, Shukla AK, et al (2012) Molecular mechanism of β-arrestin-biased agonism at
seven-transmembrane receptors. Annu Rev Pharmacol Toxicol 52:179–197
5. Laugwitz KL, Allgeier A, Offermanns S, et al (1996) The human thyrotropin receptor: a

heptahelical receptor capable of stimulating members of all four G protein families. Proc Natl
Acad Sci U S A 93(1):116–120
6. Offermanns S, Wieland T, Homann D, et al (1994) Transfected muscarinic acetylcholine
receptors selectively couple to Gi-type G proteins and Gq/11. Mol Pharmacol 45(5):890–898
7. Holloway AC, Qian H, Pipolo L, et al (2002) Side-chain substitutions within angiotensin II
reveal different requirements for signaling, internalization, and phosphorylation of type 1A
angiotensin receptors. Mol Pharmacol 61(4):768–777
8. Sagan S, Chassaing G, Pradier L, et al (1996) Tachykinin peptides affect differently the second
messenger pathways after binding to CHO-expressed human NK-1 receptors. J Pharmacol Exp
Ther 276(3):1039–1048
9. Takasu H, Gardella TJ, Luck MD, et al (1999) Amino-terminal modifications of human
parathyroid hormone (PTH) selectively alter phospholipase C signaling via the type
1 PTH receptor: implications for design of signal-specific PTH ligands. Biochemistry 38
(41):13453–13460
10. Ferguson SS (2001) Evolving concepts in G protein-coupled receptor endocytosis: the role in
receptor desensitization and signaling. Pharmacol Rev 53(1):1–24
11. Rajagopal S, Rajagopal K, Lefkowitz RJ (2010) Teaching old receptors new tricks: biasing
seven-transmembrane receptors. Nat Rev Drug Discov 9(5):373–386
12. Shenoy S, Lefkowitz RJ (2011) β-arrestin-mediated receptor trafficking and signal transduc-
tion. Trends Pharmacol Sci 32(9):521–533
13. Luttrell LM, Gesty-Palmer D (2010) Beyond desensitization: physiological relevance of
arrestin-dependent signaling. Pharmacol Rev 62(2):305–330
14. Carter AA, Hill SJ (2005) Characterization of isoprenaline- and salmeterol-stimulated inter-
actions between beta2-adrenoceptors and beta-arrestin 2 using beta-galactosidase complemen-
tation in C2C12 cells. J Pharmacol Exp Ther 315(2):839–848
15. Wisler JW, DeWire SM, Whalen EJ, et al (2007) A unique mechanism of beta-blocker action:
carvedilol stimulates beta-arrestin signaling. Proc Natl Acad Sci U S A 104(42):16657–16662
16. Chen X, Sassano MF, Zheng L, et al (2012) Structure-functional selectivity relationship
studies of β-arrestin-biased dopamine D2 receptor agonists. J Med Chem 55(16):7141–7153
17. Thurmond RL, Desai PJ, Dunford PJ, et al (2004) A potent and selective histamine H4 receptor
antagonist with anti-inflammatory properties. J Pharmacol Exp Ther 309(1):404–413
18. Semple G, Skinner PJ, Gharbaoui T, et al (2008) 3-(1H-tetrazol-5-yl)-1,4,5,6-tetrahydro-
cyclopentapyrazole (MK-0354): a partial agonist of the nicotinic acid receptor, G-protein
coupled receptor 109a, with antilipolytic but no vasodilatory activity in mice. J Med Chem
51(16):5101–5108
19. Groer CE, Tidgewell K, Moyer RA, et al (2007) An opioid agonist that does not induce
mu-opioid receptor-arrestin interactions or receptor internalization. Mol Pharmacol 71
(2):549–557
20. Violin JD, DeWire SM, Yamashita D, et al (2010) Selectively engaging β-arrestins at the
angiotensin II type 1 receptor reduces blood pressure and increases cardiac performance.
J Pharmacol Exp Ther 335(3):572–579
21. Chaudhuri TK, Paul S (2006) Protein-misfolding diseases and chaperone-based therapeutic
approaches. FEBS J 273(7):1331–1349
22. Cohen FE, Kelly LW (2003) Therapeutic approaches to protein-misfolding diseases. Nature
426(6968):905–909
23. Sato S, Ward CL, Krouse ME, et al (1996) Glycerol reverses the misfolding phenotype of the
most common cystic fibrosis mutation. J Biol Chem 271(2):635–638
24. Loo TW, Clarke DM (1997) Correction of defective protein kinesis of human P-glycoprotein
mutants by substrates and modulators. J Biol Chem 272(2):709–712
25. Morello JP, Salahpour A, Laperrière A, et al (2000) Pharmacological chaperones rescue cell-
surface expression and function of misfolded V2 vasopressin receptor mutants. J Clin Invest
105(7):887–895
26. Morello JP, Petäjä-Repo UE, Bichet DG, et al (2000) Pharmacological chaperones: a new twist
on receptor folding. Trends Pharmacol Sci 21(12):466–469
27. Bernier V, Bichet DG, Bouvier M (2004) Pharmacological chaperone action on G protein-
coupled receptors. Curr Opin Pharmacol 4(5):528–533
28. Bernier V, Morello JP, Zarruk A, et al (2006) Pharmacologic chaperones as a potential
treatment for X-linked nephrogenic diabetes insipidus. J Am Soc Nephrol 17(1):233–243
29. Conn PM, Ulloa-Aguirre A (2010) Trafficking of G protein-coupled receptors to the plasma
membrane: insights from pharmacoperone drugs. Trends Endocrinol Metab 21(3):190–197
30. Conn PM, Smithson DC, Hodder PS, et al (2014) Transitioning pharmacoperones to thera-
peutic use: in vivo proof-of-principle and design of high throughput screens. Pharmacol Res
83:38–51
31. Leidenheimer NJ, Ryder KG (2014) Pharmacological chaperoning: a primer on mechanism
and pharmacology. Pharmacol Res 83:10–19
32. Karageorgos LE, Isaac EL, Brooks DA, et al (1997) Lysosomal biogenesis in lysosomal
storage disorders. Exp Cell Res 234(1):85–97
33. Parkinson-Lawrence EJ, Shandala T, Prodoehl M, et al (2010) Lysosomal storage disease:
revealing lysosomal function and physiology. Physiology (Bethesda) 25(2):102–115
34. Parenti G, Andria G, Valenzano KJ (2015) Pharmacological chaperone therapy: preclinical
development, clinical translation, and prospects for the treatment of lysosomal storage disor-
ders. Mol Ther 23(7):1138–1148
35. Brady RO (2006) Enzyme replacement for lysosomal diseases. Annu Rev Med 57:283–296
36. Platt FM, Jeyakumar M (2008) Substrate reduction therapy. Acta Paediatr 97(457):88–93
37. Germain DP, Giugliani R, Hughes DA, et al (2012) Safety and pharmacodynamic effects of a
pharmacological chaperone on α-galactosidase A activity and globotriaosylceramide clearance
in Fabry disease: report from two phase 2 clinical studies. Orphanet J Rare Dis 7:91
38. Zimran A, Altarescu G, Elstein D (2013) Pilot study using ambroxol as a pharmacological
chaperone in type 1 Gaucher disease. Blood Cells Mol Dis 50(2):134–137
39. Germain DP, Hughes DA, Nicholls K, et al (2016) Treatment of Fabry’s disease with the
pharmacologic chaperone Migalastat. N Engl J Med 375(6):545–555
40. Conn PM, Ulloa-Aguirre A (2011) Pharmacological chaperones for misfolded gonadotropin-
releasing hormone receptors. Adv Pharmacol 62:109–141
41. Conn PM, Ulloa-Aguire A, Ito J, et al (2007) G protein-coupled receptor trafficking in health
and disease: lessons learned to prepare for therapeutic mutant rescue in vivo. Pharmacol Rev
59(3):225–250
42. Janovick JA, Maya-Nunez G, Conn PM (2002) Rescue of hypogonadotropic hypogonadism-
causing and manufactured GnRH receptor mutants by a specific protein-folding template:
misrouted proteins as a novel disease etiology and therapeutic target. J Clin Endocrinol Metab
87(7):3255–3262
43. Janovick JA, Stewart MD, Jacob D, et al (2013) Restoration of testis function in
hypogonadotropic hypogonadal mice harboring a misfolded GnRHR mutant by
pharmacoperone drug therapy. Proc Natl Acad Sci U S A 110(52):21030–21035
44. Jean-Alphonse F, Perkovska S, Frantz MC, et al (2009) Biased agonist pharmacochaperones of
the AVP V2 receptor may treat congenital nephrogenic diabetes insipidus. J Am Soc Nephrol
20(10):2190–2203
45. White E, McKenna J, Cavanaugh A, et al (2009) Pharmacochaperone-mediated rescue of
calcium-sensing receptor loss-of-function mutants. Mol Endocrinol 23(7):1115–1123
46. Janovick JA, Maya-Nunez G, Ullo-Aguire A, et al (2009) Increased plasma membrane
expression of human follicle-stimulating hormone receptor by a small molecule thienopyr
(im)idine. Mol Cell Endocrinol 298(1–2):84–88
47. Newton CL, Whay AM, McArdle CA, et al (2011) Rescue of expression and signaling of
human luteinizing hormone G protein-coupled receptor mutants with an allosterically binding
small-molecule agonist. Proc Natl Acad Sci U S A 108(17):7172–7176
48. Aronson D, Verbalis JG, Mueller M, et al (2011) Short- and long-term treatment of dilutional
hyponatraemia with satavaptan, a selective arginine-vasopressin V2 receptor antagonist: the
DILIPO study. Eur J Heart Fail 13(3):327–336
49. Feinstein TN, Yui N, Webber MJ, et al (2013) Noncanonical control of vasopressin receptor
type 2 signaling by retromer and arrestin. J Biol Chem 288(39):27849–27860
50. Moeller HB, Rittig S, Fenton RA (2013) Nephrogenic diabetes insipidus: essential insights
into the molecular background and potential therapies for treatment. Endocr Rev 34
(2):278–301
51. Treschan TA, Peters J (2006) The vasopressin system. Anesthesiology 105(3):599–612
52. Morello JP, Bichet DG (2001) Nephrogenic diabetes insipidus. Annu Rev Physiol 63:607–630
53. Bichet DG, Birnbaumer M, Lonergan M, et al (1994) Nature and recurrence of AVPR2
mutations in X-linked nephrogenic diabetes insipidus. Am J Hum Genet 55(2):278–286
54. Tsukagushi H, Matsubara H, Taketani S, et al (1995) Binding, intracellular transport and
biosynthesis-defective mutants of vasopressin type 2 receptor in patients with X-linked
nephrogenic diabetes insipidus. J Clin Invest 96(4):2043–2050
55. Ala Y, Morin D, Mouillac B, et al (1998) Functional studies of twelve mutant V2 vasopressin
receptors related to nephrogenic diabetes insipidus: molecular basis of a mild clinical pheno-
type. J Am Soc Nephrol 9(10):1861–1872
56. Bockenhauer D, Bichet DG (2014) Urinary concentration: different ways to open and close the
tap. Pediatr Nephrol 29(8):1297–1303
57. Birnbaumer M, Seibold A, Gilbert S, et al (1992) Molecular cloning of the receptor for human
antidiuretic hormone. Nature 357(6376):333–335
58. Lolait SJ, Carroll AM, McBride OW, et al (1992) Cloning and characterization of a vaso-
pressin V2 receptor and possible link to nephrogenic diabetes insipidus. Nature 357
(6376):526–529
59. Rosenthal W, Seibold A, Antaramian A, et al (1992) Molecular identification of the gene
responsible for congenital nephrogenic diabetes insipidus. Nature 359(6392):233–235
60. Tamarappoo BK, Verkman AS (1998) Defective aquaporin-2 trafficking in nephrogenic
diabetes insipidus and correction by chemical chaperones. J Clin Invest 101(10):2257–2267
61. Serradeil-Le Gal C, Lacour C, Valette G, et al (1996) Characterization of SR 121463A, a
highly potent and selective, orally active vasopressin V2 receptor antagonist. J Clin Invest 98
(12):2729–2738
62. Bockenhauer D, Carpentier E, Rochdi D, et al (2010) Vasopressin type 2 receptor V88M
mutation: molecular basis of partial and complete nephrogenic diabetes insipidus. Nephron
Physiol 114(1):1–10
63. Janovick JA, Park BS, Conn PM (2011) Therapeutic rescue of misfolded mutants: validation of
primary high throughput screens for identification of pharmacoperone drugs. PLoS One 6(7):
e22784
64. Tan CM, Nickols HH, Limbird LE (2003) Appropriate polarization following pharmacological
rescue of V2 vasopressin receptors encoded by X-linked nephrogenic diabetes insipidus alleles
involves a conformation of the receptor that also attains mature glycosylation. J Biol Chem 278
(37):35678–35686
65. Wüller S, Wiesner B, Loffler A, et al (2004) Pharmacochaperones post-translationally enhance
cell surface expression by increasing conformational stability of wild-type and mutant vaso-
pressin V2 receptors. J Biol Chem 279(45):47254–47263
66. Bernier V, Lagacé M, Lonergan M, et al (2004) Functional rescue of the constitutively
internalized V2 vasopressin receptor mutant R137H by the pharmacological chaperone action
of SR49059. Mol Endocrinol 18(8):2074–2084
67. Robben JH, Sze M, Knoers NV, et al (2007) Functional rescue of vasopressin V2 receptor
mutants in MDCK cells by pharmacochaperones: relevance to therapy of nephrogenic diabetes
insipidus. Am J Physiol Renal Physiol 292(1):F253–F260
68. Robben JH, Sze M, Knoers NV, et al (2006) Rescue of vasopressin V2 receptor mutants by
chemical chaperones: specificity and mechanism. Mol Biol Cell 17(1):379–386
69. Robben JH, Kortenoeven MLA, Sze M, et al (2009) Intracellular activation of vasopressin V2
receptor mutants in nephrogenic diabetes insipidus by nonpeptide agonists. Proc Natl Acad Sci
U S A 106(29):12195–12200
70. Auzan RJ, Ventura MA, Clauser E (2005) Mechanisms of cell-surface rerouting of an
endoplasmic reticulum-retained mutant of the vasopressin V1b/V3 receptor by a pharmaco-
logical chaperone. J Biol Chem 280(51):42198–42206
71. Hawtin SR (2006) Pharmacological chaperone activity of SR49059 to functionally recover
misfolded mutations of the vasopressin V1a receptor. J Biol Chem 281(21):14604–14614
72. Mendre C, Mouillac B (2010) Pharmacological chaperones: a potential therapeutic treatment
for conformational diseases. Med Sci (Paris) 26(6–7):627–635
73. Los EL, Deen PMT, Robben JH (2010) Potential of nonpeptide (ant)agonists to rescue
vasopressin V2 receptor mutants for the treatment of X-linked nephrogenic diabetes insipidus.
J Neuroendocrinol 22(5):393–399
74. Wesche D, Deen PMT, Knoers NV (2012) Congenital nephrogenic diabetes insipidus: the
current state of affairs. Pediatr Nephrol 27(12):2183–2204
75. Schrier RW, Gross P, Gheorghiade M (2006) Tolvaptan, a selective oral vasopressin
V2-receptor antagonist, for hyponatremia. N Engl J Med 355(20):2099–2112
76. Mouillac B, Mendre C (2014) Vasopressin receptors and pharmacological chaperones: from
functional rescue to promising therapeutic strategies. Pharmacol Res 83:74–78
77. Rahmeh R, Damian M, Cottet M, et al (2012) Structural insights into biased G protein-coupled
receptor signaling revealed by fluorescence spectroscopy. Proc Natl Acad Sci U S A 109
(17):6733–6738
78. Mary S, Damian M, Louet M, et al (2012) Ligands and signaling proteins govern
the conformational landscape explored by a G protein-coupled receptor. Proc Natl Acad
Sci U S A 109(21):8304–8309
79. Liu JJ, Horst R, Katritch V, et al (2012) Biased signaling pathways in β2-adrenergic receptor
characterized by 19F-NMR. Science 335(6072):1106–1110
Top Med Chem (2019) 30: 181–194
DOI: 10.1007/7355_2017_1
Endosomal PTH Receptor Signaling

Through cAMP and Its Consequence
for Human Medicine
Ieva Sutkeviciute, Frederic G. Jean-Alphonse, and Jean-Pierre Vilardaga
Abstract The parathyroid hormone (PTH) type 1 receptor (PTHR) is a medically

important G protein-coupled receptor (GPCR) that triggers the cAMP/PKA signal-
ing pathway in kidney and bone cells to regulate calcium ion homeostasis and bone
turnover. It has been generally assumed that the production of cAMP mediated by
GPCR and its termination take place exclusively at the plasma membrane. Recent
studies reveal that the PTHR does not always follow this conventional paradigm. In
the new model, PTH induces a prolonged cAMP response that is derived from the
internalized ligand–PTHR complex located within endosomes. This model has
been recognized as a new paradigm of GPCR signaling for peptide hormones,
and the PTHR is a prototypical example. In this chapter we discuss molecular,
structural, and cellular mechanisms responsible for this unexpected signaling pro-
cess and its biological consequences.
Keywords Arrestin, Endosomal signaling, GPCR, Hypocalcemia, PTH receptor,

Retromer
Contents
1 Introduction: The PTHR Signaling System as a Prototype . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
2 Structural Basis of Ligand Binding to PTHR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
3 Regulation of Endosomal PTHR Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
4 Consequence for Human Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
I. Sutkeviciute, F.G. Jean-Alphonse, and J.-P. Vilardaga (*)

Laboratory for GPCR Biology, Department of Pharmacology and Chemical Biology,
University of Pittsburgh School of Medicine, Pittsburgh, PA 15217, USA
e-mail: jpv@pitt.edu
182 I. Sutkeviciute et al.
1 Introduction: The PTHR Signaling System

as a Prototype
The PTHR is a family B GPCR transducing the actions of two hormones: PTH,
endocrine and homeostatic regulator of concentrations of calcium and phosphate
ions, and vitamin D in blood and extracellular fluids; and PTH-related peptide
(PTHrP), paracrine mediator for the development of bone, mammary glands, and
other tissues [1, 2]. Another important function of PTH is to promote bone forma-
tion when the hormone is daily administered [3]; severe cases of osteoporosis are
currently treated by the synthetic N-terminal fragment of the hormone, PTH1-34, to
promote trabecular and cortical bone formation and decrease fracture risk. This
anabolic action of PTH on bone, contrasts with its catabolic effect that is needed to
release Ca2+ from bone through stimulation of bone resorption. Understanding how
activation of the same receptor by its two native ligands triggers distinct biological
effects and mediates the paradoxical effects of PTH on bone mass is key not only to
discover new fundamental aspects of GPCR signaling, but also for the development
of new therapies targeting bone and mineral diseases. Recent research efforts have
discovered altered modes of cAMP signaling at the PTHR that provide a rational
explanation for the functional differences between PTH and PTHrP, and which
might account for the paradoxical actions of PTH (Fig. 1).
Studies of ligand–PTHR interaction mechanisms indicate that PTHR adopts at
least two distinct active conformations that induce distinct cAMP signaling
responses [4, 5]. One of these PTHR conformations, called R0 in reference to
earlier studies done with the corticotropin-releasing factor 1 receptor [6], is a G
protein-independent high-affinity PTHR conformation that is preferentially stabi-
lized by PTH and mediates sustained cAMP production from internalized receptors
associated within endosomes. This R0 conformation is distinct from the classical G
protein-dependent high-affinity receptor conformation, which is named RG, is
indistinguishably stabilized by PTH or PTHrP, and mediates transient cAMP
responses that originate exclusively from the plasma membrane. Two distinct
assays can differentiate ligand binding to the R0 and RG conformations of the
PTHR [7]. The first employs a cell membrane competition binding assay [5]. For
R0, [125I]-PTH(1–34) is used as a tracer radioligand with an excess of GTPγS to
prevent receptor and G-protein coupling. For RG, binding isotherms used a short-
acting PTH variant, [125I]-M-PTH [1–15], as tracer radioligand and the presence of
a high-affinity, negative-dominant GαS subunit (GαS-ND) to force receptor and G
protein coupling. The second is a live-cell FRET-based assays recording dissocia-
tion time courses of tetramethylrhodamine (TMR)-labeled ligands from PTHR
N-terminally tagged with GFP in the absence or presence of a GαS-ND [4]. The
striking differences between the two native PTHR ligands in driving either short
cAMP signaling from the plasma membrane (PTHrP) via the RG conformation or
sustained cAMP signaling from endosomes (PTH) via the R0 conformation can be
considered as a form of ligand bias. This form of biased agonism can be extended to
the vasopressin type 2 receptor (V2R), a class A GPCR, which shows similar
Endosomal PTH Receptor Signaling Through cAMP and Its Consequence for Human. . . 183
Fig. 1 Endosomal PTHR signaling: from bench-to-bedside. (a) Studies in cells lead to the
discovery that PTH, but not PTHrP, sustains cAMP production after PTHR internalization into
early endosomes. (b) This observation is motivating the development of PTH analogs able to
promote the endosomal cAMP signaling. One of them, LA-PTH, mediates prolonged hypercalce-
mic responses when injected into mice, and is now in preclinical development for eventual testing
as a treatment for hypocalcemia (c)
signaling properties in comparison with PTHR [8]. These include: (1) two native
high-affinity agonists, vasopressin and oxytocin, which trigger distinct physiolog-
ical responses via the same receptor (vasopressin has strong antidiuretic and
antinatriuretic effects whereas oxytocin has weak to no effects); (2) sustained
cAMP production that originates from early endosomes that is only mediated by
one of the two agonists, vasopressin; and (3) endosomal cAMP production is
promoted by β-arrestins and attenuated by the retromer complex together with
concomitant acidification of endosomes (see below). These similarities indicate
that persistent endosomal cAMP signaling and bias agonism as it relates to location
and duration of cAMP production are not restricted to the PTHR but are integral
signaling properties of GPCRs.
2 Structural Basis of Ligand Binding to PTHR
Despite the marked differences of the two native PTHR ligands in their cAMP
signaling modes, the available structural data, however, suggests a very similar
peptide ligand recognition mechanism. While free in solution, the bioactive parts
comprising the first 34 N-terminal amino acids of both PTH and PTHrP exist in
relatively disordered, likely flexible peptide forms (Fig. 2) as revealed by solution
NMR studies [9, 10]. Both peptides have a short α-helical motive at N-terminal side
and a more extended α-helix at the C-terminal part. The flexible loops that link
these two motives allow them to move dynamically in solution relative to each
other. X-ray crystallography data of the C-terminal PTH and PTHrP fragments,
comprising 15–34 amino acids in complex with the PTHR extracellular domain
(Fig. 3), indicate that a disorder-to-order transition occurs in these peptide regions
upon binding to the receptor [11, 12]. Even though these C-terminal fragments
share low sequence homology (<17%, Fig. 2b), their binding mode to the
N-terminal PTHR is remarkably similar. Both peptides occupy not only the same
binding site on the receptor with the hydrophobic faces of their amphipatic helices
contacting receptor, but also their sequences superimpose almost perfectly (Fig. 3).
Nonetheless, a slight deviation occurs at the C-terminal ends of the two peptides
indicating that their C-terminal extensions could take different paths along the
receptor surface. However, these C-terminal extensions have not yet been found
to have any functional role in PTHR signaling.
While the 15–34 segments of PTH and PTHrP comprise the high-affinity
receptor binding domains and thus are responsible for an initial high-affinity
receptor binding, the N-terminal peptide portions contain the signaling moieties
[2]. Thus PTHR activation can be described by a two-step model: first, the
C-terminal peptide fragment associates with the receptor, and second, a low affinity
N-terminal peptide part binding to PTHR juxtamembrane domain takes place,
which triggers receptor activation and intracellular signaling events. The structural
information of receptor activation process, however, is still limited, as the high
resolution structure of PTHR transmembrane domain (TMD) has not yet been
solved. Nonetheless, the data from extensive photoaffinity crosslinking and muta-
tional analysis studies provide an important structural insight to receptor interaction
with the signaling portions of PTH and PTHrP [13–18]. In particular, the conserved
Val2 in PTH and PTHrP, which is an essential determinant in receptor activation,
was found to bind Met425 located at the extracellular end of transmembrane helix
6 of PTHR. A further hint on a possible PTH–PTHR binding mode can be inferred
from the crystal structure of PTH1-34 peptide and the recent crystal structures of the
TMDs of two other family B GPCRs, the glucagon receptor [19] or the
corticotropin-releasing factor receptor [20]. The crystallographic data shows that
PTH1-34 exists as a slightly bent helix with two twisted amphipatic regions spanning
residues 6–20 and 21–33 (Fig. 4) [21]. Given that the crystal structure possibly
reflects the conformational state of the peptide in a protein-like environment (the
crystallization partners mimic protein surface), such a full α-helix might be a
Fig. 2 Comparison of PTH and PTHrP sequences and structures. (a) sequence alignment of PTH
and PTHrP. Amino acid color-coding: hydrophobic are light orange, polar – green, acidic – blue,
basic – red. The conserved residues are marked by asterisk. The bars below mark principal
signaling and principal binding domains. (b) Solution NMR structures of PTH (pdb 1HPH) and
PTHrP (pdb 1BZG). Three out of 10 and three out of 30 solution conformations of PTH and
PTHrP, respectively, are represented. Rainbow color scheme showing N-termini in blue and
C-termini in red
plausible conformational state of the receptor-bound hormone. This assumption is

supported by mutational studies combined with solution NMR analysis that indi-
cated at least some extent of α-helicity of the bioactive state N-terminal end of PTH
hormone [9, 22–29]. Furthermore, the disruption of α-helical conformation at the
middle region by replacing Gly12 with a helix breaker proline resulted in 1,000-
fold receptor binding affinity decrease and 3,500-fold loss of PTH mediated
adenylyl cyclase stimulation [30]. Using the aforementioned data and the structure
of the glucagon receptor TMD, which shares ~50% sequence homology with TMD
of PTHR, the overall PTH-PTHR complex may be envisioned as illustrated in
Fig. 5. This model suggests a perpendicular superposition of PTHR extracellular
domain to the membrane plane, the so-called open conformational state, in
Fig. 3 Binding modes of C-terminal PTH and PTHrP within PTHR extracellular domain. (a, b)
Three different views of PTH15-34 (a, pdb 3C4M) and PTHrP13-34 (b, pdb 3H3G) bound to PTHR
ECD. The receptor ECD is shown as a surface with carbons in grey, oxygen red, nitrogen blue, and
sulfur yellow. Peptide ligands are shown as coils with the side chains represented as sticks for those
residues that contact the receptor; the hydrophobic and hydrophilic residues are in yellow and cyan
colors, respectively. The polar contacts and van der Waals interactions are highlighted by light
blue and orange dashed lines, respectively. N-terminal and C-terminal residues of the peptides are
marked. (c) Alignment of PTH15-35 (yellow) and PTHrP13-34 (blue) bound to PTHR ECD. Arrows
mark possible directions of C-terminal peptide extensions
agreement with a recent study of full-length glucagon receptor conformational

states, which showed that peptide-bound receptor preferably assumes the open
state [31].
Fig. 4 Representation of PTH1-34 crystal structure (pdb 1ET1). Polar and hydrophobic amino
acids are shown in blue and yellow, respectively
Fig. 5 A hypothetical model of PTH1-34–PTHR complex in an open state. (a) Representation of

the model where black spheres indicate Val2 of PTH and Ala363 of glucagon receptor homolo-
gous to Met425 of PTHR. (b) Two views of surface representation of PTH1-34–PTHR complex
model. Yellow helix is PTH1-34 (pdb 1ET1) superimposed to PTH15-34 (red helix) bound to PTHR
ECD (green) that is aligned on glucagon receptor crystal structure (pdb 4L6R); the extracellular,
transmembrane and cytosolic parts of glucagon receptor are shown in orange, violet, and cyan
colors, respectively
Unlike C-terminal regions, the N-terminal segments of PTH and PTHrP possess
a relatively high sequence identity (>61%, Fig. 2b). Regardless, receptor binding
and signaling properties of the two hormones differ significantly. Since C-terminal
peptide segments bind similarly to the receptor ECD, one can anticipate that the
distinct hormone behaviors originate from differences in N-terminal segments of
the peptides. The observation that PTHrP binds the RG conformation of PTHR with
a higher affinity than the R0 conformation implies that G-protein association with
the receptor allosterically modulates the PTHrP binding site within the receptor in a
way to favor RG-selective peptide ligand binding. The presence of GS further
stabilizes PTHrP interaction with the receptor, since preventing GS coupling leads
to a rapid ligand dissociation. This is opposed by the so-called R0-selective PTHR
ligands, as PTH, which do not require coupling to GαS to stabilize a high-affinity
receptor conformation [4, 5, 32].
Taking into account the prevailing assumption that the bioactive conformation
of N-terminal signaling domain adopts an α-helical structure, the following
scenario may be inferred. Receptor association with GS creates and maintains a

partially stabilized ligand binding pocket within the receptor, which promotes
N-terminal domain folding into a bioactive helix after RG-selective peptide ligand
binds to the receptor. Subsequently, the folded bioactive α-helix makes critical
contacts with the receptor TMD switching it to fully active conformation, whereas
dissociation of GαS destabilizes these contacts and thus the bioactive peptide
conformation. On the other hand, R0-selective ligands may have a stronger intrinsic
potential to fold into bioactive α-helix upon binding to the receptor independently
of this preformed ligand binding pocket, or alternatively preexist as a bioactive
α-helix. This way R0-selective ligands form stable complexes with receptor
switching its conformation to an active state and maintaining it for extended periods
of time, thus allowing prolonged cAMP production even after receptor internaliza-
tion to endosomal compartments.
3 Regulation of Endosomal PTHR Signaling
Unexpectedly, we discovered that the general mechanism that desensitizes GPCR

signaling via β-arrestins is needed to promote endosomal cAMP production in
PTHR signaling. Internalized PTH–PTHR signaling complexes containing
β-arrestins promote rather than terminate cAMP signaling through two concomitant
biochemical events. In the first, endosomal PTH–PTHR–βarr complexes stimulate
ERK1/2 activation, leading to inhibition of the cAMP-specific phosphodiesterases
PDE4 [33]. In the second, PTH-bound PTHR–βarr complexes stabilize the associ-
ation with Gβγ subunits, which accelerates the rate of GS activation and increases
the steady-state level of activated GαS. Here, β-arrestins stabilize the G-protein
cycle, thus leading to persistent generation of cAMP by PTH [34]. Despite these
results, the detailed mechanism by which β-arrestins promotes Gs activity is largely
unknown. It is particularly intriguing how PTH–PTHR–βarr–Gβγ complexes can
couple to Gα considering the recently published structure of rhodopsin in complex
with arrestin [35]. A possibility that we are exploring is that β-arrestin forms a
dynamic interaction with PTH–PTHR complexes, which persists in endosomes for
many cycles of receptor–arrestin association and dissociation. The receptor com-
plex formed through interactions between β-arrestin and Gβγ subunits might permit
multiple rounds of GαS coupling and activation or stabilize its active state, thus
prolonging cAMP generation.
Termination of endosomal cAMP signaling correlates with the exchange at the
PTHR complex of β-arrestin for vesicle protein sorting (Vps) 26, Vps29, and/or
Vps35, which are constituent of the retromer complex well known to regulate post-
endocytic sorting of several cargo proteins to the Trans-Golgi network or receptor
recycling to the plasma membrane [33, 36–38]. Recent FRET studies show that
PTHR can closely interact with Vps29, and the striking structural similarities
between Vps26 and β-arrestins suggest that Vps26 might also directly interact
with PTHR (Fig. 6) [39]. Although the binding mechanism between PTHR and
Fig. 6 Structures of β-arrestin and Vps26. Structural alignment of inactive β-arrestin1 (blue, pdb
1G4M) and retromer subunit Vps26 (orange, pdb 2FAU)
retromer is largely undetermined, Gidon et al. showed that the disassembly of

signaling PTH–PTHR–βarr complexes and assembly of inactive PTHR–retromer
complexes are promoted by vATPase-mediated endosomal acidification [40]. The
vATPases are activated by cAMP-dependent PKA, and thereby establish a negative
feedback loop [40]. PTHR activation of cAMP signaling that differs in duration and
location of origin within the cell thus provides a potential mechanism for ligand-
directed diversification of cellular responses (Fig. 1).
4 Consequence for Human Medicine
The physiological relevance of this unexpected endosomal cAMP production in

PTHR signaling becomes to be understood. Certain synthetic long-acting PTH
(LA-PTH) analogs that show enhanced selectivity for the R0 state of PTHR
conformation and have higher efficacies than PTH to induce endosomal cAMP
signaling in cells are also inducing strikingly prolonged hypercalcemic responses
when injected into mice (Fig. 1) [41]. These R0-selective ligands have thus potential
medical applications and LA-PTH is now in preclinical development for eventual
testing as a future treatment of certain forms of hypocalcemia such as hypopara-
thyroidism [42]. Implicit with these observations is that ligands favoring acute
cAMP production generated at the plasma membrane via the RG conformation
would be more osteoanabolic [43].
5 Conclusion
Once considered to be exclusively generated at the plasma membrane, cAMP can

also be produced and sustained from endosomal membranes after receptor inter-
nalization. This new mode of cAMP signaling has been so far observed not only for
several peptide hormone receptors [44], but also for monoamine receptors such as
the dopamine D1 receptor [45]. The regulatory mechanism and biological impor-
tance of this process begin to be understood at least for the PTHR signaling system,
however, further characterization of structural and cellular mechanisms regulating
the duration of PTHR-mediated GS/cAMP signaling within endosomes are now
needed.
Acknowledgments This work was supported by the National Institutes of Health (NIH) under
Award numbers R01 DK087688 and R01 DK102495 (JPV), and the Cotswold Foundation
Fellowship Award (FJA).
References
1. Juppner H, Abou-Samra AB, Freeman M, Kong XF, Schipani E, Richards J, Kolakowski LF Jr,
Hock J, Potts JT Jr, Kronenberg HM et al (1991) A G protein-linked receptor for parathyroid
hormone and parathyroid hormone-related peptide. Science 254:1024–1026
2. Gardella TJ, Vilardaga JP (2015) International Union of Basic and Clinical Pharmacology.
XCIII. The parathyroid hormone receptors – family B G protein-coupled receptors. Pharmacol
Rev 67:310–337
3. Neer RM, Arnaud CD, Zanchetta JR, Prince R, Gaich GA, Reginster JY, Hodsman AB,
Eriksen EF, Ish-Shalom S, Genant HK, Wang O, Mitlak BH (2001) Effect of parathyroid
hormone (1-34) on fractures and bone mineral density in postmenopausal women with
osteoporosis. N Engl J Med 344:1434–1441
4. Ferrandon S, Feinstein TN, Castro M, Wang B, Bouley R, Potts JT, Gardella TJ, Vilardaga JP
(2009) Sustained cyclic AMP production by parathyroid hormone receptor endocytosis. Nat
Chem Biol 5:734–742
5. Dean T, Vilardaga JP, Potts JT Jr, Gardella TJ (2008) Altered selectivity of parathyroid
hormone (PTH) and PTH-related protein (PTHrP) for distinct conformations of the
PTH/PTHrP receptor. Mol Endocrinol 22:156–166
6. Hoare SR, Sullivan SK, Pahuja A, Ling N, Crowe PD, Grigoriadis DE (2003) Conformational
states of the corticotropin releasing factor 1 (CRF1) receptor: detection, and pharmacological
evaluation by peptide ligands. Peptides 24:1881–1897
7. Vilardaga JP, Gardella TJ, Wehbi VL, Feinstein TN (2012) Non-canonical signaling of the
PTH receptor. Trends Pharmacol Sci 33:423–431
8. Feinstein TN, Yui N, Webber MJ, Wehbi VL, Stevenson HP, King JD Jr, Hallows KR,
Brown D, Bouley R, Vilardaga JP (2013) Noncanonical control of vasopressin receptor type
2 signaling by retromer and arrestin. J Biol Chem 288:27849–27860
9. Marx UC, Austermann S, Bayer P, Adermann K, Ejchart A, Sticht H, Walter S, Schmid FX,
Jaenicke R, Forssmann WG et al (1995) Structure of human parathyroid hormone 1-37 in
solution. J Biol Chem 270:15194–15202
10. Weidler M, Marx UC, Seidel G, Schafer W, Hoffmann E, Esswein A, Rosch P (1999) The
structure of human parathyroid hormone-related protein(1-34) in near-physiological solution.
FEBS Lett 444:239–244
11. Pioszak AA, Parker NR, Gardella TJ, Xu HE (2009) Structural basis for parathyroid hormone-
related protein binding to the parathyroid hormone receptor and design of conformation-
selective peptides. J Biol Chem 284:28382–28391
12. Pioszak AA, Xu HE (2008) Molecular recognition of parathyroid hormone by its G protein-
coupled receptor. Proc Natl Acad Sci U S A 105:5034–5039
13. Bisello A, Adams AE, Mierke DF, Pellegrini M, Rosenblatt M, Suva LJ, Chorev M (1998)
Parathyroid hormone-receptor interactions identified directly by photocross-linking and
molecular modeling studies. J Biol Chem 273:22498–22505
14. Adams AE, Bisello A, Chorev M, Rosenblatt M, Suva LJ (1998) Arginine 186 in the
extracellular N-terminal region of the human parathyroid hormone 1 receptor is essential for
contact with position 13 of the hormone. Mol Endocrinol 12:1673–1683
15. Behar V, Bisello A, Bitan G, Rosenblatt M, Chorev M (2000) Photoaffinity cross-linking
identifies differences in the interactions of an agonist and an antagonist with the parathyroid
hormone/parathyroid hormone-related protein receptor. J Biol Chem 275:9–17
16. Shimizu M, Carter PH, Gardella TJ (2000) Autoactivation of type-1 parathyroid hormone
receptors containing a tethered ligand. J Biol Chem 275:19456–19460
17. Gensure RC, Carter PH, Petroni BD, Juppner H, Gardella TJ (2001) Identification of deter-
minants of inverse agonism in a constitutively active parathyroid hormone/parathyroid
hormone-related peptide receptor by photoaffinity cross-linking and mutational analysis. J
Biol Chem 276:42692–42699
18. Gensure RC, Gardella TJ, Juppner H (2001) Multiple sites of contact between the carboxyl-
terminal binding domain of PTHrP-(1–36) analogs and the amino-terminal extracellular
domain of the PTH/PTHrP receptor identified by photoaffinity cross-linking. J Biol Chem
276:28650–28658
19. Siu FY, He M, de Graaf C, Han GW, Yang D, Zhang Z, Zhou C, Xu Q, Wacker D, Joseph JS,
Liu W, Lau J, Cherezov V, Katritch V, Wang MW, Stevens RC (2013) Structure of the human
glucagon class B G-protein-coupled receptor. Nature 499:444–449
20. Hollenstein K, Kean J, Bortolato A, Cheng RK, Dore AS, Jazayeri A, Cooke RM, Weir M,
Marshall FH (2013) Structure of class B GPCR corticotropin-releasing factor receptor
1. Nature 499:438–443
21. Jin L, Briggs SL, Chandrasekhar S, Chirgadze NY, Clawson DK, Schevitz RW, Smiley DL,
Tashjian AH, Zhang F (2000) Crystal structure of human parathyroid hormone 1-34 at 0.9-A
resolution. J Biol Chem 275:27238–27244
22. Shimizu N, Guo J, Gardella TJ (2001) Parathyroid hormone (PTH)-(1-14) and -(1-11) analogs
conformationally constrained by alpha-aminoisobutyric acid mediate full agonist responses
via the juxtamembrane region of the PTH-1 receptor. J Biol Chem 276:49003–49012
23. Tsomaia N, Pellegrini M, Hyde K, Gardella TJ, Mierke DF (2004) Toward parathyroid
hormone minimization: conformational studies of cyclic PTH(1-14) analogues. Biochemistry
43:690–699
24. Barazza A, Wittelsberger A, Fiori N, Schievano E, Mammi S, Toniolo C, Alexander JM,
Rosenblatt M, Peggion E, Chorev M (2005) Bioactive N-terminal undecapeptides derived
from parathyroid hormone: the role of alpha-helicity. J Pept Res 65:23–35
25. Fiori N, Caporale A, Schievano E, Mammi S, Geyer A, Tremmel P, Wittelsberger A,
Woznica I, Chorev M, Peggion E (2007) Structure-function relationship studies of PTH
(1-11) analogues containing sterically hindered dipeptide mimetics. J Pept Sci 13:504–512
26. Caporale A, Biondi B, Schievano E, Wittelsberger A, Mammi S, Peggion E (2009) Structure-
function relationship studies of PTH(1-11) analogues containing D-amino acids. Eur J
Pharmacol 611:1–7
27. Caporale A, Fiori N, Schievano E, Wittelsberger A, Mammi S, Chorev M, Peggion E (2009)

Structure-function relationship study of parathyroid hormone (1-11) analogues containing
D-AA. Adv Exp Med Biol 611:113–114
28. Caporale A, Sturlese M, Gesiot L, Zanta F, Wittelsberger A, Cabrele C (2010) Side chain
cyclization based on serine residues: synthesis, structure, and activity of a novel cyclic
analogue of the parathyroid hormone fragment 1-11. J Med Chem 53:8072–8079
29. Cupp ME, Song B, Kibler P, Raghavender US, Nayak SK, Thomsen W, Galande AK (2013)
Investigating hydrophobic ligand-receptor interactions in parathyroid hormone receptor using
peptide probes. J Pept Sci 19:337–344
30. Chorev M, Goldman ME, McKee RL, Roubini E, Levy JJ, Gay CT, Reagan JE, Fisher JE,
Caporale LH, Golub EE et al (1990) Modifications of position 12 in parathyroid hormone and
parathyroid hormone related protein: toward the design of highly potent antagonists. Bio-
chemistry 29:1580–1586
31. Yang L, Yang D, de Graaf C, Moeller A, West GM, Dharmarajan V, Wang C, Siu FY, Song G,
Reedtz-Runge S, Pascal BD, Wu B, Potter CS, Zhou H, Griffin PR, Carragher B, Yang H,
Wang MW, Stevens RC, Jiang H (2015) Conformational states of the full-length glucagon
receptor. Nat Commun 6:7859
32. Dean T, Linglart A, Mahon MJ, Bastepe M, Juppner H, Potts JT Jr, Gardella TJ (2006)
Mechanisms of ligand binding to the parathyroid hormone (PTH)/PTH-related protein recep-
tor: selectivity of a modified PTH(1-15) radioligand for GalphaS-coupled receptor conforma-
tions. Mol Endocrinol 20:931–943
33. Feinstein TN, Wehbi VL, Ardura JA, Wheeler DS, Ferrandon S, Gardella TJ, Vilardaga JP
(2011) Retromer terminates the generation of cAMP by internalized PTH receptors. Nat Chem
Biol 7:278–284
34. Wehbi VL, Stevenson HP, Feinstein TN, Calero G, Romero G, Vilardaga JP (2013)
Noncanonical GPCR signaling arising from a PTH receptor-arrestin-Gbetagamma complex.
35. Kang Y, Zhou XE, Gao X, He Y, Liu W, Ishchenko A, Barty A, White TA, Yefanov O, Han
GW, Xu Q, de Waal PW, Ke J, Tan MH, Zhang C, Moeller A, West GM, Pascal BD, Van
Eps N, Caro LN, Vishnivetskiy SA, Lee RJ, Suino-Powell KM, Gu X, Pal K, Ma J, Zhi X,
Boutet S, Williams GJ, Messerschmidt M, Gati C, Zatsepin NA, Wang D, James D, Basu S,
Roy-Chowdhury S, Conrad CE, Coe J, Liu H, Lisova S, Kupitz C, Grotjohann I, Fromme R,
Jiang Y, Tan M, Yang H, Li J, Wang M, Zheng Z, Li D, Howe N, Zhao Y, Standfuss J,
Diederichs K, Dong Y, Potter CS, Carragher B, Caffrey M, Jiang H, Chapman HN, Spence JC,
Fromme P, Weierstall U, Ernst OP, Katritch V, Gurevich VV, Griffin PR, Hubbell WL,
Stevens RC, Cherezov V, Melcher K, Xu HE (2015) Crystal structure of rhodopsin bound to
arrestin by femtosecond X-ray laser. Nature 523:561–567
36. Burda P, Padilla SM, Sarkar S, Emr SD (2002) Retromer function in endosome-to-Golgi
retrograde transport is regulated by the yeast Vps34 PtdIns 3-kinase. J Cell Sci 115:3889–3900
37. Collins BM (2008) The structure and function of the retromer protein complex. Traffic
9:1811–1822
38. Temkin P, Lauffer B, Jager S, Cimermancic P, Krogan NJ, von Zastrow M (2011) SNX27
mediates retromer tubule entry and endosome-to-plasma membrane trafficking of signalling
receptors. Nat Cell Biol 13:715–721
39. Shi H, Rojas R, Bonifacino JS, Hurley JH (2006) The retromer subunit Vps26 has an arrestin
fold and binds Vps35 through its C-terminal domain. Nat Struct Mol Biol 13:540–548
40. Gidon A, Al-Bataineh MM, Jean-Alphonse FG, Stevenson HP, Watanabe T, Louet C,
Khatri A, Calero G, Pastor-Soler NM, Gardella TJ, Vilardaga JP (2014) Endosomal GPCR
signaling turned off by negative feedback actions of PKA and v-ATPase. Nat Chem Biol
10:707–709
41. Okazaki M, Ferrandon S, Vilardaga JP, Bouxsein ML, Potts JT Jr, Gardella TJ (2008)
Prolonged signaling at the parathyroid hormone receptor by peptide ligands targeted to a
specific receptor conformation. Proc Natl Acad Sci U S A 105:16525–16530
42. National Center for Advancing Translational Sciences (2014) Long-acting parathyroid hor-
mone analogs for treatment of hypoparathyroidism. http://www.ncats.nih.gov/research/
reengineering/bridgs/projects/parathyroid.html
43. Hattersley G, Dean T, Corbin BA, Bahar H, Gardella TJ (2015) Binding selectivity of
abaloparatide for PTH-type-1-receptor conformations and effects on downstream signaling.
Endocrinology 157(1):141–149
44. Vilardaga JP, Jean-Alphonse FG, Gardella TJ (2014) Endosomal generation of cAMP in
GPCR signaling. Nat Chem Biol 10:700–706
45. Kotowski SJ, Hopf FW, Seif T, Bonci A, von Zastrow M (2011) Endocytosis promotes rapid
dopaminergic signaling. Neuron 71:278–290
Top Med Chem (2019) 30: 195–216
DOI: 10.1007/7355_2017_20
Published online: 3 June 2017
Structure and Function Studies of GPCRs

by Site-Specific Incorporation of Unnatural
Amino Acids
Meilin Tian, Qian Wang, Chonggang Yuan, and Shixin Ye
Abstract In the past decade, genetic code expansion technology has emerged as
discovery tools in studies of GPCRs for monitoring of dynamic protein conforma-
tional changes, for the screening of ligand–protein and protein–protein interactions,
and as alternatives to conventional labeling approaches for the site-specific labeling
of GPCRs with spectroscopy probes. Interactome mapping among GPCRs, their
ligands, and interactive proteins discovered using genetically encoded photo-cross-
linking unnatural amino acids (Uaas) display methods that link substrate specificity
to binding pockets revealed by static X-ray crystal structures are inaccessible by
other methodologies. Fluorescent-based analysis to directly monitor the GPCR
conformational changes are beginning to move forward into cell-based assays for
M. Tian
Shanghai Key Laboratory of Brain Functional Genomics, School of Life Sciences, ECNU,
Shanghai, China
Institut de Biologie de l’Ecole Normale Supérieure, Ecole Normale Supérieure, Paris, France
Institut National de la Santé et de la Recherche Médicale (INSERM), U1024, Paris, France
Centre National de la Recherche Scientifique (CNRS), UMR 7238, 8197 Paris, France
Q. Wang
Origins of Cancer Program, Centenary Institute, Sydney Medical School, University of
Sydney, Camperdown, NSW, Australia
C. Yuan
Shanghai Key Laboratory of Brain Functional Genomics, School of Life Sciences, ECNU,
Shanghai, China
S. Ye (*)
Institut National de la Santé et de la Recherche Médicale (INSERM), U1024, Paris, France
Centre National de la Recherche Scientifique (CNRS), UMR 7238, 8197 Paris, France
Laboratory of Computational and Quantitative Biology (LCQB), Institute of Biology, Paris-
Seine, University of Pierre and Marie Curie, Paris, France
e-mail: yelehman@biologie.ens.fr
196 M. Tian et al.
high-throughput drug screening platforms. This review details the significant pro-
gress in Uaa containing GPCRs discovery platforms, as well as advances in
understanding the structure activity relationship of GPCRs in the “post structural
biology” era.
Keywords Fluorescent assay, Genetic code expansion, GPCR crystal structures,

GPCR interactome, Photocrosslinking, Spectroscopic analysis, Structure activity
relationship
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196
2 Genetic Code Expansion Methodology Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
3 Genetically Encoding Uaas to Study GPCR Structure and Function Relationship . . . . . . . 199
3.1 Mapping Receptor and Peptide Ligand Interactions via Crosslinking UAAs . . . . . . 200
3.2 Mapping Receptor and Small-Molecule Ligand Interactions via
Crosslinking UAAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
3.3 Mapping Other Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
3.4 Detecting Dynamic Changes via Spectroscopic UAAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
3.5 Probing Helical Backbone Structure at Conserved Proline Sites . . . . . . . . . . . . . . . . . . . . 209
4 Conclusions and Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212
1 Introduction
G protein-coupled receptors (GPCRs) are seven transmembrane-spanning proteins

belonging to the largest family of cell-surface receptors. GPCRs elicit cellular
responses to multiple diverse stimuli and play essential roles in human health and
have important clinical implications in various diseases. Upon binding to their
cognate ligand, GPCRs typically signal via heterotrimeric GTP-binding proteins
(G proteins), which are comprised of an α-subunit (Gα) and a tightly associated
beta and gamma-subunits (Gßν) (Fig. 1). To ensure that signals are of the appropriate
magnitude and duration, GPCR signaling is tightly regulated through a balance
between activation involving G proteins and desensitization signaling associated
with ß-arrestin. The process of GPCR desensitization involves (1) receptor phosphor-
ylation by GRK, which uncouples the receptor from G proteins and (2) ß-arrestin-
binding. Therefore, the signaling is an orchestration of multiple distance temporal
events that occur at the level of the GPCRs, G proteins or ß-arrestin, and downstream
effector molecules. Over the past decade, significant advances in the structural
biology of GPCRs and their signaling partners have provided a wealth of information
about the molecular architectures of GPCRs and receptor/partner complexes at
atomic resolution [1–4]. However, to understand the dynamics of GPCR signaling
remains challenging. A toolbox of chemical biology approaches centered on the
genetic code expansion (GCE) technology has enabled the incorporation of unnatural
amino acids (Uaas) serving as unique physical and chemical probes into proteins [5–
9]. These probes allow for monitoring receptor conformational changes and real-time
detection of signaling complexes. In this review, we will discuss the development of
Structure and Function Studies of GPCRs by Site-Specific Incorporation of. . . 197
Fig. 1 GPCR signaling via balanced interactions with G proteins and β-arrestin. Upon binding to
their cognate ligand such as an agonist from extracellular side, GPCR is activated and signals via
heterotrimeric GTP-binding protein (composed of α, β, and γ subunits) and coupling with the GDP
that is released from the intracellular side. The process of GPCR desensitization involves
(1) receptor phosphorylation by GRK, which uncouples the receptor from G proteins and (2) -
β-arrestin-binding
GCE technology and their applications in addressing specific questions related to

GPCRs signaling currently inaccessible to other methods.
2 Genetic Code Expansion Methodology Development
Misacylated Suppressor tRNA By contrast to the conventional site-directed muta-

genesis, the genetic code expansion enables the site-specific incorporation of a Uaa
into a protein. The common strategy utilizes misacylated suppressor tRNA in
response to the amber stop codon, one of the three stop codons serving as a stop
signal, to cause read-through in protein translation. During translation, the ribosome
“reads” the UAG stop codon by the aminoacylated suppressor tRNA in competi-
tions with the terminating process. Protein translation continues and produces a
full-length protein with the Uaa inserted at a specified position. The generation of
misacylating tRNAs was based on the semi-chemical synthesis method worked out
by Hecht and co-workers [10], and later refined by Schultz and co-workers
[11]. While promising, early demonstrations were only achieved through in vitro
translation. In the following decade, efforts have been devoted to advancing the
technique in live cells. The first cellular system demonstrated to be compatible with
Uaa mutagenesis is Xenopus laevis oocytes [12]. Since then, varieties of Uaas have
been introduced into ligand-gated ion channels through a chemically modified
suppressor tRNA [13, 14]. The key advantage of Xenopus oocytes in UAA incor-
poration is as little as ten attomol of proteins can be detected using whole-cell
electrophysiological analyses. For any other cellular systems, the limiting factor is
the suppressor tRNA, which is consumed as a “stoichiometric reagent” in the
process, therefore imposing limitation of the stop codon suppression methodology.
198 M. Tian et al.
Orthogonal RS/tRNA Pairs and Uaas To engineer tRNA synthetases amino-

acylating the suppressor tRNA in situ became an attractive solution for efficient
Uaa incorporation in living cells [6, 15, 16]. In 2001, Schultz and co-workers
pioneered in the development of orthogonal tRNACUA/Tyr-tRNA synthetase pairs
(derived from an archaebacterium Methanococcus jannaschii) that could introduce
Uaas in E. coli [15] and conceptualized the genetic code expansion. Their success
relied on the development of positive and negative selection cycles, which allowed
the identification of orthogonal pairs that do not react to endogenous tRNA and
aminoacyl-tRNA synthetases (aaRS). Four main orthogonal aminoacyl-tRNA syn-
thetases have been developed since then: (1) the Methanococcus jannaschii
Tyr-tRNA synthetase (MjTyrRS)/tRNACUA pair, (2) the E. coli Tyr-tRNA synthe-
tase (EcTyrRS)/tRNACUA pair, (3) the E. coli Leu-tRNA synthetase (EcLeuRS)/
tRNACUA pair, and (4) the pyrrolysyl-tRNA synthetase (PylRS)/tRNACUA pairs
from certain Methanosarcina. The general rule for a successful orthogonal pair of
suppressor tRNA and aaRS is that the suppressor tRNA is not a substrate of
endogenous synthetases; the aaRS neither takes natural amino acids as substrates,
nor aminoacylates the endogenous tRNAs [17], although minor degree of crosstalk
between engineered and endogenous aaRS/tRNA/amino acids have been well-
documented. This review focuses primarily on the (EcTyrRS)/tRNACUA and
(PylRS)/tRNACUA pairs. The former one is the most widely used in eukaryotic
cells such as yeast, Xenopus oocytes, and mammalian cells compatible for efficient
expression of GPCR. For the (PylRS)/tRNACUA pair, although currently there are
few examples in studies of GPCRs, the major advantage is its high substrate side-
chain promiscuity, which leads to the successful encoding of Uaas with varieties of
chemical functionalities such as bioorthogonal handles [5, 18–20]. Researches on
the development and application of these pairs have been extensively reviewed
elsewhere [6, 7].
Transient Expansion The basic approach of incorporating Uaas into a GPCR was
established in mammalian HEK293 cells [21] and Saccharomyces cerevisiae [22]
using the (EcTyrRS)/tRNACUA pair through transient transfection (Fig. 2). In the
experimental procedure, plasmids encoding the gene of GPCR with an amber stop
codon, EcTyrRS, and the suppressor tRNACUA were transiently delivered to the
cells. Uaas were provided to the growth medium. Varieties of tyrosine and
pyrrolysine analogues have been successfully incorporated into GPCRs in response
to the amber stop codon. GCE methodology has also been established in animals
including Caenorhabditis elegans [23], Drosophila melanogaster [24], and mice
tissues [25–28]. Interests in modifying other components of the protein translation
apparatus have also gained momentum. Since suppression is in competition with
releasing factor (RF) to stop translation, strategies have evolved to modulate RF
levels [29] and EF-Tu [30] to increase the efficiency of Uaa incorporation during
protein translations by minimizing the competition processes.
Heritable Expansion By genetically integrating orthogonal RS and suppressor
tRNA into the genome mammalian cells [31], Caenorhabditis elegans [32],
Danio rerio [33], and Mus musculus [33, 34], stable cell lines or transgenic species
Fig. 2 Structure and function studies of GPCRs by site-specific incorporation of Uaas. Uaas
grouped according to their chemical properties, such as crosslinkers, spectroscopic probes, and
bioorthogonal handles. The genetic encoding of a Uaa (red star) into a GPCR is achieved by the
co-transfection of three plasmids that encoding the GPCR with the amber stop codon (TAG) at a
specific site (red dot) of interest, suppressor tRNA, and the aminoacyl tRNA-synthetase (aaRS).
The aaRS aminoacylates the suppressor tRNA in the presence of the Uaa. The aminoacylated
suppressor tRNA recognizes the UAG in the mRNA of GPCR, and participates in the protein
translation and read-through the stop codon to generate full-length functional GPCR. Successful
incorporation of various Uaas has been developed for cellular systems (in cellulo) including yeast,
Xenopus laevis oocytes, and mammalian cells. Transgenic animals including C. elegans, Dro-
sophila, zebra fish, and mice have been generated by stably integrating the aaRS/tRNA gene into
their genomes to achieve heritable genetic code expansion
with a heritable “expanded genetic code” have been successfully created. Those
animal models should allow diverse areas of GPCR biology to be investigated in a
system-wide manner, that heretofore have been limited mainly to the studies of
proteins in cell-based model systems.
3 Genetically Encoding Uaas to Study GPCR Structure

and Function Relationship
A network of interactions (interactome) underlines all GPCR signaling processes

involving ligands, GPCRs, and their signaling protein partners. Ligand binding
induces GPCRs to undergo a conformational change at the intracellular side, which
lead to receptor interactions with various cytosolic proteins and regulation by post-
translational modification such as phosphorylation and ubiquitination [35, 36]. The
dynamic nature of interactome makes the comprehensive mapping challenging.
Combining with GCE and photo-crosslinking Uaas, Sakmar, Huber, and colleagues
have developed a powerful technique to discover and characterize GPCR–ligand
interactions in the setting of living, intact cells [37, 38]. In the methodology, photo-
crosslinking Uaas have been incorporated at specific sites into the ligands or
200 M. Tian et al.
receptors and they are used in native cellular environments to identify specific
ligand–protein or protein–protein interactions. Along with the benefit of mapping
native interactions, light induced crosslinking offers high spatial and temporal
resolution to monitor interaction events. Spectroscopic techniques such as FTIR,
NMR, and fluorescent spectroscopy provide dynamic information of interactomes.
The p-azido-L-phenylalanine (AzF) as an infrared probe and F2Y as an NMR probe
have been successfully incorporated into GPCRs to provide functional insights.
3.1 Mapping Receptor and Peptide Ligand Interactions via

Crosslinking UAAs
The expression of crosslinking Uaas such as AzF, Bpa, tmdPhe, and Ffact provides
a means to capture a covalent GPCR–ligand complex (Fig. 2). Among those Uaa
AzF, p-benzoyl-L-phenylalanine (Bpa) [39], and tmdPhe [40] are all photo-induced
crosslinkers, whereas p-20 -fluoroacetylphenylalanine (Ffact) is a proximity induced
crosslinker which will react to nearby Cys [8]. The mapping of ligand-binding sites
has been successfully demonstrated on chemokine [41, 42], neurokinin [43], Ste2p
[22], and corticotropin releasing factor receptors [44, 45]. Photo-crosslinking
experiments were carried out in live cells prior to identification of crosslinked
products. When crosslinks occur, the site of the linkage on the receptor is known
since the photoreactive amino acid was genetically encoded at a specific known site
in the primary structure. In the case of chemokine and corticotropin releasing factor
receptors, crystal structure data or homology models were used to interpret quan-
titative crosslinking data.
3.1.1 CXCR4 and T140 Peptide Antagonist
CXC chemokine receptor 4 (CXCR4) belongs to the family A GPCR. It mediates

directed cell migration in development and during inflammation and cancer metas-
tasis, also known to be a co-receptor for cellular HIV-1 entry. T140, a 14-residue
cyclic peptide CXCR4 antagonist, blocks HIV-1 entry. Although earlier strategies
were able to resolve a peptide fragment of CXCR4 specifically bounds to T140, it
was not known how T140 binds the full-length CXCR4 to exert the mode of action.
To probe the site-specific binding interface between CXCR4 and T140, Huber,
Sakmar, and colleagues developed Bpa photo-crosslinking technology in CXCR4
to trap fluorescein-labeled T140 analogue [41]. Eight positions in CXCR4 were
chosen based on the decrease in T140 inhibition when mutated into Ala. After Bpa
were incorporated into the receptor, a unique UV-light-dependent crosslink specif-
ically between residue F189 and T140 peptide was found. Computational modeling
from the crystal structure of CXCR4 bound to CVX15 (a 16-residue peptide with
high homology to T140) was carried out to predict the possible orientations of Bpa.
Distance between the center of mass of the reactive carbonyl in Bpa to the nearest
atom in the CVX15 peptide for each of the possible orientations of Bpa at each
position was calculated. Bpa at position F189 was the only site to have a reasonably
high probability of being within 2–5 Å from the peptide, being within crosslinking
distance. The main ligand-binding pocket for family A GPCRs is defined as the
pocket between the extracellular segments of transmembrane helices (TM) III, V,
and VI. The CXCR4_CVX15 crystal structure shows that F189 is within extracel-
lular loop 2 (EC2) of CXCR4, which borders the main ligand-binding pocket of
CXCR4 and lies within a defined distance from bound T140. This study was the first
demonstration to combine Uaa site-directed photo-crosslinking technology with
available crystal structures to develop accurate models of GPCR signaling com-
plexes or “signalosomes.”
3.1.2 Neurokinin (NK) Receptors and Neuropeptide Substance P (SP)
The NK receptors belong to the rhodopsin-like class A 7-transmembrane receptors

and are divided into three subtypes: NK1, NK2, and NK3. NK1 is widely expressed
in both central and peripheral nervous systems. SP is a neuropeptide that mediates
numerous physiological responses, including transmission of pain and inflamma-
tion through the NK1 receptor. Previous mutagenesis studies and photoaffinity
labeling using ligand analogues suggested that the binding site for SP includes
multiple interactions in the outer N-terminal (Nt) segment and the second extra-
cellular loop (EC2) of NK1, but the overall results have not led to a satisfactory
refined model of how SP binds to the NK1 receptor. To map precisely the NK1
residues that interact with SP, Bpa was incorporated at 11 selected positions in the
Nt tail and 23 positions in the EC2 [43]. The 34 NK1-Bpa mutants were expressed
in mammalian HEK293 cells and characterized the ability to crosslink to a fluores-
cently labeled SP analog. UV activation of the NKI-SP complexes were carried out
to induce crosslinking where the Bpa was in close proximity to the bound
SP. Notably, 10 of the NK1-Bpa in the Nt segment and 4 of those with Bpa in
EC2 loop crosslinked efficiently to SP, indicating that these 14 sites are juxtaposed
to SP in the ligand-bound receptor. These results show that two distinct regions of
the NK1 receptor possess multiple determinants for SP binding.
3.1.3 Ste2p and Tridecapeptide Pheromone α-Factor
Ste2p is the GPCR for the tridecapeptide pheromone α-factor of Saccharomyces

cerevisiae. Although the receptor-pheromone pair has been used extensively as a
paradigm for investigating GPCR structure and function, the precise binding sites
between α-factor and the receptor remain elusive. Becker and colleagues have
established the Bpa incorporation into Ste2p using live yeast [22]. To enhance the
Bpa take-up, a dipeptide containing Bpa has been designed and demonstrated to be
effectively enhancing the delivery of Bpa into the cell. Several of the expressed
202 M. Tian et al.
Bpa-substituted Ste2p receptors exhibited high-affinity ligand binding, and incor-

poration of Bpa into Ste2p influenced biological activity as measured by growth
arrest of whole cells in response to α-factor. Incorporation of Bpa into Ste2p was
verified by mass spectrometric analysis, in addition, two Bpa-Ste2p mutants (F55 in
the TM1 and Y193 in EC2) were able to selectively capture α-factor after
photoactivation. This is one of the first experimental evidences successfully dem-
onstrating a photo-crosslinking Uaa replacement in a GPCR expressed in its native
environment and the use of a mutated receptor to photocapture a peptide ligand.
3.2 Mapping Receptor and Small-Molecule Ligand

Interactions via Crosslinking UAAs
3.2.1 CCR5 and Maraviroc
GPCR-mediated signaling can be affected by allosteric modulators. CC chemokine

receptor 5 (CCR5), similar to CXCR4, belongs to family A GPCRs and is the
primary co-receptor required for HIV-1 cellular entry and molecular target for
maraviroc. Although maraviroc has been demonstrated as the first GPCR-specific
HIV-1 entry inhibitor, its precise receptor drug binding interactions had not been
defined before the X-ray structure of the CCR5-maraviroc complex became avail-
able [46]. Using the same photo-crosslinking approach, Huber, Sakmar, and col-
leagues have incorporated Bpa and AzF in CCR5 to obtain structural information
about the allosteric binding site of maraviroc [39, 42]. Based on the earlier proposed
models of the maraviroc binding site in CCR5 and molecular dynamics simulations,
eight sites in CCR5 were chosen and mutated into Bpa or AzF. After UV
crosslinking, the radioactivity of [3H]-labeled maraviroc was quantified to probe
the binding since tritium is a sensitive detection tag for small-scale cell-based
assays. Significant tritium signals were detected in the CCR5-maraviorc complex
when AzF replaced I28 or W86, and Bpa replaced I28 or F109 after photo-
crosslinking. The crosslinking results were used to evaluate a model of the
CCR5-maraviroc complex and confirmed the early hypothesis of maraviroc binding
within the transmembrane helix bundle. Among the 11 key residues reported to be
critical for maraviroc binding in the high-resolution X-ray structure of CCR5-
maraviroc complex [46] came out after the crosslinking study, W86 and F109
were identified by the photo-crosslinking study. Interestingly, I28 was not identified
in the structure complex to be within crosslinking distance of maraviroc. The
authors already noticed this by making modeling before the complex structure
being available and provided a plausible explanation to suggest an additional
“docking site” site on the extracellular surface of CCR5 [42], which might be
relevant for its function as an HIV-1 entry blocker. This example demonstrated
the crosslinking method to be complementary to X-ray structural studies of
receptor-ligand complexes.
3.2.2 Corticotropin Releasing Factor Receptor Type 1 (CRF1R)

and Urocortin-1
Class B GPCRs are a family of 15 peptide receptors of high pharmacological

relevance to widespread diseases, such as diabetes and osteoporosis. Molecular
determinants regulating the activation of class B GPCRs by native peptide agonists
are largely unknown. To investigate the interaction between the corticotropin
releasing factor receptor type 1 (CRF1R) and its native 40-mer peptide ligand
Urocortin-1 directly (Ucn1) in mammalian cells, Wang and colleagues have thor-
oughly mapped the interactomes by combining AzF photo-crosslinking and novel
proximity induced ligation through Ffact [44, 45]. AzF was first genetically incor-
porated into the N-terminal domain of the receptor to map the Ucn1 binding
interface on CRF1R, based on previous studies of photoreactive Ucn ligands in
the receptors [47]. If the ligand is proximal to the AzF in the associated complex, it
is covalently captured by the crosslinking moiety upon UV irradiation and can be
detected in correspondence of the molecular weight (MW) of the receptor-ligand
adduct in western blot (WB). Among 145 CRF1R-AzF mutants analyzed, Ucn1-
specific bands were detected at the MW of the receptor-ligand adduct for
35 mutants, indicating that Ucn1 crosslinked at these receptor sites.
To pinpoint specific pairs of ligand and receptor residues proximal to each other
and position Ucn1 in the receptor-binding pocket, Ffact was incorporated into
CRF1R at the 23 sites that yielded the most intense signals in AzF-photo-
crosslinking. Cys was introduced into Ucn1 by substituting the hydrophilic resi-
dues. The click reaction occurs only when the Cys and Ffact are in close reciprocal
proximity and forms a covalent bond stable in reducing SDS-PAGE. Among a total
of 115 combinations, nine spatial constraints between specific residue pairs in
CRF1R and Ucn1 derived from the Cys-Ffact click experiments were used in
energy-based conformational modeling. The data were analyzed in the context of
the recently resolved crystal structure of CRF1R transmembrane domain and
existing extracellular domain structures, yielding a complete conformational
model for the peptide-receptor complex [48]. This model not only provides an
unprecedented panoramic map of the receptor–ligand interactions, but also yields
molecular insights on the mechanism of receptor activation and the basis for
discrimination between agonist and antagonist function in class B GPCRs.
3.3 Mapping Other Interactions

3.3.1 CXCR4 with mAb 12G5
Monoclonal antibodies (mAbs), which have emerged as an important class of

therapeutic biologicals, have demonstrated antiviral activity and have been
advanced for clinical applications in HIV-1 fusion and entry inhibition. Using
CXCR4 as a model system, Sakmar and co-workers introduced AzF at various
204 M. Tian et al.
positions in EC2 [49]. By using targeted loss-of-function studies and photo-

crosslinking in whole cells in a microplate-based format, the interactions of the
CXCR4-AzF mutants with mAb 12G5 were mapped. They have found that 12G5
crosslinked primarily to residues S178, Y184, and F189 in EC2 of CXCR4.
Mapping of the data to the crystal structure of CXCR4 showed a distinct mAb
epitope footprint with the photo-crosslinked residues clustered around the loss-of-
function sites. This whole-cell enzyme-linked immunosorbent assay provides a new
strategy for creating epitope maps of monoclonal antibodies that bind to GPCRs.
3.3.2 CCR5 with mAb PRO 140 and 2D7
Using the same immunosorbent assay, the epitope interactions between CCR5 and
PRO 140 together with CCR5 and mAb 2D7 have been studied in parallel
[49]. PRO 140 is a humanized mAb that inhibits human immunodeficiency virus-
1 cellular entry. Strikingly, PRO 140 and 2D7 produced distinct crosslinking
patterns on EC2 of CCR5, with PRO 140 crosslinked primarily to residues L174
and H175 at the aminoterminal end of EC2, and 2D7 crosslinked mainly to residues
Q170, Y176, and Y184. These results were mapped to the recent crystal structure of
CCR5 in complex with maraviroc [46], showing crosslinked residues at the tip of
the maraviroc binding crevice formed by EC2. As a strategy for mapping mAb
epitopes on GPCRs, this method is complementary to loss-of-function mutagenesis
results and should be especially useful for studying mAbs with discontinuous
epitopes.
3.3.3 Photobridges
Although the high efficiency of incorporating photo-crosslinkers through the GCE

in GPCRs has not yet allow the expression and purification of enough mutants for
X-ray crystallographic study, two studies conducted on photo-crosslinked proteins
expressed in E. coli revealed the actual structures of the “photobridges.” Yokoyama
and co-workers obtained the crystal structure of the crosslinked complex of the liver
oncoprotein gankyrin encoded with Bpa and the C-terminal domain of S6
proteasomal protein at 2.05 Å resolution [48]. The structure revealed that the
carbonyl group of the benzophenone of Bpa inserted at the specific site in gankyrin
formed a covalent bond exclusively with the Ca atom of Glu356 in S6C, showing
the high selectivity of Bpa. In the study conducted by Tippmann, Jones, and
co-workers, the crosslinked bond between AzF inserted in a specific site in GFP
and the chromophore has been determined by the crystal structure solved at 1.45 Å
resolution [50]. It revealed the nitrene of AzF attacks the meta carbon of the
chromophore phenol (from the residue of Y66) moiety to form an N-phenyl
crosslinking bond.
3.4 Detecting Dynamic Changes via Spectroscopic UAAs
Several amino acids carrying specific biophysical probes can also be directly
introduced into targeted positions in the proteins and used as photoreactive labels.
These include fluorescent probes, NMR probes, and infrared probes that can report
on the location of proteins within cells or the chemical environment of the region
of a protein in which they are installed. They can be useful probes of protein
localization and trafficking, protein conformation changes, and protein–protein
interactions.
3.4.1 Ligand-Induced Helical Movements in Rhodopsin Tracked by

Infrared Uaa
To study conformational dynamics of rhodopsin by photoactivation, the unique

infrared vibrational spectrum of the azido group was probed in a series of site-
specific AzF mutants. Upon photoactivation, the endogenous retinal group in
rhodopsin is isomerized from the cis to the trans form. Although the isomerization
coupled to a series of rearrangements in the transmembrane helices has been
extensively studied [51], the precise temporal sequence and the mechanism of the
resulting protein conformational changes were unclear. Fourier-transform infrared
(FTIR)-difference spectra were combined to show that the electrostatic environ-
ments of some residues change very early in the activation process [52], which has
proven to be a powerful tool for analyzing the function, mechanism, and dynamics
of proteins in membrane environments. Therefore, the azido label used in this study
senses in particular the polarity of its environment and the presence of electric fields
via the vibrational Stark effect, which is the shifting and splitting of spectral lines of
the azido probe due to the electric field change triggered by the protein conforma-
tional changes. AzF was further incorporated at five sites in helices 5 and 6 of
rhodopsin in mammalian cells, purified and reconstituted in lipid bilayer. The time-
resolved FTIR spectra around 2,100 cm1 report the dynamics of local chemical
environment that the azido group experiences as rhodopsin conformational transi-
tions from an inactive to an active conformation upon exposure to light. The FTIR
experiments provide first evidence that helix rearrangement occurs, and more
precisely a small rotation of helix 6 and movement of the cytoplasmic side of
helix 5 at the Meta I stage, and that receptor activation is defined by a final
movement of helix 6 [53]. Thus, the introduction of IR-active probe into proteins
can allow the precise dissection of the structural intermediates during the activation
of signaling. The combination of Uaa as biophysical probes with spectroscopic
techniques can be generally adapted to fluorescence, nuclear magnetic resonance
(NMR), and electron paramagnetic resonance (EPR) spectroscopy.
206 M. Tian et al.
3.4.2 Conformational Dynamics of Ghrelin Receptor (GhrR) Detected

by Site-Specific Fluorescent Labeling
Ligand-Induced Conformational Dynamics
Uaas such as AzF, AcF, and BCNF can serve as bioorthogonal handles to site-
specifically attach fluorescent labels to probe ligand-directed structural changes in
GPCRs. GhrR is a GPCR that binds ghrelin and plays a role in energy homeostasis
and regulation of body weight. It is associated with various physiological processes
including appetite control and food intake. Together with its endogenous ligand
ghrelin, GhrR is a promising drug target for metabolic disorders such as obesity.
Park and co-workers have demonstrated how conformational dynamics can be
measured in GhrR and Ghr by FRET analysis [54, 55]. AzF was site-specifically
introduced to GhrR in mammalian cell membranes and bioorthogonal labeled with
an Alexa647 using strain-promoted [3 + 2] alkyne-azide cycloaddition (SpAAC) at
six different sites. The Ghr was labeled with fluorescein at Lys-20. Alexa657 on the
GhrR and fluorescein in the ligand Ghr form a robust FRET-pair. In addition,
homogenous time-resolved fluorescence (HTRF) technology to reflect structural
integrity was developed to monitor ligand binding and ligand-dependent confor-
mational changes. It was found that GhrR-180AzF-Alexa647 was the most suitable
and non-perturbing tagging site to monitor ligand binding. Using GhrR-146AzF-
Alexa647, when adding Ghr or Abbott-13d, a small-molecule inverse agonist, there
was distinct HTRF signals reflecting specific conformations of GhrR induced by
two ligands. The approach enables monitoring dynamic intra- and intermolecular
interactions of GhrR with different ligands, providing a useful fluorescent-based
assay to study ligand-induced receptor conformations that are relevant to drug
design and discovery.
Dynamic Transition of the Receptor:Gq Complex
GhrR is also named as growth hormone secretagogue receptors (GHSR). In the

brain they are located in the hypothalamic ventromedial nucleus and arcuate
nucleus, as well as in ventral tegmental area dopamine neurons projecting to the
nucleus accumbens [56]. The neuroendocrine peptide hormone ghrelin acts through
GhrR to growth hormone secretion and food intake. GHSR-1a is a transcript variant
in the brain excising an intron and encodes the functional receptor that responds to
the agonist ghrelin. To demonstrate how GHSR-1a interacts with its G protein
partners, Damien and co-workers combined GHSR-1α labeling through site-specific
incorporation of AzF at F71 in the intracellular site of TM1 [57]. Alexa488 was
labeled using SpAAC reaction. The α subunit of the G protein trimer was labeled at
the N-term with a fluorophore Lumi-Tb through a classical reaction with its
N-hydroxysuccinimide (NHS) derivative at neutral pH. The GHSR-1α and the G
protein form a donor and acceptor pair that enables Lanthanide resonance energy
transfer (LRET) analysis. It was found that there were two equally populated recep-
tor:G protein complexes in the absence of ligand Ghr, whereas in the presence of full
agonist MK0677, the distribution of the two populations has changed suggesting
MK0677 triggers similar effect as the endogenous ligand Ghr. In contrast, in the
presence of antagonist GMV3011, there was no difference observed compared to the
ligand-free state. Strikingly, in the presence of inverse antagonist SPA, no significant
signal of the receptor:G protein complex could be measured, suggesting SPA disso-
ciates the complex. These data provide direct evidence of a mechanism for ghrelin
receptor-mediated Gq signaling in which transition of the receptor from an inactive to
an active conformation is accompanied by a rearrangement of a preassembled
receptor:G protein complex, ultimately leading to G protein activation and signaling.
Uaa Mediated Site-Specific Fluorescent Labeling
GCE to introduce bioreactive functionality into proteins in combination with

fluorescent labeling could overcome certain limitations of conventional GFP label-
ing such as interference with protein function to allow for site-specific labeling of
proteins. There are two kinds of approaches: (1) fluorescent Uaas directly serving as
the probe, (2) Uaas carrying bioorthogonal handles, which after the incorporation
of such Uaas into the protein, a fluorescent probe carrying a complementary
bioorthogonal function is site-specifically reacted with Uaas modified proteins.
One successful study using the first approach to label GPCR is the incorporation
of a fluorescent Ala derivative, 3-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-2,3-
diaminopropionic acid (NBD-Dap) into the NK2 receptors [58]. Using membrane
preparations of receptors expressed in Xenopus oocytes, this study determined
intermolecular distances between a fluorescently labeled antagonist and the fluo-
rescent NBD-Dap incorporated at several positions in the protein by measuring
fluorescence resonance energy transfer. Although a powerful demonstration, the
second approach to label receptors offers several advantages: (1) it enables a
modular approach for installing diverse probes with a single genetic system similar
to cysteine-mediated labeling chemistry; (2) compared to cysteine chemistry, it
circumvents the interference of endogenous cysteine residues, thereby enabling;
(3) it removes limitations that the translational machinery may place on the size of
fluorescent probes that can be used. Using this strategy, Sakmar, Huber, and
co-workers have pioneered in demonstrating the feasibility of site-specific labeling
of GPCRs through AzF directed bioorthogonal labeling [38, 59]. First, Saranga
et al. utilized the Staudinger ligation specific to azido moiety to conjugate a
triarylphosphine-conjugated FLAG peptide in to CCR5 [60, 61]. They have set
up a whole-cell-based ELISA approach to detect the modified AzF-CCR5 using
anti-FLAG mAb. Through optimized conditions, they managed to achieve labeling
and detection of CCR5 in living cells. They also demonstrated a preparative
strategy to obtain pure bioorthogonally modified GPCRs suitable for single-
molecule detection fluorescence experiments [62]. Using rhodopsin, the prototyp-
ical family A GPCR, as the model system, Tian et al. demonstrated the successful
208 M. Tian et al.
site-specific labeling of rhodopsin-AzF mutants by strain-promoted [3 + 2] azide–

alkyne cycloaddition (SpAAC) reaction with fluorophores carrying dibenzo-
cyclooctyne (DIBO). They further used rhodopsin-Alexa488 to measure the kinet-
ics of ligand uptake in a membrane-mimicking bicelle system [63–65]. These
studies demonstrate “a promising approach for labeling GPCRs in cells for study
in their native contexts” [66].
3.4.3 Desensitization Through Phosphorylation Induced Beta-Arrestin-

1 Interaction Mapped by NMR
Post-translational modifications through phosphorylation modulate the GPCR

desensitization process. Genetic code expansion has demonstrated successful incor-
porations of modified Tyr, including Tyr-nitration and Tyr-sulfation [6]. Since the
modification is introduced in a site-specific manner, the function of the modification
can be directly correlated. β-arrestins are adaptor proteins that function to regulate
G protein-coupled receptor (GPCR) signaling and trafficking. β-arrestins are ubiq-
uitously expressed and function to inhibit GPCR/G protein coupling, a process
called desensitization, and promote GPCR trafficking and arrestin-mediated sig-
naling. These interactions are facilitated by a conformational change in β-arrestin
that is thought to occur upon binding to a phosphorylated activated GPCR. Crystal
structures, one of a pre-activated splice variants and one bound to a GPCR
phosphopeptide, provided insights into the conformational changes upon phosphate
recognition. Scanning mutagenesis and spectroscopic studies complete the picture
of arrestin activation and receptor binding. Although the C-tail exchange mecha-
nism has been proposed, by which the C-tail of arrestin is released from its basal
conformation and replaced by the phosphorylated GPCR C-terminus with three
positively charged clusters could act as conserved arrestin phosphosensors, how the
specific phospho-barcodes control the precise interaction with arrestin remains to
be determined.
To demonstrate how distinct receptor phospho-barcodes are translated to specific
β-arrestin conformations and direct selective signaling, Wang, Sun, and co-workers
have analyzed V2-vasopressin receptor carboxy-terminal–phosphopeptide (V2Rpp)
binding to β-arrestin-1 by combining genetic code expansion and NMR spectroscopy
[13]. Uaa 3,5-difluorotyrosine (F2Y) was incorporated into β-arrestin-1 to determine
the interaction with phosphorylated V2Rpp peptides. Fifteen sites were selected
β-arrestin-1 to cover all phosphate-binding sites based on the crystal structure of
the V2Rpp/β-arrestin-1 complex. Conformational changes of β-arrestin induced by
the binding with phosphorylated V2Rpp peptides are revealed by fluorine-19 nuclear
magnetic resonance (19F-NMR) spectroscopy. Although all functional phospho-
peptides interact with a common phosphate-binding site in β-arrestin-1, distinct
phospho-interaction patterns between V2Rpp and GRK–phosphopeptides (GRKpps)
are deciphered by 19F-NMR. With its phosphate-binding concave surface, β-arrestin-
1 “reads” the message in the receptor phospho-C-tails and induces distinct structural
arrestin states and functions. It was also observed that only clathrin recognizes and
stabilizes GRK2-specific β-arrestin-1 conformations. Therefore, those multiple phos-

phate binding sites in the arrestin and phospho selective mechanism for arrestin
conformation enable arrestin to recognize abundant phosphorylation states of numer-
ous GPCRs.
3.5 Probing Helical Backbone Structure at Conserved

Proline Sites
Uaa mutagenesis offers a powerful way to make precise changes in the amino acid.
Dougherty, Lester, and co-workers have conducted a series of elegant Uaa muta-
genesis studies on ligand-gated ion channels [14, 67]. One of the examples they
have applied in GPCRs is the understanding of functional roles of proline in
Dopamine 2 (D2) receptor, which is a subtype of dopamine receptor and the main
receptor for all antipsychotic drugs [68]. Among 20 natural amino acids, proline
stands apart from the other amino acids. Its cyclic side chain uniquely shapes
protein structure and facilitates protein dynamics. To examine the functional roles
of five conserved TM proline residues in the D2 receptor, Uaas such as α-hydroxy
acids and proline analogues were employed. Proline analogues that vary the size of
the ring or introduce substituents can probe tolerance for subtle changes to the
proline side chain as well as cis-trans isomerization, which is critical for receptor
activation. It was found that the well-known tendency of proline to disrupt helical
structure is important at all sites in the D2 receptors, while no evidence was found
for a functional role for backbone amide cis-trans isomerization, another feature
associated with proline. Among all selected proline sites, four of them showed the
backbone hydrogen bond donor is critical for maintaining helical stability and
function. However, at one site in the TM5, a substituent on the backbone N appears
to be essential for proper function. Interestingly, the pattern in functional conse-
quences is mirrored in the pattern of structural distortions seen in recent GPCR
crystal structures.
4 Conclusions and Outlook
In this review, we described key examples of Uaas that can be incorporated in

GPCRs and their diverse applications. All GPCR processes are governed by ligand-
binding and protein–protein interactions that range from strong and effectively
permeate to weak and transient. Own to the eliminations of traditional mutagenesis
techniques, however, the vast majority of research has focused on strong interac-
tions and neglected weak transient contacts. Mapping the ligand-binding interac-
tions on a particular GPCR with genetically encoded crosslinking Uaas based on
available crystal structures or homology models has advanced significantly to
210 M. Tian et al.
understanding the transient nature of GPCR signaling complexes. Although varie-

ties of photo-crosslinking Uaas have been developed with different advantages and
disadvantages, up to know, AzF and Bpa are the most popular Uaas applied on six
GPCRs and eight types of GPCR–ligand complexes (see Table 1 for an overview)
due to an efficient expression protocol established in yeast [22] and HEK293
mammalian cells [16, 21, 44]. Another important factor to consider is the site in
the protein to introduce the Uaa which has to be in close vicinity to its binding
partner satisfying within 3–4 Å distance criteria to react with the ligands upon UV
Table 1 Published studies on encoding photo-crosslinking Uaas in GPCRs

Interactive Uaa Selected Success
GPCR partner incorporation sites Effective sites rate (%) Reference
Ste2p α-Factor Bpa 8 F55, Y193 25 [22]
ligand
CXCR4 T140 Bpa 8 F189 12.5 [41]
T140 AzF 8 F189 12.5 [41]
12G5 AzF 19 S178, Y184, F189 15.8 [48]
CCR5 Maraviroc Bpa 8 I28, F109 25 [42]
Maraviroc AzF 8 I28, W86 25 [42]
2D7 AzF 25 Q170, Y176, 12 [48]
Y184
PRO 140 AzF 25 L174, H175 8 [48]
NK1 SP Bpa 34 11L, 12S, 14N, 41.2 [43]
15I, 16S, 17T,
18N, 19T, 20S,
21E, E172, M174,
M181, I182
CRF1R Ucn1 AzF 145 Y73, E109, K110, 24.1 [44]
K111, K113,
Y116, A119,
N123, Y124,
W169, V172,
V176, T192,
Y195, N196,
H199, F260,
G261, K262,
P264. G265,
Y267, D269,
Y270, Q273.
L329, F330, F331,
V332, N333,
R341, F344, I345,
N348, S349
F fact 23 Q273, L329, 30.4 [45]
F330, N333, I345,
N348, S349
GRB2 EGFR, tmdPhe 10 F108, K109 20 [40]
SHC
activation [48]. From the available studies, the success rate of identifying a
crosslinkable site in a receptor to a ligand ranges from 8 to ~40%. Compared to
studies using Uaas as spectroscopic probes currently applied to five GPCRs
(Table 2), the success rate for effective labeling is in general higher (~47 to
~86%) than crosslinking. These examples demonstrate the power of Uaa mediated
labeling approach to uncover functional sites in a receptor and also dramatically
expanded the list of sites where conformational changes of the proteins occur.
Methodological advances in Uaa mutagenesis to obtain site-specifically modi-
fied proteins have facilitated to define a range of biological questions in GPCRs.
The technical convenience of the genetic code expansion approach has already
popularized the methodology and broadens the applications [6, 67, 69]. Uaas have
allowed detailed studies of receptors and proteins with exquisite molecular preci-
sion in a life cellular environment. However, we note that the use of GCE has thus
far been trisected to cells in tissue culture. The development of the transgenic
animals with expanded genetic code will broadly enable the studies of GPCRs in
multicellular organisms [23, 24, 32–34]. In the future, it may be possible to
incorporate photo-crosslinking Uaas in a living animal and determine the nature
Table 2 Published studies on GPCRs via spectroscopic Uaas

Interactive Uaa Selected Effective Success
GPCR partner incorporation Label sites sites rate (%) Reference
Rhodopsin AzF Azido 5 Y136, V227, 80 [49]
V250, M253
GhrR Ghr, Abbott- AzF Alexa647 14 Y106, C146, 85.7 [51]
13d V153, G158,
L162, V180,
E185, F203,
A204, L239,
F290, Q299
GHS-R1a Ghr, AzF Alexa488 1 F71 [52]
MK0677
GMV3011,
SPA
NK2 NKA, NBD-Dap NBD- 2 R103, F248 [53]
heptapeptide Dap
Rhodopsin AzF Alexa488 3 Y102, S144, [60]
V173
CCR5 AzF FLAG- 15 I23, N24, 80 [56]
Phos V25, K26,
Q27, T65,
A129, I217,
K219, L222,
C224, E262
V2Rpp β-arrestin-1 F2Y 19
F 15 R7, K11, 46.7 [62]
Y21, Y63,
K138, R165,
K294
212 M. Tian et al.
of the GPCRs signaling in vivo. New applications of Uaas will also emerge with the
diverse GCE technology approaches and optimization [28, 31, 70, 71]. We foresee
the incorporation of more efficient photo-crosslinker tmdPhe to trap GPCR
and novel partners, as demonstrated by Sakamoto, Yokoyama, and co-workers in
GRB2. By combining mass-spectrometry, two signaling-associated proteins (GIT1
and AF6) and the heterogeneous nuclear ribonucleoproteins F, H1, and H2 were
identified as novel direct binders of GRB2 which were previously unknown
[40]. Recent reports demonstrated that trans-cyclooctenes (e.g., BCNK) in combi-
nation with fluorescent tetrazine conjugates allow fast and efficient cell-based
labeling of various genetically modified proteins via inverse electron-demand
DielsAlder cycloaddition reaction (IEDDA) [18, 19, 71]. We also anticipate the
development of phosphorylated Uaas in mammalian cells for the elucidation of
signal transduction pathways in GPCRs and other spectroscopic probes such as
spin-labels reporting directly on GPCRs dynamics. Finally, the incorporation of
photo-active UAAs will allow the generation of light-sensitive receptors which
have potential in “optogenetics” [25, 26, 72, 73].
Acknowledgements We are grateful for M. Kazmi, T. He, T. Huber, and T.P. Sakmar for their
supports and advice. Financial support was provided by the Chinese Scholars Council (CSC
fellowship to M.T.), the Agence Nationale de la Recherche of France (ANR-JCJC grant to S.
Y.), and the National Natural Science Foundation of China (31528007 to S.Y. and C.Y.).
References
1. Rosenbaum DM, Rasmussen SG, Kobilka BK (2009) The structure and function of G-protein-
coupled receptors. Nature 459:356–363
2. Shoichet BK, Kobilka BK (2012) Structure-based drug screening for G-protein-coupled
receptors. Trends Pharmacol Sci 33:268–272
3. Granier S, Kobilka B (2012) A new era of GPCR structural and chemical biology. Nat Chem
Biol 8:670–673
4. Xiang J et al (2016) Successful strategies to determine high-resolution structures of GPCRs.
5. Daggett KA, Sakmar TP (2011) Site-specific in vitro and in vivo incorporation of molecular
probes to study G-protein-coupled receptors. Curr Opin Chem Biol 15:392–398
6. Chin JW (2014) Expanding and reprogramming the genetic code of cells and animals. Annu
Rev Biochem 83:379–408
7. Neumann-Staubitz P, Neumann H (2016) The use of unnatural amino acids to study and
engineer protein function. Curr Opin Struct Biol 38:119–128
8. Wang L (2016) Genetically encoding new bioreactivity. New Biotechnol. doi:10.1016/j.nbt.
2016.10.003
9. Grunbeck A, Sakmar TP (2013) Probing G protein-coupled receptor ligand interactions with
targeted photoactivatable cross-linkers. Biochemistry 52:8625–8632
10. Heckler T, Chang L, Zama Y, Naka T, Hecht S (1984) Preparation of ’2,(’3)-O-acyl-pCpA
derivatives as substrates for T4 RNA ligase-mediated “chemical aminoacylation”. Tetrahedron
40:87–94
11. Noren CJ, Anthony-Cahill SJ, Griffith MC, Schultz PG (1989) A general method for site-
specific incorporation of unnatural amino acids into proteins. Science 244:182
12. Nowak MW et al (1995) Nicotinic receptor-binding site probed with unnatural amino-acid-
incorporation in intact-cells. Science 268:439–442
13. Yang F et al (2015) Phospho-selective mechanisms of arrestin conformations and functions
revealed by unnatural amino acid incorporation and 19F-NMR. Nat Commun 6:8202
14. Beene DL, Dougherty DA, Lester HA (2003) Unnatural amino acid mutagenesis in mapping
ion channel function. Curr Opin Neurobiol 13:264–270
15. Wang L, Schultz PG (2001) A general approach for the generation of orthogonal tRNAs. Chem
Biol 8:883–890
16. Sakamoto K et al (2002) Site-specific incorporation of an unnatural amino acid into proteins in
mammalian cells. Nucleic Acids Res 30:4692–4699
17. Davis L, Chin JW (2012) Designer proteins: applications of genetic code expansion in cell
biology. Nat Rev Mol Cell Biol 13:168–182
18. Nikić I et al (2014) Minimal tags for rapid dual-color live-cell labeling and super-resolution
microscopy. Angew Chem Int Ed 53:2245–2249
19. Lang K, Chin JW (2014) Cellular incorporation of unnatural amino acids and bioorthogonal
labeling of proteins. Chem Rev 114:4764–4806
20. Tian H, Fürstenberg A, Huber T (2017) Labeling and single-molecule methods to monitor G
protein-coupled receptor dynamics. Chem Rev 117:186–245
21. Ye S et al (2008) Site-specific incorporation of keto amino acids into functional G protein-
coupled receptors using unnatural amino acid mutagenesis. J Biol Chem 283:1525–1533
22. Huang L-Y et al (2008) Unnatural amino acid replacement in a yeast G protein-coupled
receptor in its native environment. Biochemistry 47:5638–5648
23. Greiss S, Chin JW (2011) Expanding the genetic code of an animal. J Am Chem Soc
133:14196–14199
24. Bianco A, Townsley FM, Greiss S, Lang K, Chin JW (2012) Expanding the genetic code of
Drosophila melanogaster. Nat Chem Biol 8:748–750
25. Kang JY et al (2013) In vivo expression of a light-activatable potassium channel using
unnatural amino acids. Neuron 80:358–370
26. Zhu SJ et al (2014) Genetically encoding a light switch in an ionotropic glutamate receptor
reveals subunit-specific interfaces. Proc Natl Acad Sci U S A 111:6081–6086
27. Ernst RJ et al (2016) Genetic code expansion in the mouse brain. Nat Chem Biol 12:776–778
28. Zheng Y, Lewis Jr TL, Igo P, Polleux F, Chatterjee A (2017) Virus-enabled optimization and
delivery of the genetic machinery for efficient unnatural amino acid mutagenesis in mamma-
lian cells and tissues. ACS Synth Biol 6:13–18
29. Ryu Y, Schultz PG (2006) Efficient incorporation of unnatural amino acids into proteins in
Escherichia coli. Nat Methods 3:263–265
30. Park H-S et al (2011) Expanding the genetic code of Escherichia coli with phosphoserine.
Science 333:1151–1154
31. Elsässer SJ, Ernst RJ, Walker OS, Chin JW (2016) Genetic code expansion in stable cell lines
enables encoded chromatin modification. Nat Methods 13:158–164
32. Parrish AR et al (2012) Expanding the genetic code of Caenorhabditis elegans using bacterial
aminoacyl-tRNA synthetase/tRNA pairs. ACS Chem Biol 7:1292–1302
33. Chen Y et al (2017) Heritable expansion of the genetic code in mouse and zebrafish. Cell Res
27:294–297
34. Han S et al (2017) Expanding the genetic code of Mus musculus. Nat Commun 8:14568
35. Reiter E, Ahn S, Shukla AK, Lefkowitz RJ (2012) Molecular mechanism of β-arrestin-biased
agonism at seven-transmembrane receptors. Annu Rev Pharmacol Toxicol 52:179–197
36. Marchese A, Trejo J (2013) Ubiquitin-dependent regulation of G protein-coupled receptor
trafficking and signaling. Cell Signal 25:707–716
37. Huber T, Naganathan S, Tian H, Ye S, Sakmar TP (2013) Unnatural amino acid mutagenesis of
GPCRs using amber codon suppression and bioorthogonal labeling. Methods Enzymol
520:281–305
214 M. Tian et al.
38. Huber T, Sakmar TP (2014) Chemical biology methods for investigating G protein-coupled
receptor signaling. Chem Biol 21:1224–1237
39. Grunbeck A, Huber T, Sakmar TP (2013) Mapping a ligand binding site using genetically
encoded photoactivatable crosslinkers. Methods Enzymol 520:307–322
40. Hino N et al (2011) Genetic incorporation of a photo-crosslinkable amino acid reveals novel
protein complexes with GRB2 in mammalian cells. J Mol Biol 406:343–353
41. Grunbeck A, Huber T, Sachdev P, Sakmar TP (2011) Mapping the ligand-binding site on a G
protein-coupled receptor (GPCR) using genetically encoded photocrosslinkers. Biochemistry
50:3411–3413
42. Grunbeck A et al (2012) Genetically encoded photo-cross-linkers map the binding site of an
allosteric drug on a G protein-coupled receptor. ACS Chem Biol 7:967–972
43. Valentin-Hansen L et al (2014) Mapping substance P binding sites on the neurokinin-1
receptor using genetic incorporation of a photoreactive amino acid. J Biol Chem
289:18045–18054
44. Coin I et al (2013) Genetically encoded chemical probes in cells reveal the binding path of
urocortin-I to CRF class B GPCR. Cell 155:1258–1269
45. Xiang Z et al (2013) Adding an unnatural covalent bond to proteins through proximity-
enhanced bioreactivity. Nat Methods 10:885–888
46. Tan Q et al (2013) Structure of the CCR5 chemokine receptor–HIV entry inhibitor maraviroc
complex. Science 341:1387–1390
47. Kraetke O et al (2005) Photoaffinity cross-linking of the corticotropin-releasing factor receptor
type 1 with photoreactive urocortin analogues. Biochemistry 44:15569–15577
48. Sato S et al (2010) Crystallographic study of a site-specifically cross-linked protein complex
with a genetically incorporated photoreactive amino acid. Biochemistry 50:250–257
49. Ray-Saha S, Huber T, Sakmar TP (2014) Antibody epitopes on G protein-coupled receptors
mapped with genetically encoded photoactivatable cross-linkers. Biochemistry 53:1302–1310
50. Reddington SC et al (2013) Different photochemical events of a genetically encoded phenyl
azide define and modulate GFP fluorescence. Angew Chem Int Ed 52:5974–5977
51. Sakmar TP, Menon ST, Marin EP, Awad ES (2002) Rhodopsin: insights from recent structural
studies. Annu Rev Biophys Biomol Struct 31:443–484
52. Ye S, Huber T, Vogel R, Sakmar TP (2009) FTIR analysis of GPCR activation using azido
probes. Nat Chem Biol 5:397–399
53. Ye S et al (2010) Tracking G-protein-coupled receptor activation using genetically encoded
infrared probes. Nature 464:1386–1389
54. Park M et al (2015) Bioorthogonal labeling of ghrelin receptor to facilitate studies of ligand-
dependent conformational dynamics. Chem Biol 22:1431–1436
55. Park M, Tian H, Naganathan S, Sakmar TP, Huber T (2015) Quantitative multi-color detection
strategies for bioorthogonally labeled GPCRs. Methods Mol Biol 1335:67–93. G protein-
coupled receptors in drug discovery: methods and protocols
56. Malenka R, Nestler E, Hyman S (2009) Neural and neuroendocrine control of the internal
milieu. In: Molecular pharmacology. A foundation for clinical neuroscience, 2nd edn.
McGraw-Hill Medical, New York, pp. 265–266
57. Damian M et al (2015) Ghrelin receptor conformational dynamics regulate the transition from
a preassembled to an active receptor: Gq complex. Proc Natl Acad Sci 112:1601–1606
58. Turcatti G et al (1996) Probing the structure and function of the tachykinin neurokinin-2
receptor through biosynthetic incorporation of fluorescent amino acids at specific sites. J Biol
Chem 271:19991–19998
59. Tian H, Sakmar TP, Huber T (2016) A simple method for enhancing the bioorthogonality of
cyclooctyne reagent. Chem Commun 52:5451–5454
60. Naganathan S, Ye S, Sakmar TP, Huber T (2013) Site-specific epitope tagging of G protein-
coupled receptors by bioorthogonal modification of a genetically encoded unnatural amino
acid. Biochemistry 52:1028–1036
61. Naganathan S, Grunbeck A, Tian H, Huber T, Sakmar TP (2013) Genetically-encoded

molecular probes to study G protein-coupled receptors. J Vis Exp e50588
62. Naganathan S et al (2015) Multiplex detection of functional G protein-coupled receptors
harboring site-specifically modified unnatural amino acids. Biochemistry 54:776–786
63. Tian H, Sakmar TP, Huber T (2012) Site-specific labeling of genetically encoded azido groups
for multicolor, single-molecule fluorescence imaging of GPCRs. Methods Cell Biol
117:267–303
64. Tian H, Sakmar TP, Huber T (2015) Micelle-enhanced bioorthogonal labeling of genetically
encoded azido groups on the lipid-embedded surface of a GPCR. Chembiochem 16:1314–1322
65. Tian H et al (2014) Bioorthogonal fluorescent labeling of functional G-protein-coupled
receptors. Chembiochem 15:1820–1829
66. Manglik A, Kobilka B (2014) The role of protein dynamics in GPCR function: insights from
the β 2 AR and rhodopsin. Curr Opin Cell Biol 27:136–143
67. Pless SA, Ahern CA (2013) Unnatural amino acids as probes of ligand-receptor interactions
and their conformational consequences. Annu Rev Pharmacol Toxicol 53:211–229
68. Van Arnam EB, Lester HA, Dougherty DA (2011) Dissecting the functions of conserved
prolines within transmembrane helices of the D2 dopamine receptor. ACS Chem Biol
6:1063–1068
69. Krall N, da Cruz FP, Boutureira O, Bernardes GJ (2016) Site-selective protein-modification
chemistry for basic biology and drug development. Nat Chem 8:103–113
70. Herner A, Lin Q (2016) Photo-triggered click chemistry for biological applications. Top Curr
Chem 374:1
71. Serfling R, Coin I (2016) Incorporation of unnatural amino acids into proteins expressed in
mammalian cells. Methods Enzymol 580:89–107
72. Klippenstein V, Plested AJ (2014) Probing the channel gating of a glutamate receptor with a
photoactive unnatural amino acid. Biophys J 106:29A
73. Tian M, Ye S (2016) Allosteric regulation in NMDA receptors revealed by the genetically
encoded photo-cross-linkers. Sci Rep 6:34751
Top Med Chem (2019) 30: 217–252
DOI: 10.1007/7355_2017_2
Fluorescent-Based Strategies to Investigate

G Protein-Coupled Receptors: Evolution
of the Techniques to a Better Understanding
Orestis Faklaris, Joyce Heuninck, Amandine Falco, Elise Goyet,

Jurriaan M. Zwier, Jean-Philippe Pin, Bernard Mouillac, Julie Perroy,
and Thierry Durroux
Abstract G protein-coupled receptors are key proteins in the regulation of most of

the physiological responses. Their conformations are generally oscillating between
inactive and active forms leading to the activation of no, a few, or many signaling
pathways. Although receptors can spontaneously adopt these various conforma-
tions, their interactions with ligands, other G protein-coupled receptors, or intra-
cellular proteins (G proteins, arrestins, etc.) can stabilize one of these
conformations, leading to specific cellular responses. The identification of the
partners interacting with the G protein-coupled receptors and the dynamics of
these interactions is therefore crucial to fully understand receptor functioning.
Although it is crucial, it remains nevertheless ambitious and difficult to achieve
this goal. In the last two decades, various technical strategies have been developed
to investigate molecular complexes and their dynamics. In this review, we will
focus on recent technological breakthroughs in fluorescent-based techniques and
their impact on the understanding of G protein-coupled receptor functioning. We
will give particular attention to resonance energy transfer-based strategies, their
advantages, and drawbacks and to other microscopy based techniques which are
efficient to investigate stability, mobility, and dynamics of molecular complexes at
the cell surface.
O. Faklaris
ImagoSeine core facility – Institut Jacques Monod – Université Paris Diderot/CNRS – UMR
7592, 15 rue Hélène Brion, Paris Cedex 13 75205, France
J. Heuninck, A. Falco, E. Goyet, J.-P. Pin, B. Mouillac, J. Perroy, and T. Durroux (*)
IGF, CNRS, INSERM, Université de Montpellier, Montpellier 34094, France
Université Montpellier 1 and 2, Montpellier, France
e-mail: thierry.durroux@igf.cnrs.fr
J.M. Zwier
Cisbio Bioassays, BP 84175, Codolet 30200, France
218 O. Faklaris et al.
Keywords G-protein coupled receptors, Luminescence-based strategy

developments, Protein–protein interactions, Resonance energy transfer, Single
particule tracking, Spatio-temporal network
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218
2 Resonance Energy Transfer Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220
2.1 Principle of FRET . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220
2.2 BRET Strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
2.3 Time-Resolved FRET Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
2.4 Labeling Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226
2.5 Relevance of Time-Resolved FRET Strategies to Study GPCRs . . . . . . . . . . . . . . . . . . . 228
3 New Strategies to Investigate G Protein-Coupled Receptors into More Details . . . . . . . . . . 231
3.1 Microscopy vs. Plate-Reader . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
3.2 Resonance Energy Transfer Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232
3.3 Complementary Fluorescence-Based Techniques to Study GPCR . . . . . . . . . . . . . . . . . . 239
4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245
1 Introduction
Getting a thorough understanding of the role of a molecule in a cellular process is often

an ambitious and long-term project. It requires the identification of the interacting
partners, their location, and their dynamics. Such goals are often out of reach for
many molecules since it requires important technological developments. In the present
review, we will focus on recent technological breakthroughs in fluorescent-based
techniques made in the last two decades, which give the opportunity to investigate in
more detail the spatio-temporal network of G protein-coupled receptors (GPCRs).
GPCRs are a success story since they constitute the largest membrane protein
family. They participate to the control of all physiological functions and logically
constitute the target of about 30% of the drugs on the market. Paradoxically, their
functioning is far from being well understood and recent investigations show that
the regulation of their activity is ever more complex than initially thought.
GPCRs, first considered as monomeric seven-transmembrane proteins, play an
important role in the transduction of the signal. They generally exhibit a high
selectivity for a small number of natural ligands, which induce conformational
changes of the receptor. These changes trigger in a second time the interaction of
the receptor with a G protein and the activation of a signaling pathway. During the
last decades, this scheme of transduction has been revised many times and GPCRs
appear now as complex signal integrating platforms.
Firstly, it has been shown that GPCRs are able to interact together to form homo- or
hetero-oligomers depending on whether the receptors are similar or not. The nature of
the interacting GPCRs can impact either the binding properties of the GPCR of interest
or its coupling properties [1]. Hence it can influence the interaction of GPCRs with
Fluorescent-Based Strategies to Investigate G Protein-Coupled Receptors:. . . 219
others partners such as G proteins or β-arrestins. The relevance of the existence of GPCR
oligomers in physiology remains a matter of debate since most of the experiments have
been done on GPCRs over-expressed in cell lines. The direct interaction of GPCRs
expressed in native tissues has only been demonstrated for a small number of GPCRs
[2–4]. Moreover, first considered as stable complexes, GPCR oligomers are now
described as dynamic complexes oscillating between monomeric, dimeric, and higher
order oligomeric forms. One of the parameters driving the oligomerization process
seems to be the GPCR density. It is noteworthy that the receptor density at the cell
membrane is not homogeneous and depends on the clustering of receptors in micro- or
nano-domains in the membrane. Therefore, cellular processes facilitating the migration
of GPCRs to high-density domains would probably have a positive impact on GPCR
oligomerization.
Secondly, GPCRs have been shown to couple simultaneously to various signal-
ing pathways and not only to a unique one. This process named “functional
selectivity” or “differential coupling” can be influenced by two main parameters:
(1), the receptor location. For example, targeting of the oxytocin receptor inside or
outside caveolar domains modulates the coupling to G proteins and leads to
different patterns of ERK1/2 and EGFR activation [5]; (2), and the nature of the
ligand. Some ligands called full agonists induce the activation of all the signaling
cascades while others, biased agonists, activate only a small number of signaling
pathways [6].
Thirdly, GPCRs internalize after their activation by agonists. Various routes of
internalization can be followed by the receptor, either a short route leading to the
recycling of the receptor to the membrane or a long route resulting in receptor degra-
dation. A receptor can follow one or the other route depending on its interacting
partner [7].
These data fully illustrate that the understanding of the GPCR functioning
requires the identification of its interacting partners, the location of the complexes,
and the investigation of their dynamics.
About two decades ago, interactions between GPCRs and various partners were
suggested by western blot and co-immunoprecipitation-based approaches. Despite
the existence of false-positives or false-negatives, these techniques are efficient to
demonstrate the presence of molecules within the same molecular complex but
cannot prove the direct interactions between the partners. In the same manner,
molecule co-location in microscopy was often considered as an evidence of the
existence of a molecular complex. Although these techniques were clearly inade-
quate to precisely investigate complex existence or dynamics, they undoubtedly
provide the biological basis encouraging the development of new strategies.
In the 1990s, resonance energy transfer (RET) based strategies became reference
techniques to investigate direct interactions between molecules. In most of the
variants of the RET techniques – F€orster (or fluorescence) RET (FRET), biolumi-
nescence RET (BRET), and time-resolved FRET (TR-FRET) – the signal only
occurs if the partners are in close proximity, compatible with a direct interaction.
The last two methods exhibit a high signal-to-noise ratio, explaining their success to
investigate either GPCR oligomerization or signaling pathway activation. These

methods have recently been adapted to microscopy.
Although these techniques are very efficient for sensing close molecular inter-
actions, complementary techniques are needed to prove GPCR oligomerization
and/or dynamics. The last two decades, original microscopy (single-particle track-
ing, super-resolution microscopy – PALM) and spectroscopy techniques (FCS)
have thrown light on the dynamics and stoichiometry of GPCRs. Table 1 summa-
rizes the various techniques developed during the last two decades and proposes an
overview on the evolution of GPCR oligomerization studies that will be examined
in this review.
2 Resonance Energy Transfer Strategies
2.1 Principle of FRET
FRET was first formalized by Theodor F€orster in the middle of the twentieth
century [8]. FRET strategies are based on a non-radiative resonance energy transfer
between a donor and an acceptor. The efficiency of the transfer is regulated by three
parameters: (1), the energy compatibility between the donor and the acceptor. It
correlates to the importance of the overlap of the emission spectrum of the donor
and the excitation spectrum of the acceptor; (2), the orientation of the probes: the
RET is maximal and minimal when the transition dipole moments of the probes are
parallel and perpendicular, respectively; (3), the distance between the probes. The
energy transfer occurs only if the fluorophores are in a close proximity and the
efficiency of the transfer is inversely proportional to the sixth power of the distance
(r). The efficiency is given by:
R60
E¼
R60 þ r 6
where R0 is the F€orster distance and corresponds to the distance for which E is
equal to 0.5. Although R0 depends on the pair of fluorophores, it is generally in the
range of 30–80 Å. Because the RET occurs only if the donor and the acceptor are in
very close proximity, it is generally considered as a strong indicator of direct
interactions of molecules which carry the fluorophores.
The first FRET experiments performed on GPCRs were based on receptors fused
to fluorescent proteins. Although these fluorescent proteins are bright and the
labeling of receptors very efficient, the signal-to-noise ratio obtained with fluores-
cent protein pairs such as BFP/GFP or CFP/YFP as donor/acceptor is generally
weak. Various factors can impact this ratio: (1), the autofluorescence of the
biological preparation or the medium; (2), the direct excitation of the acceptor at
the donor excitation wavelength; (3), the emission of the donor at emission
Table 1 Overview of fluorescent techniques used to study GPCR oligomerization
aTechniques Format Advantages Drawbacks Results for GPCRs Perspectives
Preliminary Fluorescence Microscopy Compatible with Diffraction-limited res- Imaging of receptor Renewal interest
colocalization direct or indirect olution unable to char- colocalization for super-
fluorescent labeling of acterize direct (~250 nm) resolution
GPCRs interactions microscopy
Co- Blot Detect and identify No characterization of Receptor interactions
immunoprecipitation receptor interactions direct interactions in larger protein
in native tissue between receptors complexes
FRET-based FRET with fluores- Plate-reader Direct labeling with – No direct measure- – GPCR oligomeri-
cent proteins (FPs) Microscopy FPs ment of FRET zation
Detect direct interac- – Use of indirect – Receptor confor-
tions (<10 nm) measures (pbFRET. . .) mational changes
BRET Plate-reader Higher signal speci- No distinction between – GPCR oligomeri- Development of
ficity through surface and intra- zation brighter luciferases
bioluminescence cellular receptor – Signaling protein
interactions recruitment
Microscopy – High signal-to- – Not compatible with – GPCR oligomeri-
noise ratio wild type GPCRs zation
– Analysis of intra- – GPCR signaling
cellular complexes
– Multiplexing
measurement
Time-resolved Plate-reader – Simple measure – Indirect labeling • GPCR oligomeri- Development of
Fluorescent-Based Strategies to Investigate G Protein-Coupled Receptors:. . .
FRET of TR-FRET step zation: new cryptates of

– Study of native – Intracellular label- – With covalent lanthanides
receptors with fluo- ing requires cell labeling Application on
rescent ligands permeabilization – Ex vivo with fluo- native tissues
rescent ligands
(continued)
221
Table 1 (continued)
222
aTechniques Format Advantages Drawbacks Results for GPCRs Perspectives

– Multiplexing • Binding assays
measurement • Signaling assays
Microscopy – Multicolor – Imaging of GPCR
TR-FRET imaging oligomers
– Compatible with – Evidence of
kinetics experiments GPCR oligomer
internalization
Complementary FRAP Microscopy Image receptor diffu- Measurement averaged Study of oligomer
sion in live cells over numerous fluores- stability
cent receptors
FCS Microscopy Additional informa- Diffraction-limited res- Receptor diffusion,
tion brought by cross- olution unable to char- complex stoichiome-
correlation acterize direct try, effect of ligand
calculations interactions binding
SPT Microscopy Tracking of individ- Adapt fluorescent Surface receptors
ual receptor diffusion labeling/density (co-) diffusion
Improved spatial Resolution still unable Conclude to dimer-
resolution to characterize direct ization
interactions (~30 nm) Measure monomer–
dimer equilibrium
sptFRET Microscopy Characterize direct Not yet implemented Potentially confirm
interactions for single for GPCR direct interactions
complexes oligomerization indicated by SPT
PALM Microscopy – Super resolution Potential photophysical Existence of GPCR Combination with
microscopy artifacts (for example, monomers other techniques:
– Study of protein molecule blinking) sptFRET or
organization in dense sptPALM
samples
FRET fluorescence resonance energy transfer, FP fluorescent protein, pbFRET photobleaching FRET, BRET bioluminescence resonance energy transfer,
TR-FRET time-resolved FRET, FRAP fluorescence recovery after photobleaching, FCS fluorescence correlation spectroscopy, SPT single particle tracking,
O. Faklaris et al.
sptFRET single-particle tracking FRET, PALM photo-activation light microscopy, sptPALM single-particle tracking PALM
wavelength of the acceptor; (4), the dynamic FRET resulting from random collision
of donor and acceptor diffusing in the medium.
2.2 BRET Strategy
Two alternative strategies were developed to increase the signal-to-noise ratio. The
first one is BRET. BRET is a naturally occurring phenomenon. The photoprotein
Renilla luciferase (Rluc) purified from sea pansy (Renilla reniformis) emits blue
light upon oxidation of its substrate. When GFP and Rluc are associated, GFP
accepts the energy from Rluc and emits a green light.
The difference between FRET and BRET stands on the nature of the donor entity.
The BRET donor is a bioluminescent protein, classically a luciferase. Hence, by
opposition to FRET, BRET does not require any light stimulation of the donor.
Instead, the light emitted by catalytic oxidation of the luciferase’s substrate initiates
the BRET process. In the absence of fluorescence excitation, BRET circumvents all
the drawbacks linked to the use of light stimulation such as autofluorescence of the
cells, direct excitation of the acceptor entity by the light used to excite the donor, or
photobleaching of the fluorophores. Consequently, the noise of the BRET is dramat-
ically reduced compared to FRET, which confers to this technology an excellent
signal-to-noise ratio [9]. Beside its excellent sensitivity, BRET offers an advanta-
geous method to study protein–protein interactions in living cells without the pho-
totoxic effects of prolonged light illuminations or undesirable activation of
photosensitive biological processes. As a counterpart, a second atypical property of
the BRET technology also arises from the donor nature: the low light intensity
intrinsic to the bioluminescent process. This limitation has hampered for a while
the use of BRET in microscopy to locate precisely protein–protein interactions at a
subcellular level. However, recent advances in physical developments, and in par-
ticular the use of sensitive cameras, have overcome this difficulty (see Sect. 3.2.1).
Taking advantage of the spectral properties of BRET-compatible donor and
acceptor entities, several generations of BRET have been developed and combined
(Fig. 1). As for FRET, the only absolute requirement when choosing efficient BRET
pairs stands on a strong overlap between the donor emission and acceptor excitation
spectra. BRET is a ratio-metric measurement of the light emitted by the acceptor
over the light emitted by the donor. The ratio resulting from the energy transfer per
se can be easily distinguished from the basal BRET signal coming from the
overflow of the donor light into the acceptor channel.
The first generation of BRET, BRET1, uses the energy transfer between Rluc as
donor and YFP as acceptor [10, 11]. Upon catalytic oxidation of the Rluc substrate,
Coelenterazine H, a blue light is emitted (Emission peak centered on 480 nm).
Concomitantly, the non-radiative energy transfer excites the YFP, which in turn
emits light at its characteristic wavelength (Emission peak at 535 nm). BRET2
relies on the energy transfer between Rluc oxidation of the substrate DeepBlueC
(Em peak ¼ 395 nm) as donor and GFP2 as acceptor (Em peak ¼ 510 nm) [12]. The
BRET 3
BRET 1
Donor Acceptor
emission emission
Light intensity
BRET 2
400 480 510 530 570 600
Wavelength (nm)
Fig. 1 Schematic representation illustrating the various BRET generations. The normalized
emission spectra of donors and acceptors are drawn in dashed and solid lines, respectively.
BRET1 consists in the transfer of energy resulting from the oxidation of Coelenterazine H by
Rluc (light-blue line, Em peak ¼ 480 nm) to the acceptor YFP (yellow line, EM peak ¼ 535 nm). It
is noteworthy that the donor and acceptor emissions significantly overlap. BRET 2 results from the
energy transfer between the Rluc oxidation of DeepBlue C as donor (dark-blue line, Em
peak ¼ 395 nm) and the GFP variant, GFP2, as acceptor (green line, Em peak ¼ 510 nm).
BRET 3 relies on the combination of Rluc–Coelenterazine H (light-blue line Em peak ¼ 480 nm)
as donor and a red shifted acceptor, mOrange (orange line, Em peak ¼ 562 nm). Note that the
donor emission intensity in BRET2 is lower than for BRET1 and 3. Moreover, the spectral
separation between donor and acceptor emission is substantially improved in BRET2 and
3 (115 nm and 82 nm, respectively) compared to BRET1 (55 nm)
spectral separation between donor and acceptor emission is substantially improved

in BRET2 compared to BRET1, which significantly decreases the basal BRET
signal coming from the donor overflow in the acceptor detection channel, improv-
ing the detection of subtle BRET variations. However, the disadvantage of BRET2
compared to BRET1 is the 100 times lower intensity of the donor-emitted light,
which hampers to work with low protein expression levels. By combining the Rluc–
Coelenterazine H (Em peak ¼ 480 nm) as donor and an acceptor with an emission
peak shifted to the red (Em peak ¼ 562 nm), mOrange, BRET3 allows to work with
low protein expression levels using an increased spectral separation of donor and
acceptor emission spectra [13]. Thus, BRET1 and 3 will be preferred to monitor
interactions between proteins expressed at low levels, and BRET2 and 3 will favor
the detection of subtle BRET changes. Interestingly, different BRET generations
can be successfully combined to monitor several interactions at the same time
[14, 15]. Additionally, bimolecular-fluorescence complementation-BRET (BiFC-
BRET) [16, 17], bimolecular luminescence complementation-BiFC (BiLC-BiFC)
[18, 19], and complemented donor-acceptor-RET (CODA-RET) [20] can be used to
detect interactions between high order protein complexes.
Numerous efforts have been made to develop new BRET donors that would emit
more light. Donor emission can indeed be significantly improved by using Rluc
mutants with improved quantum efficiency and/or stability, such as Rluc8 [21]. The
nature of the substrate can also slightly affect the intensity or duration of light
emission [22, 23]. Recently, a smaller luciferase Nano Luciferase (Nluc), described
to be much brighter than Rluc8 [24, 25], has proven to significantly enhance the
resolution of BRET imaging [26].
2.3 Time-Resolved FRET Strategies
Time-resolved FRET methods constitute a second elegant strategy to improve the

signal-to-noise ratio [27–30]. It hinges on three aspects. Firstly, in time-resolved
FRET, the donors are cryptates or chelates of lanthanide ions, generally europium
or terbium. Eu3+ and Tb3+ display original luminescent properties since they have a
very long-lived emission, in the range of 1 ms, i.e., about 100,000 times greater than
conventional fluorophores (in the 10 ns range). It is noteworthy that lanthanide
emissions are not strictly speaking fluorescence [29, 30] but follow the same rules
regarding energy transfer and this is the reason why their luminescence energy
transfer is assimilated to FRET. These long-lived emissions give the opportunity to
introduce a delay between the excitation of the sample and the fluorescent mea-
surement. During this delay, all the short-lived fluorescence will be eliminated and
only long-lived fluorescence will be measured in the time window (Fig. 2). More-
over, europium and terbium cryptates are excited between 300 and 350 nm, display
very large Stoke shifts, and have multiple emission. Indeed, for example, Lumi4-
terbium (Lumi4®-Tb) that we often use displays four emission bands around
490, 550, 585, and 620 nm. Lumi4-Tb is therefore compatible with various accep-
tors to perform FRET, not only with fluorescein- or d2-like fluorophores but also
with non-conventional fluorophores such as quantum dots that emit at 605 and
705 nm (see Fig. 2). Finally, emission of lanthanide cryptates is weakly polarized. It
is due to complex energy transfer between the cryptate itself and terbium or
europium. Therefore, the absence of polarization makes that time-resolved FRET
is hardly sensitive to the relative orientation of the donor and the acceptor. It is
noteworthy that the cryptate plays different roles: the role of an antenna to increase
the emission yield of lanthanides [29, 30]; the role of a linker to graft lanthanides on
various molecules, and the role of a cage to protect lanthanides from water
quenching [29, 30].
The time selectivity, the spectral compatibility, and the weak dependence on the
relative orientation contribute to notably increasing the signal-to-noise ratio and
therefore the sensitivity of TR-FRET-based approaches.
Short-lived fluorescence:
(free acceptor, autofluorescence, …) Long-lived
fluorescence
Fluorescence
Principle of FRET
Time-Resolved FRET
Free donor
Time
Excitation Emission
Setup Flash lamp or Time delay Fluorescent measurement

Laser 337 nm (Pulse generator *) (PMT or Camera in Multigatemode *)
or 349 nm* DCM Em. Filter Em. Filter Em. Filter
Plate reader T400LP 520/15 * 605/15 * 700/75 *
Microscope *
Absorption / Fluorescence
Green acceptor QD605 Red acceptor/ QD710

FRET
30000
20000
10000
0
300 350 400 450 500 550 600 650 700 750
wavelength (nm)
Fig. 2 Principle of time-resolved FRET: time-resolved FRET is based on two properties of the
luminescence of europium and terbium cryptates: their long-lived emission and their spectral
compatibility with different fluorophores. Europium and terbium cryptates display a long-lived
fluorescence (in the range of 1 ms). The time delay between the excitation of the sample and the
measurement of the fluorescence in the time window (upper panel, yellow window) allows the
separation of the short-lived fluorescence (red line) due to the direct excitation of the acceptor or
the autofluorescence of the sample, and the long-lived fluorescence due to free donor emission
(light blue line) or FRET between the donor and the acceptor (dark blue line). Terbium cryptate
displays four bands of emission (lower panel) and is therefore compatible to transfer its energy not
only to fluorescein-like and d2-like fluorophores but also to 607 and 710 quantum dots (QD).
Specification of excitation and emission filters and dichroic mirrors have been chosen in order to
avoid as much as possible contamination of the emission of the donor or the acceptor in the
acceptor or donor fluorescence channel, respectively. The specifications indicated on the diagram
are those chosen for the microscope setup
2.4 Labeling Strategies
Depending on the RET strategies, various experimental methods exist to label mole-
cules of interest. Lanthanide cryptates or conventional organic fluorophores can be
linked by chemical means. Such strategies have been developed to synthesize, for
example, fluorescent analogs of cAMP or IP1 [31] to develop assays to measure second
messengers. Chemical approaches have also been used to synthesize fluorescent ligands,
which in turn bind to the receptor with a high affinity and therefore allow a specific
non-covalent receptor labeling [32–35]. Surprisingly, although no general conclusion
can be drawn from a few examples, the size of the fluorophores, which can be larger than
the one of the ligand, is not necessarily prejudicial to get a fluorescent ligand exhibiting a
high affinity for the receptor. By contrast, the nature of the fluorophore is important, for
example, the substitution of fluorescein to d2 can dramatically increase or decrease
ligand affinity for the receptor. Fluorescent ligand-based assays have been used to
investigate receptor binding properties (Tag-lite® assays) [35–38] or receptor oligomer-
ization [2] (see below).
Covalent receptor labeling can also be achieved by using Flash and Reash
strategies. They consist in integrating in the receptor a sequence containing four
cysteines, CCXXCC, which will make covalent bonds with green or red fluorescent
arsenic derivatives [39–41].
Non-covalent labeling can also be achieved with antibodies which specifically
recognize the protein of interest. Antibodies can easily be labeled with organic
fluorophores or lanthanide cryptates. The ratio of the number of fluorophores per
antibody is usually greater than 1, leading in theory to a signal amplification.
However not all fluorophores on antibodies are necessarily engaged in a FRET.
Moreover, antibody labeling with fluorophores is usually a random labeling. It
means that variations in the labeling can be observed from one antibody molecule to
another. Moreover, antibodies are large molecules and their binding on the protein
of interest can generate a steric hindrance, which can be prejudicial to the interac-
tion of other partners. To circumvent this issue, smaller antibodies, such as
nanobodies produced by camelids, can be an excellent alternative to label proteins.
Biomolecular engineering approaches have also been used to label proteins
covalently. Luminescent (BRET) and fluorescent (BRET and FRET) proteins can
be fused either to the N- or C-termini of the proteins. It has been shown that for
GPCRs the fusion at one or the other terminus does not have an impact on receptor
pharmacology. Interestingly, fluorescent proteins such as the green fluorescent
protein (GFP) and its mutants can also be inserted in loops of proteins because of
the barrel structure in which the N- and the C-terminus are very close. This
possibility has been used to perform intramolecular FRET within GPCRs to study
receptor signaling, for example [42]. By contrast, the labeling of molecules with
lanthanide cryptates or organic fluorophores cannot be entirely encoded by the cell
and it thus requires an additional step such as the incubation of the cell with
lanthanide-derivatized substrates. The strategy consists in using self-labeling pro-
teins (also called suicide enzymes) such as SNAP-, CLIP-, or Halo-Tags, which
have the capacity to transfer a chemical group from a substrate to themselves [43–
47]. Providing fluorescent substrates to these self-labeling proteins allows covalent
receptor labeling. This strategy is particularly efficient to label membrane proteins
fused to the self-labeling protein on their extracellular terminus and has been
successfully used on G protein-coupled receptors [48–51], tyrosine kinase recep-
tors, and ionic channels [36]. It is also interesting to note that various self-labeling
proteins and specific substrates for each of them have been developed [52, 53],
giving the opportunity to label simultaneously different proteins with different
fluorophores. The selectivity of the fluorescent substrates for the different self-
labeling proteins is such that when differently tagged receptors are co-expressed in
cells, the labeling of these receptors can be performed in a single step.
Because substrates for luminescent proteins are cell permeant, BRET strategies
can be efficiently used to investigate intracellular complexes but do not allow the
exclusive labeling of receptors targeted to the cell surface (both cell surface and
intracellular receptor interactions will be monitored at the same time). By contrast,
time-resolved FRET strategies are not convenient to study intracellular complexes
in living cells since their substrates are usually not cell permeant, but they turn out
to be efficient to label the fraction of proteins targeted to the cell surface and not
those retained into the cells.
2.5 Relevance of Time-Resolved FRET Strategies to Study

GPCRs
In the next paragraphs, only major results will be reported since time-resolved
FRET strategies to study GPCRs have already been reviewed extensively [54–
57]. The different applications of TR-FRET in GPCR studies are illustrated in
Fig. 3.
2.5.1 Binding Assays
Fluorescent binding assays have been developed as alternatives to radioactive

binding assays. The first assays were based on the measurement of fluorescence
GPCR oligomerizaon GPCR binding assays GPCR signaling assays GPCR internalizaon
self-labeling proteins
ligands
second
messengers
homo- or hetero-oligomers hetero-oligomers
Fig. 3 Time-resolved FRET assays have been developed to investigate different steps of a GPCR
life. Firstly, FRET can occur between two receptors to prove the existence of homo- or hetero-
oligomers. Secondly, FRET can occur between a fluorescent ligand and a fluorescent receptor to
investigate binding properties of a receptor or a hetero-oligomer. Thirdly, time-resolved FRET can
be used to investigate the activation of signaling pathways by the receptor such as second
messenger production or protein phosphorylation. Finally, time-resolved FRET can also be used
to measure receptor internalization
intensity of the fluorescent ligand bound onto the receptors. They require washing
steps to separate the free ligand fraction from the ligand bound to the receptors.
These tests generally lack sensitivity except when the fluorophore is a lanthanide
chelate [58]. It is noteworthy that the linkage of some conventional fluorophores
can dramatically increase the hydrophobicity of ligands and therefore their
non-specific binding. Other approaches based on fluorescence anisotropy or polar-
ization have been implemented. They do not require washing steps and are there-
fore homogeneous assays, but their sensitivity can be strongly impacted by
non-specific binding [59]. To circumvent these drawbacks, FRET-based binding
assays have been developed. The FRET occurs between a tagged receptor (SNAP-,
CLIP-, or Halo-tag receptor) and a fluorescent ligand. The addition of an unlabeled
competitor results in the modulation of the FRET signal, a decrease if the compet-
itors are orthosteric ligands or negative allosteric modulators, or an increase in the
case of positive allosteric modulators. The signal due to non-specific binding is thus
very low since the probability that a ligand binds non-specifically in close proximity
to a receptor is very low. Although various fluorophore pairs can be used as donor
and acceptor, these assays were principally developed with Lumi4-Tb as donor,
although the use of Eu3+ complexes works as well [51]. They were implemented for
many GPCRs [35, 37, 38, 60–62] proving their reliability and also on tyrosine
kinases and ionic channels [36]. More recently, these assays were used to investi-
gate binding parameters such as association and dissociation kinetics [63]. These
TR-FRET assays display a high signal-to-noise ratio and are therefore compatible
with high-throughput screening. More recently, the technique has been slightly
modified to be able to investigate the pharmacology of receptor oligomers; the
TR-FRET signal occurs between a fluorescent ligand bound to a first receptor and a
second tagged receptor [37]. It constitutes to our knowledge the only assays to
investigate binding properties of hetero- and homo-oligomers. BRET assays based
on the same principle, i.e., an energy transfer between a tagged receptor and a
fluorescent ligand, were also recently developed [64, 65]. In addition, FRET
binding assays based on conformational changes of GPCR dimers have been
developed. It has been shown that full agonists, partial agonists, or allosteric
modulators induced specific variations of FRET between a donor and an acceptor,
each carried by one protomer within a dimer [66]. It is noteworthy that these assays
cannot be used on wild-type receptors or on native tissues since they require the
labeling of the tagged receptor.
2.5.2 Receptor Oligomerization
The concept of the GPCR oligomerization emerged more than two decades ago.
The first arguments suggesting the existence of oligomers were based on western
blotting, co-immunoprecipitation experiments, and on mutant receptor
re-complementation by transmembrane domain swapping between receptors. The
first two techniques suffer from two main disadvantages. The first one is that they
do not prove direct interactions between the partners and, second, the observation of
high molecular bands corresponding to receptor complexes on blots is dependent on
the nature of detergents used, leading to false-positive and/or false-negative results.

BRET, FRET, and TR-FRET analyses were used to demonstrate interactions of
GPCRs. Indeed, as mentioned above, energy transfer only occurs when partners are
in close proximity (generally less than 80 Å) and the existence of a RET signal is
often interpreted as a direct interaction between partners. However, this interaction
is not necessarily stable over time. These transient interactions can either corre-
spond to physiological processes such as an adaptation of the GPCR oligomeriza-
tion to a cellular context or to random collisions of receptors diffusing into the
membrane. It is noteworthy that GPCR oligomerization is at least in part depending
on receptor density at the membrane [67]. Various strategies have been used to
demonstrate the specificity of the signal. The most straightforward method is to find
a receptor, which does not interact with the receptor of interest as a negative
control. Such a strategy has been used for class C metabotropic glutamate receptors,
which form covalent dimers. It has been shown that mGlu 1 and mGlu 5 are not able
to interact with other mGlu receptors [68, 69]. Finding such a negative control is,
however, much more difficult for class A GPCRs and because of their common
general structure, increasing receptor density generally results in the observation of
a RET signal between receptors. To demonstrate the specificity of the interaction in
BRET experiments, saturation experiments of the BRET signal were performed.
This consists in expressing the receptor labeled with the acceptor at various
densities while keeping the receptor-donor expression constant. The hyperbolic
curve resulting from the plot of the BRET signal as a function of the acceptor/donor
expression ratio should saturate if the interaction between receptors is specific, the
plateau being reached as soon as all the donors are linked to the acceptor [7]. A
similar strategy can be used in TR-FRET experiments or alternatively, FRET
efficacy can also be plotted as a function of receptor density [2, 49].
Generally, the strategies mentioned above are not adequate to study the oligo-
merization of wild-type receptors expressed in native tissues since they are based on
the fusion of receptors with different tags. As mentioned above, an alternative
consists in labeling the receptor with specific fluorescent ligands. These methods
have been used to evidence the existence of homo-oligomers of the oxytocin
receptor in the mammary gland of lactating rats; the TR-FRET signal is abolished
in the presence of an excess of unlabeled ligands. Moreover, this difference in
TR-FRET signal observed with the fluorescent agonists and antagonists strongly
suggests that the observed TR-FRET signal is not due to receptor collisions. Similar
approaches have been used to prove the existence of dopamine D2-ghrelin recep-
tors in hypothalamic neurons [4] and dopamine D2-adenosine A2A receptor hetero-
oligomers in the rat striatum [3].
2.5.3 Receptor Signaling
At least, four ways can be used to investigate receptor signaling. Firstly, receptor
activation can be investigated when observing receptor conformational modifica-
tion during ligand binding. For example, structural insights into biased GPCR
signaling have been revealed by TR-FRET on purified vasopressin V2 receptors.

Secondly, the signaling of receptors can also be investigated when following the
recruitment of proteins – such as G proteins or β-arrestins – by the receptor
[70]. The RET occurs between the receptor and the recruited proteins, and generally
increases when the receptor is activated. By contrast, receptor signaling can also be
investigated by following the dissociation of complexes such as G proteins acti-
vated by the receptor. In this case, Gα on the one hand and Gβγ on the other hand
are labeled with a donor and an acceptor tag, respectively. Dissociation of the G
proteins consecutive to receptor activation leads to a decrease of the RET signal. It
is noteworthy that mild permeabilization is required to get proteins labeled in the
TR-FRET mode [71]; BRET is therefore more convenient. Thirdly, receptor acti-
vation can be followed with assays based on the measurement of the production of
second messengers or the level of protein phosphorylation. These assays can be
compatible with kinetic analyses [26, 72, 73] or can be end-point assays, which
require cell lysis [31]. Finally a TR-FRET assay has also been developed to
investigate receptor internalization [74–77]. It is based on the measurement of a
FRET signal between a tagged receptor and a free acceptor present in the extracel-
lular medium. This FRET signal can be observed as long as the receptor remains at
the cell surface. The energy transfer is interrupted as soon as the receptor is
internalized.
3 New Strategies to Investigate G Protein-Coupled

Receptors into More Details
3.1 Microscopy vs. Plate-Reader
Many plate-readers have been designed to monitor the RET signal, making strat-
egies based on BRET and time-resolved FRET relevant strategies to study GPCRs.
The success of these approaches is due to their sensitivity and their robustness.
Paradoxically, despite their good signal-to-noise ratio, BRET and TR-FRET strat-
egies did not conquer the field of microscopy until recently.
As all techniques that give access to a single-element analysis, RET-based
microscopy offers the possibility to explore the heterogeneity in a population
while plate-reader-based techniques average responses of a whole population.
Because oligomer formation depends on receptor density [67] and because transient
transfection leads to a large heterogeneity in receptor density, great variations from
one cell to another can be observed in oligomer density. It is noteworthy that, in
stable cell lines, receptor density may be dependent on cell confluence and therefore
variation in oligomer density can also be observed in stable cell lines. Secondly, as
already mentioned, various studies have reported that monomers, homo- or hetero-
oligomers can exhibit different pharmacological properties [1] or behavior [69].
Moreover, most of the native tissues display cellular heterogeneity. Investigating

receptor oligomerization can therefore be a great challenge in these models.
Because technologies using plate-readers average the signal, it is very difficult to
evidence the existence of oligomers in cells, which constitute a minor population in
the tissues. By contrast, RET-based microscopy can discriminate positive cells
(cells expressing the receptor of interest) from negative ones. These techniques
display a spatial resolution which is good enough to allow locating receptor
oligomers in cells or tracking receptor oligomers during their internalization or
their recycling to the membrane.
Complementary techniques offer a higher spatial or temporal resolution, giving
the possibility to track a single particle in a cell and can therefore be more relevant
to investigate native tissues.
3.2 Resonance Energy Transfer Microscopy
3.2.1 BRET Microscopy
One significant advantage of BRET over FRET resides in the absence of external
light excitation to initiate BRET. As mentioned above, BRET circumvents cell
autofluorescence, direct excitation of the acceptor fluorophore by external excita-
tion light, and donor fluorophore photobleaching. This results in a higher signal-to-
noise ratio and facilitates analysis of the signals making BRET a technology of
choice for measurements using microplate readers [9]. These physical properties
also apply at the single-cell level. Thus, for decades, development of efficient
BRET imaging has been a goal to improve the detection of protein–protein inter-
action dynamics at subcellular level in living cells.
The major challenge of BRET imaging comes from the difficulty in detecting the
low number of photons emitted by the donor at the single-cell level. Today, the
enhanced sensitivity of microscopy, electron multiplying cooled charge-coupled
device (EMCCD) cameras and improved bioluminescence probes facilitate lumi-
nescence imaging at the single-cell level [78, 79].
We have developed and optimized a setup for BRET imaging at the subcellular
level [78, 79]:
– A standard inverted fluorescence microscope (Axiovert 200M, Zeiss) is modi-
fied such that all light-emitting diodes are taken out and the light path is blocked
with a 1.5 m optical fiber to limit optical interferences.
– A black box protects the microscope from ambient light. Images are recorded
with a 40 or 63 objective. Identification of transfected cells necessitates the
use of two-color detection. Exciter HQ480/40 and emitter HQ525/50 are used
for YFP, whereas exciter HQ540/40 and emitter HQ600/50 allow DsRed detec-
tion. A BRET experiment, i.e., without photoexcitation but in the presence of a
luciferase substrate, requires the selection of specific emission wavelengths:
480 nm (filter D480/60 nm) for the donor (Rluc or Nluc) and 535 nm (filter
HQ535/50 nm) or 562 nm (filter HQ585/40 nm) for the YFP or mOrange
acceptor, respectively.
– Images are collected with a camera (Evolve, Roper scientific) equipped with an
EMCCD detector, back-illumination, and On-chip Multiplication Gain, which is
mounted on the camera base-port of the microscope.
The spatial resolution of BRET imaging allows to easily locate oligomers in
large and stable cellular compartments, but it reaches its limits for monitoring
BRET signals in small and labile structures. Indeed, the detection of the low-light
donor emission necessitates long acquisition times, which compromise the tempo-
ral resolution of BRET images and restrict the use of BRET to protein–protein
interactions that are stable for more than 1 min. Furthermore, also the spatial
resolution of BRET images is highly dependent on the acquisition time, since, in
living cells, the complexes under study will move during the acquisition. Thus
technical improvements to reduce the acquisition time would not only increase the
temporal resolution but also the spatial resolution, by restricting movements of
protein complexes during shorter acquisition times.
Application of BRET Imaging
The physical properties of Nluc, especially its stability and luminescence effi-
ciency, have dampened the field of bioluminescence these last months (see [80]
for review). Hence, Nluc improves the detection of gene expression, protein
stability, and protein–ligand interaction. The brightness of Nluc also opens up
new possibilities for bioluminescence imaging. For example, it has been used to
track viral infection [81] or tumor growth [82] and monitor disease progression.
Nluc was then successfully used to increase the sensitivity of BRET-based assays in
high-throughput screening [83] or to study protein–protein interaction dynamics
[84, 85] in plate format assays [84, 85]. Finally, by defining the experimental
conditions to accurately record the BRET signal at the single-cell level, we recently
characterized the benefit of Nluc for BRET imaging and defined the possibilities
and limits of the assay [26]. We further took profit of Nluc to improve a BRET
biosensor of ERK activity at the subcellular level in living cells, which was
sensitive enough to report ERK activation by endogenous NMDA receptors in
dendritic spines of hippocampal neurons. Hence, the use of Nluc has significantly
improved BRET imaging. This brighter luciferase shortens the acquisition time to
hundreds of milliseconds, enhancing the spatial and temporal resolution while the
sensitivity is increased and the dynamic window enlarged. Consequently, the
dynamics of subtle and transient BRET signal can be followed in small subcellular
compartments [26].
BRET imaging can be performed with Rluc, Rluc8, and Nluc as donor, com-
bined with YFP variants or mOrange as acceptor. Dual, simultaneous, recordings
can be performed with BRET1 and 3 [70]. So far, intramolecular BRET imaging
can be used to monitor the activation in space and time of specific signaling
pathways, such as ERK [26, 73] or IL-1beta processing [86]. Intermolecular
BRET imaging has been applied to study the interactions between metabotropic
and ionotropic receptors [87], receptor interactions with cytosolic scaffolding pro-
teins [87, 88], β-arrestin recruitment to GPCRs [70, 78], and interactions between
nuclear receptors [89]. It is worth noting that BRET imaging experiments were also
efficiently performed at the cellular level in plant seedlings. The weak light derived
from bioluminescence did not photobleach the sample nor cause autofluorescence,
a particularly acute issue in plant cells because of the presence of chlorophyll
[90]. Similarly, the bioluminescent process is well suited to locate deep signals
within small living subjects [21].
3.2.2 Multicolor Time-Resolved FRET Microscopy
The second approach is the time-resolved FRET-based microscopy [69, 91–93]. As

for BRET microscopy, the setup results from homemade development, limiting its
use in laboratories. This microscopy is based on the use of a cryptate of terbium,
Lumi4-terbium (Lumi4-Tb), as donor. As in plate-readers, the technique is based on
a time delay between the excitation of the sample and the recording of the FRET
signal.
The setup consists of:
– A UV light source: Three solutions have been proposed: (1), UV LED source
emitting at 665 nm and which is directly attached to the epi-illumination port of
the microscope [92, 94]; (2), laser excitation at 349 nm [69, 95]; (3), lamp can
constitute a source of UV but rise/fall times of illumination are probably longer
than those obtained with a laser.
– A camera: because of their sensitivity, intensified cameras have been used to
image biological samples [69, 92, 94, 95]. Comparison of intensified camera and
camera in multigate mode has been carried out. The multigate mode camera can
store up to 4,000 images and it contributes to increase substantially the signal-to-
noise ratio. As expected, intensified cameras exhibit a higher sensitivity, a faster
image acquisition but a lower spatial resolution. Therefore, the choice of the
camera should be determined in function of the experiment. Image acquisition is
synchronized to the laser pulse and a delay of 10 μs is used to eliminate all short-
lived fluorescence [69, 94].
– TR-FRET probe pairs: as mentioned above, one of the major drawbacks of
FRET microscopy with fluorescent proteins is the contamination of FRET
with fluorescence of the donor or the acceptor. This means that the acquired
FRET images do not correspond to the real FRET signal. By contrast, in
TR-FRET microscopy, cryptates or chelates of terbium can be associated with
various acceptors to perform energy transfer experiments, because of their long-
lasting emission, their important Stokes shift, and several emission peaks. The
most commonly used acceptors are fluorescein-like and d2-like acceptors [96];
both types of fluorophores have excitation spectra which overlap with the
emission peaks of Lumi4-Tb at 490 and 620 nm, respectively. Contamination
of Lumi4-Tb at the emission wavelength of the d2-like probe is reduced and
therefore the emission filter can have a large bandpass, 75 nm in our setup. By
contrast, when using a fluorescein-like acceptor, a narrow bandpass (15 nm) for
the emission filter has to be selected to avoid important bleed-through of the
donor at the FRET wavelength. One consequence is that green FRET is less
intense than red FRET. In these conditions, the bleed-through of Lumi4-Tb in
the green (panel b) and red (panel c) FRET channels is less than 3% of its
emission intensity at 550 nm [69] (Fig. 4). When considering the emission peak
550 and 585, Lumi4-Tb could also be associated with other acceptors such as
quantum dots [91]. However, once again, it is important to use an emission filter
a b
c d
300
Lumi4-Tb
green
(Fluo - Bg) / Bg
200 red
100
0
0 50 100 150
Pixel
Fig. 4 The sensitivity of the FRET strategy in microscopy is generally dependent on the
contamination of the signal by non-specific luminescent or fluorescent signals. One of them is
the contamination due to the bleed-through of the donor at the acceptor emission wavelength. In
time-resolved FRET, the emission of the donor at the acceptor wavelength is very small and
generally negligible compared to the FRET signal; at its worse, it does not exceed 4% of the
luminescence measure at 550 nm. (a) Excitation: 349 nm, emission: 550 nm; (b) excitation:
349 nm, emission: 520 nm; (c) excitation: 349 nm, emission: 700 nm; (d) fluorescence intensity
along the line at the various emission wavelength
with a narrow bandpass to reduce bleed-through of Lumi4-Tb in the FRET

channels. For example, we used quantum dot 605 nm which is particularly
suitable since it displays a bright fluorescence and a narrow emission spectrum.
Therefore using a 605/15 nm bandpass emission filter allows reducing contam-
ination of Lumi4-Tb at 605 nm (<4% of the emission of Lumi4-Tb at 550 nm)
without decreasing drastically the fluorescence emission amplitude. It should be
noted that quantum dots are large compared to organic fluorophore and their size
can be prejudicial in some cases to conclude that the FRET is specific and
therefore the interactions between partners as well [69].
One particular advantage of MC-TFM over the other energy transfer strategies in
microscopy is the possibility to combine various acceptors to detect different
complexes in the same sample [69]. As an example, fluorescein, d2, and QD605
have been associated to detect three types of receptor homodimers present in three
different cell populations.
Application of MC-TFM
BRET and TR-FRET microscopy remain quite confined probably because no commer-
cial setups are available yet and homemade adaptations of the microscope have to be
carried out. Miller and his collaborators have shown that the TR-FRET-based method
can be used to image intracellular complexes composed of a protein fused to
Escherichia coli dihydrofolate reductase (eDHFR) and labeled with TMP-Lumi4-Tb
and a second protein fused to green fluorescent protein [92]. Because TMP-Lumi4-Tb is
not cell permeant, it can be delivered into the cell after a mild permeabilization of the cell
or after using osmotic lysis or pinocytic vesicle techniques [92]. Hildebrandt and
collaborators described the potential application of the TR-FRET-based technique for
diagnostics [95].
Finally, we used MC-TFM to investigate GPCR oligomers [69]. Receptors were
fused to a self-labeling protein and receptor oligomerization was imaged after one
receptor-labeling step by incubating cells in the presence of substrates derivatized
with a donor or an acceptor. We first reported that the signal we observed is not due
to a dynamic TR-FRET signal resulting from random collision between receptors
diffusing in the membrane but to a specific interaction between receptors. To prove
this, we have cotransfected glutamate mGlu1 and mGlu2 at the same density. We
imaged the TR-FRET signal corresponding to the mGlu1 and mGlu2 homodimers
and observed a significant signal. By contrast, only a faint TR-FRET corresponding to
the heterodimer can be imaged proving that the signal observed for the homodimers did
not correspond to random collision. The strategy has also been used to image class A
receptor oligomers: dopamine D2, vasopressin V1a and V2 [69], CXCR4 and CXCR7
chemokine receptors (Fig. 5).
In a next step, we focused on receptor oligomer internalization. We showed that
GPCRs can be internalized as oligomers and that a dissociation of oligomers in
a Exc: 349 nm d Exc: 349 nm

Em: 550 nm Em: 550 nm
HALO HALO SNAP SNAP
HALO-CXCR4 SNAP-CXCR7
b Exc: 490 nm e Exc: 620 nm

Em: 520 nm Em: 700 nm
HALO HALO SNAP SNAP
c Exc: 349 nm f Exc: 349 nm

Em: 520 nm Em: 700 nm
FRET FRET
HALO HALO SNAP SNAP
Fig. 5 Multicolor time-resolved FRET microscopy, MC-TFM, is a relevant strategy to investigate

receptor oligomerization. Various strategies can be used, based either on fluorescent ligands or on
fluorescent receptors. In this late case, receptors are fused to self-labeling protein (also called
suicide enzymes) at the N-terminus; Chimeric receptors can therefore be labeled when cells are
incubated in the presence of a specific substrate. CXCR4 and CXCR7 have been fused to a Halo-
tag and SNAP-tag, respectively. Cells expressing Halo-CXCR4 or SNAP-CXCR7 were incubated
in the presence of a mix of Halo-Lumi4-terbium and Halo-green for CXCR4 (panels a–c) and
SNAP-Lumi4-terbium and SNAP-red for CXCR7 (panels d–f). Emission of the donor, the
acceptor, and the FRET signal were, respectively, imaged using the following excitation/emission
wavelength: at 349/550 nm for Lumi4-Terbium emission (panels a, d); 490/520 nm for fluorescein
emission (panel b) and 620/700 for d2 emission (panels e); 349/520 nm for green FRET (the
acceptor is fluorescein) (panel c); 349/700 nm for the red FRET (the acceptor is d2) (panel f);
Scale ¼ 25 μm
monomers is not a prerequisite for their internalization. Indeed, after the addition of
a quencher in the extracellular medium, we were still able to see a punctuated
TR-FRET labeling. Because, as mentioned above, a FRET occurs only if partners
are closer than 8 nm, this is usually interpreted as a direct interaction between the
partners. Therefore, we confirmed the existence of internalized oligomers and thus
validated previous hypotheses based on co-location or co-internalization experi-
ments [1, 7, 97–100].
Finally, we demonstrated the existence of cross-regulation between GPCRs
regarding the internalization processes. Indeed, vasopressin stimulation of cells
co-expressing vasopressin V1a and V2 receptors results in a strong internalization
of V2 homomers, a significant internalization of V1a/V2 heteromers and a low V1a

homomer internalization. The addition of a selective V2 receptor antagonist,
SR121463, blocks the V2 homomer internalization but restores the one of the
V1a homomer. In these conditions, V1a/V2 heteromer internalization still occurs
suggesting that the activation of only one protomer within the heteromer is suffi-
cient to induce internalization processes.
3.2.3 Advantages of BRET- and MC-TRF-Based Microscopy

Approaches Over Conventional FRET Microscopy
First FRET experiments in microscopy have been based on energy transfer between
fluorescent proteins, the most often used pairs being Cyan and Yellow fluorescent
proteins as donor and acceptor, respectively. As mentioned above, although these
proteins are very bright, the overlap between the excitation and the emission spectra
constitute a limit to observe in a simple way images corresponding to a real FRET.
The detection of FRET is based on a mathematical analysis to discriminate fluo-
rescent contamination due to the direct excitation of the acceptor and emission of
the donor at the emission wavelength of the acceptor from the FRET resulting from
a real energy transfer from the donor to the acceptor. This overlap prevent using
multiple pairs to detect various complexes simultaneously. An alternative strategy
can be photobleaching the donor or the acceptor and measuring the variation in the
emitted fluorescence of the acceptor or the donor, respectively [101]. This approach
is not compatible with kinetic experiments.
BRET microscopy allows bypassing these limits [78]. This approach exhibits a
very good signal-to-noise ratio since the excitation of the donor results from a
chemical process preventing any direct excitation of the acceptor. Despite this high
ratio, BRET signals were very small and an intensified camera was needed to detect
them. The discovery of brighter luminescent proteins such as the Nluc opens new
perspectives since sensitivity and spatial resolution of BRET microscopy are
dramatically increased and the combination of BRET1 and BRET3 gives the
possibility to track two protein complexes simultaneously [26].
In contrast to FRET, MC-TFM displays many advantages: (1) the time selectiv-
ity and spectral compatibility between donor and acceptor gives the possibility to
image the FRET signal without acceptor or donor fluorescence normalization or
photobleaching steps; it is therefore simple to obtain images corresponding to the
TR-FRET signal; (2) the different emission peaks give the possibility to image
various complexes labeled with a unique donor and multiples acceptors. We took
advantage of this property to image GPCR homo- and hetero-oligomers in the same
samples; (3) the resolution of MC-TFM is clearly compatible with the identification
of endocytic vesicles. It is noteworthy that the choice of the parameters to acquire
images with an excellent resolution, and especially when using the multigate mode
of the camera, is at the expense of the acquisition rate. As mentioned above, using
an intensified camera speeds up acquisition but decreases the resolution; (4) because
a large variety of molecules can be labeled either with a donor or an acceptor,
MC-TFM can be used to image various complexes such as receptor or ligand/

receptor complexes; (5) Time-resolved FRET strategies are really convenient to
label membrane proteins especially when one extremity is extracellular. It allows
discriminating receptors retained in intracellular compartments from those targeted
to the cell surface. Conversely, these strategies are less efficient to label intracel-
lular molecules because mild permeabilization [92] or cell-penetrating peptide-
based strategies [102] are required to make the substrates of the self-labeling
proteins enter the cell.
3.3 Complementary Fluorescence-Based Techniques

to Study GPCR
RET-based microscopy suffers from three main drawbacks. Firstly, the rate of
image acquisition is not compatible with fast kinetic analysis of the dynamics or
the stability of the complexes. Secondly, the sensitivity of the RET techniques as
described above is not compatible with a single-molecule analysis. Finally, as
mentioned above, the spatial resolution of MC-TFM is compatible with the detec-
tion of organelles such as endosome or lysosomes but it does not allow detecting
smaller structures such as membrane microdomains which are not larger than 50 nm
[103] and therefore not compatible with the limits of the resolution of optical
microscopy (250 nm).
The methods we focus on in the following part are particularly efficient to
investigate stability, mobility, and dynamics of molecular complexes at the cell
surface. Their interest to investigate GPCRs functions will mainly be considered on
receptor oligomerization issues.
In recent years the debate has focused on deciding whether GPCRs naturally
form oligomers or remain monomers and whether these dimers or higher order
oligomers play important roles in GPCR functions [49, 104, 105]. FRET-based
techniques and crystallography results showed that GPCRs can exist in both
monomeric and oligomeric structures [106, 107]. However, classic FRET tech-
niques are still limited to prove the existence of the oligomeric state. Recently,
original microscopy and spectroscopy techniques have allowed researchers to dwell
deeper in the dynamics of GPCRs and bring complementary information on the
GPCR oligomerization.
3.3.1 FRAP to Investigate GPCR Oligomer Stability
One of the first attempts to bring evidence to the equilibrium between GPCR
oligomers was performed by Dorsch and collaborators [108]. The method consists
in investigating the fluorescence recovery after photobleaching (FRAP) of one
receptor when its potential partner is immobilized. Technically, a receptor is
fused either with an extracellular YFP or an intracellular CFP. A fraction of the

receptors on the cell surface was immobilized by crosslinking with an antibody that
recognized the YFP, and the mobility of the non-cross-linked remainder (CFP) was
measured by FRAP. The method was used to investigate β1- and β2-adrenergic
receptor homo-oligomers. Fluorescence recovery is faster for β1-adrenergic recep-
tors than for β2-adrenergic receptors suggesting a greater stability for β2-adrenergic
receptor homomers than β1-adrenergic ones. These results are in accordance with
the observation that β2-adrenergic receptors in cardiac myocytes present a tendency
to aggregate in caveolae compartments, exhibiting a more limited diffusion com-
pared to β1-adrenergic receptors [109].
3.3.2 FCS to Study GPCR Mobility
Fluorescence correlation spectroscopy (FCS) is widely used for studying the

dynamics of molecules and was recently applied to quantitatively study GPCR
oligomerization. Labeled receptors are excited in a reduced confocal volume and
the diffusion of individual receptors is studied. The fluctuations of the fluorescence
signal coming from the labeled receptor in the defined volume are measured. The
diffusion parameters and the average brightness of the molecules entering and
exiting the volume are then calculated, giving information on the molecular aggre-
gation state of the receptors and their mobility.
Ilien et al. observed a twofold increase of the brightness of GFP-tagged muscarinic
M1 receptors in the presence of pirenzepine, concluding that pirenzepine binding on the
receptor induced its dimerization [110]. As mentioned in the study, the results are at
variance with other data that reported no effect of antagonists or agonists on muscarinic
oligomerization [111, 112]. However, ligand-induced association and dissociation of
oligomers have also been described for other class A GPCRs [113].
FCS is also a useful tool to investigate the diffusion and subsequently the aggregation
state of the receptors. A1-adenosine receptors were labeled with a fluorescent antagonist
[114]. FCS showed a fast diffusing ligand-bound population (0.9 μm2/s) associated with
single receptors and a slower one at 0.05 μm2/s, associated with receptor oligomers or
receptor aggregates in membrane microdomains. Diffusion coefficients of adenosine A3
receptors expressed in CHO cells incubated in the presence of a fluorescent agonist and
antagonist were also precisely measured by two research groups [115, 116]. They found
two diffusing populations (fast at 2.4 and slow at 0.13 μm2/s), attributing the slow
population to oligomers. It should be mentioned that the variation in the diffusion
coefficients between monomers and dimers can be difficult to detect. However, Bridon
and collaborators succeeded in demonstrating that homodimers and heterodimers can
have different diffusion coefficients [117].
FCS was adapted to follow simultaneously various complexes. The method,
called fluorescence cross-correlation spectroscopy (FCCS), examines fluorescence
variations at two different wavelengths to measure co-diffusion of two labeled
molecules. The technique was first applied to GPCRs to study the homo- and
hetero-oligomerization of somatostatin receptors (SSTRs) using fluorescent ligands
labeled with two different fluorophores (FITC and Texas Red) [118]. The authors
concluded that the SSTR1 receptor did not form homo-oligomers but hetero-
oligomerized with SSTR5 upon somatostatin addition. By contrast, SSTR5 was
able to form homo-oligomers in the presence of the agonist. This was the first
evidence of ligand-dependent GPCR oligomerization, which is not the case for all
GPCRs of the class A. Recently, Comar and collaborators obtained information on
the dynamic equilibrium between the monomeric and the dimeric state of the opsin
receptor by using this technique [119]. The amount of dimers increased in a linear
manner with the square of the monomer concentration. Similar results were dem-
onstrated for the N-formyl peptide receptor (FPR) – a chemoattractant GPCR –
using single-particle tracking (SPT) techniques [120].
A complementary approach based on two-photon fluctuation microscopy has
been recently implemented. Two-photon scanning number and brightness (sN&B)
has a very high resolution and an extremely low autofluorescence (due to the
non-linear nature of the IR excitation). sN&B provides absolute quantitative infor-
mation such as the spatial distribution, the stoichiometry, and the dynamics of the
complexes in specific subcellular compartments. This novel variation of fluores-
cence fluctuation microscopy was used to assess the number and stoichiometry of
protein complexes in living neurons [121].
To conclude, FCS methods provide quantitative information on the dynamics of
the molecules at a high temporal resolution and do not require single-molecule
conditions, as it is the case for single-particle tracking (SPT) methods. However,
they are often complicated in terms of hardware, data analysis, and interpretation.
For instance, the differences between the diffusion coefficients of monomers and
dimers are very small and noise can influence the measured fluctuation signals.
3.3.3 SPT to Study GPCR Oligomerization Dynamics in Living Cells
SPT is a suitable technique for determining whether GPCRs form oligomers and for
studying their dissociation kinetics into monomers. If the labeling of GPCRs is
efficient, this technique allows tracking of all receptors at the plasma membrane,
studying their diffusion, their collisions, and their association/dissociation times
and phases. This technique can be performed either in “Total Internal Reflection
Microscopy” (TIRF) mode to really image membrane receptors with a high signal-
to-noise ratio, or in classic epifluorescence mode, for intracellular GPCRs not yet
targeted to the cell surface or internalized. In principle, by identifying fluorescent
spots corresponding to single diffraction limited fluorescent molecules, this tech-
nique determines the position of the fluorophores with a high precision and then
allows following their diffusion in time. The only limiting factor here is to have a
sufficiently dispersed receptor distribution, in order to locate every spot individu-
ally (a few receptors per μm2).
Hern and collaborators performed the first single-molecule imaging of GPCRs
with M1 muscarinic acetylcholine receptors (a class A GPCR) [122]. Performing
two-color single-molecule imaging they found that M1 muscarinic receptor
molecules form dimers. They identified that 10% of the labeled receptors diffused
in pairs, 10% dissociated and re-associated rapidly in pairs, and 80% diffused as a
monomeric state. More interestingly, they demonstrated that the dimer dissociates
in monomers in 0.7 s at 23 C in living cells. Kasai and collaborators characterized
the GPCR monomer–dimer equilibrium by developing a quantitative single-
molecule methodology for the N-formyl peptide receptor (FPR) [120]. They
labeled the receptor with a fluorescent dye using a 1:1 ratio and studied the
co-localization times, when two fluorescent spots co-localized and diffused
together. The distribution of the co-localization duration was fitted by a single
exponential decaying function, providing the lifetime of the dimer state. They
revealed that monomers convert into dimers every 150 ms and dimers dissociate
into monomers in 91 ms. Ligation of the receptor did not change the monomer–
dimer equilibrium.
This work was followed by the studies of Calebiro and collaborators [67], which
revealed that both β1-adrenergic and β2-adrenergic receptors form transient
homodimers with a lifetime of 4 s. The receptors were SNAP-tagged and studied
in living cells at 20.5 C. The difference of the lifetime value compared to the
previous studies (6 longer than that of M1 muscarinic acetylcholine receptors and
40 longer than that of FPR dimers) could be due to the lower temperature, the
different methodologies or the different molecular interactions in the dimeric, or
higher order oligomeric state of the adrenergic receptors.
The above SPT techniques allow characterizing the GPCR dynamics and observing
the transient states of monomers and dimers. However, these techniques are limited
when the labeled molecules are tightly concentrated in the diffraction limited fluores-
cence spot (density higher than approximately 10 molecules per μm2). In this particular
case, SPT methods can be combined with super-resolution techniques, such as PALM
and sptPALM to overpass the diffraction limit. SPT has also been combined with FRET
to study the dynamics of GPCR activation [123]. It has been shown that most of the
metabotropic glutamate receptors oscillate between a resting and an active conformation
on a sub-millisecond timescale. Differences in agonist efficacies stem from different
abilities to shift the conformational equilibrium towards the fully active state, rather than
from the stabilization of alternative static conformations.
3.3.4 Spatial Mapping of the Local Oligomers in GPCRs
As mentioned above, due to the diffraction, the resolution of optical microscopy is

limited. Recently, some techniques have overcome this barrier, called super-
resolution techniques. Photo-activation light microscopy (PALM) improves the
resolution up to ten times and determines the location of single molecules with a
precision of 10–20 nm. PALM is based on the photoactivation or photoswitching
and subsequent bleaching of sparse fluorescent proteins in the sample, thus sepa-
rating temporally the molecules and distinguishing them in space [124]. PALM is
often combined with TIRF. TIRF microscopy allows a very fine excitation of the
basal cell membrane (100 nm), where only a few labeled receptors may be present,
increasing the signal-to-noise ratio.
PALM is an extremely useful tool for studying protein organization in dense
samples, i.e., a GPCR density greater than one molecule per 200 nm2, which can
also be found in some membrane compartments under physiological conditions.
However, potential photophysical artifacts can influence the results. For example,
blinking molecules, which can be counted more than one time and consequently be
interpreted as a cluster, are a common issue for this technique. Various methods
have been developed to quantify the results and overcome these issues, like the pair
correlation method [125] or the DBSCAN method [126]. However, in these cases,
some parameters have to be initially chosen (like the radius and minimal amount of
detected molecules within the radius in order to consider a cluster) and these
parameters can influence the results as well. Thus, performing control experiments
is of crucial importance for quantitative PALM measurements.
PALM experiments have shown that β2-adrenergic and M3 acetylcholinergic
receptors do not form oligomers of an order higher than a tetramer in cell lines like
HeLa and CHO cells, even when expressed at high density [127, 128]. To inves-
tigate the real stoichiometry of the receptors and organization in dimers, trimers, or
tetramers, supplementary techniques are necessary, such as SPT. In cell lines
similar to cardiomyocytes (H9c2), higher order oligomers of β2-AR were identified
[128], suggesting the cell type might influence GPCR oligomerization. Addition-
ally, the same study demonstrated that GPCR oligomerization was not associated
with lipid rafts. These results are in accordance with those obtained in HEK293
cells [103].
An important issue is the interactions within dimers and oligomers. Patoway
et al. demonstrated that M3 acetylcholinergic receptors might exist in a stable
dimeric unit and reversibly form tetramers, but not monomers or trimers [129],
underlying the strong interactions within the dimers. Studies with class C GPCR at
GABAB receptor showed that GABAB heterodimers are stable because of strong
non-covalent interactions, whereas oligomeric complexes rely on weaker and
transient interactions between heterodimers [130].
It becomes then crucial to understand really the functions of the oligomer. The
use of supplementary techniques to sense interactions at a molecular scale is
necessary, like single-particle tracking FRET (sptFRET) and single-particle track-
ing PALM (sptPALM).
3.3.5 Studying the Diffusion and Interactions in Dense Samples

(sptPALM/sptFRET)
Unlike ensemble FRET measurements, sptFRET is able to resolve the heterogene-

ity among a population of receptors, by measuring the donor changes of fluores-
cence intensity or emission lifetime, at a single-molecule level. Sakon et al. have
been able to measure sptFRET at the cell surface [131]. Irannejad et al. [132]
suggested that ligands do not alter the dimer distribution. A combination of PALM
and sptFRET method showed that asymmetric hetero-oligomeric complexes could

be formed by the use of receptor mutants that alter the protomers at the functional
level and that this impacted receptor signaling [133]. It becomes then clearer that
GPCRs in vivo may function as dimers or higher oligomer structures.
The crucial point here is on the one hand to locate precisely where the oligomers
exist in the cell or tissue and then to determine the functions of the hetero- and
homodimers. SPT combined with FRET and PALM could provide answers to these
questions. A proof of principle of two-color sptPALM was performed on β2-ARs
heterodimerising with TfR receptors [134]. More studies are necessary, but the
experimental tools exist and are continuously improved.
4 Conclusion
The emergence of new strategies to track molecules at the cell surface or into the
cells opens new perspectives in biology and more specifically in the GPCR studies,
especially because of the recent development of new devices such as more sensitive
plate-readers or new types of microscopy and the synthesis of brighter fluorescent
or luminescent proteins.
All the steps of the GPCR life cycle can now be investigated (Fig. 6). Indeed,
these technological developments give access to fine molecular location in (sub)
Receptor oligomerization Receptor compartimentation

BRET TR-FRET SMPT - FCCS Receptor clustering
Ligand binding
g Oligomer stability
BRET TR-FRET High resolution microscopy
SMPT - FCCS
G prot.
Protein recruitment
BRET TR-FRET Internalization
TR-FRET
+ BRET
2nd messengers
(AMPc, IPx …)
+
2nd messenger
Receptor oligomerization in Protein kinase
production
intracellular compartments
BRET TR-FRET
BRET
protein protein
Phosphorylation
BRET
y P
TR-FRET
Fig. 6 Different techniques which can be used to investigate the different steps of a GPCR life
cellular compartments and to the identification of partners interacting with the

molecules of interest. Different techniques allow simultaneous tracking of molec-
ular complexes and thus a better understanding of molecular network dynamics in
cells, giving crucial answers to their role in cell physiology. One challenge for the
next few years remains to implement these techniques in native tissues to investi-
gate at a single-cell and protein level the different cell responses and the role of
GPCRs.
Acknowledgments This work was supported by research grants from the Centre National de la
Recherche Scientifique, Institut National de la Santé (to J.-P.P., B.M., J.P., T.D.). This work was
also supported by the European Research Council (ERC) under the European Union’s Horizon
2020 research and innovation programme (to JP, grant agreement No. 646788), the Agence
Nationale de la Recherche (to JP, ANR-13-JSV4-0005-01) and the Reǵion Languedoc-Roussillon
(Chercheur d’Avenir), by the European Consortium Oncornet (HORIZON 2020 MSCA–ITN–
2014–ETN–Project 641833 ONCORNET (to J.H., and J.-P.P. and T.D.).
References
1. Jordan BA, Devi LA (1999) G-protein-coupled receptor heterodimerization modulates recep-

tor function. Nature 399:697–700
2. Albizu L, Cottet M, Kralikova M, Stoev S, Seyer R, Brabet I, Roux T, Bazin H, Bourrier E,
Lamarque L, Breton C, Rives ML, Newman A, Javitch J, Trinquet E, Manning M, Pin JP,
Mouillac B, Durroux T (2010) Time-resolved FRET between GPCR ligands reveals oligo-
mers in native tissues. Nat Chem Biol 6:587–594
3. Fernandez-Duenas V, Taura JJ, Cottet M, Gomez-Soler M, Lopez-Cano M, Ledent C,
Watanabe M, Trinquet E, Pin JP, Lujan R, Durroux T, Ciruela F (2015) Untangling
dopamine-adenosine receptor-receptor assembly in experimental parkinsonism in rats. Dis
Model Mech 8:57–63
4. Kern A, Albarran-Zeckler R, Walsh HE, Smith RG (2012) Apo-ghrelin receptor forms
heteromers with DRD2 in hypothalamic neurons and is essential for anorexigenic effects of
DRD2 agonism. Neuron 73:317–332
5. Rimoldi V, Reversi A, Taverna E, Rosa P, Francolini M, Cassoni P, Parenti M, Chini B
(2003) Oxytocin receptor elicits different EGFR/MAPK activation patterns depending on its
localization in caveolin-1 enriched domains. Oncogene 22:6054–6060
6. Luttrell LM (2014) Minireview: more than just a hammer: ligand “bias” and pharmaceutical
discovery. Mol Endocrinol 28:281–294
7. Terrillon S, Barberis C, Bouvier M (2004) Heterodimerization of V1a and V2 vasopressin
receptors determines the interaction with beta-arrestin and their trafficking patterns. Proc Natl
Acad Sci U S A 101:1548–1553
8. F€orster T (1948) Zwischenmolekulare Energiewanderung und Fluoreszenz. Annalen des
Physik (Leipzig) 2:55–75
9. Boute N, Jockers R, Issad T (2002) The use of resonance energy transfer in high-throughput
screening: BRET versus FRET. Trends Pharmacol Sci 23:351–354
10. Angers S, Salahpour A, Joly E, Hilairet S, Chelsky D, Dennis M, Bouvier M (2000) Detection
of beta 2-adrenergic receptor dimerization in living cells using bioluminescence resonance
energy transfer (BRET). Proc Natl Acad Sci U S A 97:3684–3689
11. Xu Y, Piston DW, Johnson CH (1999) A bioluminescence resonance energy transfer (BRET)
system: application to interacting circadian clock proteins. Proc Natl Acad Sci U S A
96:151–156
12. Dionne P, Mireille C, Labonte A, Carter-Allen K, Houle B, Joly E, Taylor SC, Menard L
(2002) BRET2: efficient energy transfer from Renilla luciferase to GFP2 to measure protein-
protein interactions and intracellular signaling events in live cells. In: van Dyke K, van Dyke
C, Woodfork K (eds) Luminescence bio/technology: instruments and applications. CRC
Press, Boca Ranton, pp 539–555
13. De A, Ray P, Loening AM, Gambhir SS (2009) BRET3: a red-shifted bioluminescence
resonance energy transfer (BRET)-based integrated platform for imaging protein-protein
interactions from single live cells and living animals. FASEB J 23:2702–2709
14. Breton B, Sauvageau E, Zhou J, Bonin H, Le Gouill C, Bouvier M (2010) Multiplexing of
multicolor bioluminescence resonance energy transfer. Biophys J 99:4037–4046
15. Perroy J, Pontier S, Charest PG, Aubry M, Bouvier M (2004) Real-time monitoring of
ubiquitination in living cells by BRET. Nat Methods 1:203–208
16. Heroux M, Hogue M, Lemieux S, Bouvier M (2007) Functional calcitonin gene-related
peptide receptors are formed by the asymmetric assembly of a calcitonin receptor-like
receptor homo-oligomer and a monomer of receptor activity-modifying protein-1. J Biol
Chem 282:31610–31620
17. Navarro G, Carriba P, Gandia J, Ciruela F, Casado V, Cortes A, Mallol J, Canela EI, Lluis C,
Franco R (2008) Detection of heteromers formed by cannabinoid CB1, dopamine D2, and
adenosine A2A G-protein-coupled receptors by combining bimolecular fluorescence com-
plementation and bioluminescence energy transfer. Sci World J 8:1088–1097
18. Drinovec L, Kubale V, Nohr Larsen J, Vrecl M (2012) Mathematical models for quantitative
assessment of bioluminescence resonance energy transfer: application to seven transmem-
brane receptors oligomerization. Front Endocrinol 3:104
19. Guo W, Urizar E, Kralikova M, Mobarec JC, Shi L, Filizola M, Javitch JA (2008) Dopamine
D2 receptors form higher order oligomers at physiological expression levels. EMBO J
27:2293–2304
20. Urizar E, Yano H, Kolster R, Gales C, Lambert N, Javitch JA (2011) CODA-RET reveals
functional selectivity as a result of GPCR heteromerization. Nat Chem Biol 7:624–630
21. De A, Loening AM, Gambhir SS (2007) An improved bioluminescence resonance energy
transfer strategy for imaging intracellular events in single cells and living subjects. Cancer
Res 67:7175–7183
22. Levi J, De A, Cheng Z, Gambhir SS (2007) Bisdeoxycoelenterazine derivatives for improve-
ment of bioluminescence resonance energy transfer assays. J Am Chem Soc
129:11900–11901
23. Otto-Duessel M, Khankaldyyan V, Gonzalez-Gomez I, Jensen MC, Laug WE, Rosol M
(2006) In vivo testing of Renilla luciferase substrate analogs in an orthotopic murine model
of human glioblastoma. Mol Imaging 5:57–64
24. Hall MP, Unch J, Binkowski BF, Valley MP, Butler BL, Wood MG, Otto P, Zimmerman K,
Vidugiris G, Machleidt T, Robers MB, Benink HA, Eggers CT, Slater MR, Meisenheimer PL,
Klaubert DH, Fan F, Encell LP, Wood KV (2012) Engineered luciferase reporter from a deep
sea shrimp utilizing a novel imidazopyrazinone substrate. ACS Chem Biol 7:1848–1857
25. Machleidt T, Woodroofe CC, Schwinn MK, Mendez J, Robers MB, Zimmerman K, Otto P,
Daniels DL, Kirkland TA, Wood KV (2015) NanoBRET – a novel BRET platform for the
analysis of protein-protein interactions. ACS Chem Biol 10:1797–1804
26. Goyet E, Bouquier N, Ollendorff V, Perroy J (2016) Fast and high resolution single-cell
BRET imaging. Sci Rep 6:28231
27. Bazin H, Trinquet E, Mathis G (2002) Time resolved amplification of cryptate emission: a
versatile technology to trace biomolecular interactions. J Biotechnol 82:233–250
28. Mathis G (1995) Probing molecular interactions with homogeneous techniques based on rare
earth cryptates and fluorescence energy transfer. Clin Chem 41:1391–1397
29. Selvin PR (2002) Principles and biophysical applications of lanthanide-based probes. Annu
Rev Biophys Biomol Struct 31:275–302
30. Zwier JM, Bazin H, Lamarque L, Mathis G (2014) Luminescent lanthanide cryptates: from
the bench to the bedside. Inorg Chem 53:1854–1866
31. Trinquet E, Fink M, Bazin H, Grillet F, Maurin F, Bourrier E, Ansanay H, Leroy C,
Michaud A, Durroux T, Maurel D, Malhaire F, Goudet C, Pin JP, Naval M, Hernout O,
Chretien F, Chapleur Y, Mathis G (2006) D-myo-inositol 1-phosphate as a surrogate of D-
myo-inositol 1,4,5-tris phosphate to monitor G protein-coupled receptor activation. Anal
Biochem 358:126–135
32. Durroux T, Peter M, Turcatti G, Chollet A, Balestre MN, Barberis C, Seyer R (1999)
Fluorescent pseudo-peptide linear vasopressin antagonists: design, synthesis, and applica-
tions. J Med Chem 42:1312–1319
33. Mouillac B, Manning M, Durroux T (2008) Fluorescent agonists and antagonists for vaso-
pressin/oxytocin G protein-coupled receptors: usefulness in ligand screening assays and
receptor studies. Mini Rev Med Chem 8:996–1005
34. Terrillon S, Cheng LL, Stoev S, Mouillac B, Barberis C, Manning M, Durroux T (2002)
Synthesis and characterization of fluorescent antagonists and agonists for human oxytocin
and vasopressin V(1)(a) receptors. J Med Chem 45:2579–2588
35. Zwier JM, Roux T, Cottet M, Durroux T, Douzon S, Bdioui S, Gregor N, Bourrier E,
Oueslati N, Nicolas L, Tinel N, Boisseau C, Yverneau P, Charrier-Savournin F, Fink M,
Trinquet E (2010) A fluorescent ligand-binding alternative using Tag-lite(R) technology. J
Biomol Screen 15:1248–1259
36. Blanc E, Wagner P, Plaisier F, Schmitt M, Durroux T, Bourguignon JJ, Partiseti M, Dupuis E,
Bihel F (2015) Design and validation of a homogeneous time-resolved fluorescence cell-
based assay targeting the ligand-gated ion channel 5-HT3A. Anal Biochem 484:105–112
37. Hounsou C, Margathe JF, Oueslati N, Belhocine A, Dupuis E, Thomas C, Mann A, Ilien B,
Rognan D, Trinquet E, Hibert M, Pin JP, Bonnet D, Durroux T (2015) Time-resolved FRET
binding assay to investigate hetero-oligomer binding properties: proof of concept with
dopamine D1/D3 heterodimer. ACS Chem Biol 10:466–474
38. Loison S, Cottet M, Orcel H, Adihou H, Rahmeh R, Lamarque L, Trinquet E, Kellenberger E,
Hibert M, Durroux T, Mouillac B, Bonnet D (2012) Selective fluorescent nonpeptidic
antagonists for vasopressin V(2) GPCR: application to ligand screening and oligomerization
assays. J Med Chem 55:8588–8602
39. Ju W, Morishita W, Tsui J, Gaietta G, Deerinck TJ, Adams SR, Garner CC, Tsien RY,
Ellisman MH, Malenka RC (2004) Activity-dependent regulation of dendritic synthesis and
trafficking of AMPA receptors. Nat Neurosci 7:244–253
40. Rahmeh R, Damian M, Cottet M, Orcel H, Mendre C, Durroux T, Sharma KS, Durand G,
Pucci B, Trinquet E, Zwier JM, Deupi X, Bron P, Baneres JL, Mouillac B, Granier S (2012)
Structural insights into biased G protein-coupled receptor signaling revealed by fluorescence
spectroscopy. Proc Natl Acad Sci U S A 109:6733–6738
41. Zurn A, Klenk C, Zabel U, Reiner S, Lohse MJ, Hoffmann C (2010) Site-specific, orthogonal
labeling of proteins in intact cells with two small biarsenical fluorophores. Bioconjug Chem
21:853–859
42. Reiner S, Ambrosio M, Hoffmann C, Lohse MJ (2010) Differential signaling of the endog-
enous agonists at the beta2-adrenergic receptor. J Biol Chem 285:36188–36198
43. Juillerat A, Gronemeyer T, Keppler A, Gendreizig S, Pick H, Vogel H, Johnsson K (2003)
Directed evolution of O6-alkylguanine-DNA alkyltransferase for efficient labeling of fusion
proteins with small molecules in vivo. Chem Biol 10:313–317
44. Juillerat A, Heinis C, Sielaff I, Barnikow J, Jaccard H, Kunz B, Terskikh A, Johnsson K
(2005) Engineering substrate specificity of O6-alkylguanine-DNA alkyltransferase for spe-
cific protein labeling in living cells. Chembiochem 6:1263–1269
45. Keppler A, Gendreizig S, Gronemeyer T, Pick H, Vogel H, Johnsson K (2003) A general
method for the covalent labeling of fusion proteins with small molecules in vivo. Nat
Biotechnol 21:86–89
46. Gronemeyer T, Chidley C, Juillerat A, Heinis C, Johnsson K (2006) Directed evolution of

O6-alkylguanine-DNA alkyltransferase for applications in protein labeling. Protein Eng Des
Sel 19:309–316
47. Keppler A, Pick H, Arrivoli C, Vogel H, Johnsson K (2004) Labeling of fusion proteins with
synthetic fluorophores in live cells. Proc Natl Acad Sci U S A 101:9955–9959
48. Comps-Agrar L, Kniazeff J, Norskov-Lauritsen L, Maurel D, Gassmann M, Gregor N,
Prezeau L, Bettler B, Durroux T, Trinquet E, Pin JP (2011) The oligomeric state sets
GABA(B) receptor signalling efficacy. EMBO J 30:2336–2349
49. Maurel D, Comps-Agrar L, Brock C, Rives ML, Bourrier E, Ayoub MA, Bazin H, Tinel N,
Durroux T, Prezeau L, Trinquet E, Pin JP (2008) Cell-surface protein-protein interaction
analysis with time-resolved FRET and snap-tag technologies: application to GPCR oligo-
merization. Nat Methods 5:561–567
50. Maurel D, Kniazeff J, Mathis G, Trinquet E, Pin JP, Ansanay H (2004) Cell surface detection
of membrane protein interaction with homogeneous time-resolved fluorescence resonance
energy transfer technology. Anal Biochem 329:253–262
51. Delbianco M, Sadovnikova V, Bourrier E, Mathis G, Lamarque L, Zwier JM, Parker D (2014)
Bright, highly water-soluble triazacyclononane europium complexes to detect ligand binding
with time-resolved FRET microscopy. Angew Chem Int Ed Engl 53:10718–10722
52. Gautier A, Juillerat A, Heinis C, Correa Jr IR, Kindermann M, Beaufils F, Johnsson K (2008)
An engineered protein tag for multiprotein labeling in living cells. Chem Biol 15:128–136
53. Zhang Y, So MK, Loening AM, Yao H, Gambhir SS, Rao J (2006) HaloTag protein-mediated
site-specific conjugation of bioluminescent proteins to quantum dots. Angew Chem Int Ed
Engl 45:4936–4940
54. Cottet M, Faklaris O, Falco A, Trinquet E, Pin JP, Mouillac B, Durroux T (2013) Fluorescent
ligands to investigate GPCR binding properties and oligomerization. Biochem Soc Trans
41:148–153
55. Lohse MJ, Nuber S, Hoffmann C (2012) Fluorescence/bioluminescence resonance energy
transfer techniques to study G-protein-coupled receptor activation and signaling. Pharmacol
Rev 64:299–336
56. Milligan G (2013) The prevalence, maintenance, and relevance of G protein-coupled receptor
oligomerization. Mol Pharmacol 84:158–169
57. Vischer HF, Castro M, Pin JP (2015) G protein-coupled receptor multimers: a question still
open despite the use of novel approaches. Mol Pharmacol 88:561–571
58. Handl HL, Vagner J, Yamamura HI, Hruby VJ, Gillies RJ (2005) Development of a
lanthanide-based assay for detection of receptor-ligand interactions at the delta-opioid recep-
tor. Anal Biochem 343:299–307
59. Albizu L, Teppaz G, Seyer R, Bazin H, Ansanay H, Manning M, Mouillac B, Durroux T
(2007) Toward efficient drug screening by homogeneous assays based on the development of
new fluorescent vasopressin and oxytocin receptor ligands. J Med Chem 50:4976–4985
60. Emami-Nemini A, Roux T, Leblay M, Bourrier E, Lamarque L, Trinquet E, Lohse MJ (2013)
Time-resolved fluorescence ligand binding for G protein-coupled receptors. Nat Protoc
8:1307–1320
61. Leyris JP, Roux T, Trinquet E, Verdie P, Fehrentz JA, Oueslati N, Douzon S, Bourrier E,
Lamarque L, Gagne D, Galleyrand JC, M’Kadmi C, Martinez J, Mary S, Baneres JL, Marie J
(2011) Homogeneous time-resolved fluorescence-based assay to screen for ligands targeting
the growth hormone secretagogue receptor type 1a. Anal Biochem 408:253–262
62. Oueslati N, Hounsou C, Belhocine A, Rodriguez T, Dupuis E, Zwier JM, Trinquet E, Pin JP,
Durroux T (2015) Time-resolved FRET strategy to screen GPCR ligand library. Methods Mol
Biol 1272:23–36
63. Klein Herenbrink C, Sykes DA, Donthamsetti P, Canals M, Coudrat T, Shonberg J,
Scammells PJ, Capuano B, Sexton PM, Charlton SJ, Javitch JA, Christopoulos A, Lane JR
(2016) The role of kinetic context in apparent biased agonism at GPCRs. Nat Commun
7:10842
64. Stoddart LA, Johnstone EK, Wheal AJ, Goulding J, Robers MB, Machleidt T, Wood KV, Hill
SJ, Pfleger KD (2015) Application of BRET to monitor ligand binding to GPCRs. Nat
Methods 12:661–663
65. Stoddart LA, White CW, Nguyen K, Hill SJ, Pfleger KD (2016) Fluorescence- and biolumi-
nescence-based approaches to study GPCR ligand binding. Br J Pharmacol 173(20):3028–3037
66. Doumazane E, Scholler P, Fabre L, Zwier JM, Trinquet E, Pin JP, Rondard P (2013)
Illuminating the activation mechanisms and allosteric properties of metabotropic glutamate
receptors. Proc Natl Acad Sci U S A 110:E1416–E1425
67. Calebiro D, Rieken F, Wagner J, Sungkaworn T, Zabel U, Borzi A, Cocucci E, Zurn A, Lohse
MJ (2013) Single-molecule analysis of fluorescently labeled G-protein-coupled receptors
reveals complexes with distinct dynamics and organization. Proc Natl Acad Sci U S A
110:743–748
68. Doumazane E, Scholler P, Zwier JM, Trinquet E, Rondard P, Pin JP (2011) A new approach
to analyze cell surface protein complexes reveals specific heterodimeric metabotropic gluta-
mate receptors. FASEB J 25:66–77
69. Faklaris O, Cottet M, Falco A, Villier B, Laget M, Zwier JM, Trinquet E, Mouillac B, Pin JP,
Durroux T (2015) Multicolor time-resolved Forster resonance energy transfer microscopy
reveals the impact of GPCR oligomerization on internalization processes. FASEB J
29:2235–2246
70. Pradhan AA, Perroy J, Walwyn WM, Smith ML, Vicente-Sanchez A, Segura L, Bana A,
Kieffer BL, Evans CJ (2016) Agonist-specific recruitment of arrestin isoforms differentially
modify delta opioid receptor function. J Neurosci 36:3541–3551
71. Ayoub MA, Trinquet E, Pfleger KD, Pin JP (2010) Differential association modes of the
thrombin receptor PAR1 with Galphai1, Galpha12, and beta-arrestin 1. FASEB J
24:3522–3535
72. Jiang LI, Collins J, Davis R, Lin KM, DeCamp D, Roach T, Hsueh R, Rebres RA, Ross EM,
Taussig R, Fraser I, Sternweis PC (2007) Use of a cAMP BRET sensor to characterize a novel
regulation of cAMP by the sphingosine 1-phosphate/G13 pathway. J Biol Chem
282:10576–10584
73. Xu C, Peter M, Bouquier N, Ollendorff V, Villamil I, Liu J, Fagni L, Perroy J (2013) REV, a
BRET-based sensor of ERK activity. Front Endocrinol (Lausanne) 4:95
74. Alvarez-Curto E, Prihandoko R, Tautermann CS, Zwier JM, Pediani JD, Lohse MJ,
Hoffmann C, Tobin AB, Milligan G (2011) Developing chemical genetic approaches to
explore G protein-coupled receptor function: validation of the use of a receptor activated
solely by synthetic ligand (RASSL). Mol Pharmacol 80:1033–1046
75. Levoye A, Zwier JM, Jaracz-Ros A, Klipfel L, Cottet M, Maurel D, Bdioui S, Balabanian K,
Prezeau L, Trinquet E, Durroux T, Bachelerie F (2015) A broad G protein-coupled receptor
internalization assay that combines SNAP-tag labeling, diffusion-enhanced resonance energy
transfer, and a highly emissive terbium cryptate. Front Endocrinol (Lausanne) 6:167
76. Roed SN, Nohr AC, Wismann P, Iversen H, Brauner-Osborne H, Knudsen SM, Waldhoer M
(2015) Functional consequences of glucagon-like peptide-1 receptor cross-talk and traffick-
ing. J Biol Chem 290:1233–1243
77. Roed SN, Wismann P, Underwood CR, Kulahin N, Iversen H, Cappelen KA, Schaffer L,
Lehtonen J, Hecksher-Soerensen J, Secher A, Mathiesen JM, Brauner-Osborne H, Whistler
JL, Knudsen SM, Waldhoer M (2014) Real-time trafficking and signaling of the glucagon-
like peptide-1 receptor. Mol Cell Endocrinol 382:938–949
78. Coulon V, Audet M, Homburger V, Bockaert J, Fagni L, Bouvier M, Perroy J (2008)
Subcellular imaging of dynamic protein interactions by bioluminescence resonance energy
transfer. Biophys J 94:1001–1009
79. Perroy J (2010) Subcellular dynamic imaging of protein-protein interactions in live cells by
bioluminescence resonance energy transfer. Methods Mol Biol 591:325–333
80. England CG, Ehlerding EB, Cai W (2016) Imaging the biodistribution and performance of
transplanted stem cells with PET. J Nucl Med 57(9):1331–1332
81. Karlsson EA, Meliopoulos VA, Savage C, Livingston B, Mehle A, Schultz-Cherry S (2015)
Visualizing real-time influenza virus infection, transmission and protection in ferrets. Nat
Commun 6:6378
82. Germain-Genevois C, Garandeau O, Couillaud F (2016) Detection of brain tumors and
systemic metastases using NanoLuc and Fluc for dual reporter imaging. Mol Imaging Biol
18:62–69
83. Boute N, Lowe P, Berger S, Malissard M, Robert A, Tesar M (2016) NanoLuc luciferase – a
multifunctional tool for high throughput antibody screening. Front Pharmacol 7:27
84. Robertson DN, Sleno R, Nagi K, Petrin D, Hebert TE, Pineyro G (2016) Design and
construction of conformational biosensors to monitor ion channel activation: a prototype
FlAsH/BRET-approach to Kir3 channels. Methods 92:19–35
85. Shigeto H, Ikeda T, Kuroda A, Funabashi H (2015) A BRET-based homogeneous insulin
assay using interacting domains in the primary binding site of the insulin receptor. Anal
Chem 87:2764–2770
86. Compan V, Baroja-Mazo A, Bragg L, Verkhratsky A, Perroy J, Pelegrin P (2012) A
genetically encoded IL-1beta bioluminescence resonance energy transfer sensor to monitor
inflammasome activity. J Immunol 189:2131–2137
87. Moutin E, Raynaud F, Roger J, Pellegrino E, Homburger V, Bertaso F, Ollendorff V,
Bockaert J, Fagni L, Perroy J (2012) Dynamic remodeling of scaffold interactions in dendritic
spines controls synaptic excitability. J Cell Biol 198:251–263
88. Moutin E, Raynaud F, Fagni L, Perroy J (2012) GKAP-DLC2 interaction organizes the
postsynaptic scaffold complex to enhance synaptic NMDA receptor activity. J Cell Sci
125:2030–2040
89. Mulero M, Perroy J, Federici C, Cabello G, Ollendorff V (2013) Analysis of RXR/THR and
RXR/PPARG2 heterodimerization by bioluminescence resonance energy transfer (BRET).
PLoS One 8:e84569
90. Xu X, Soutto M, Xie Q, Servick S, Subramanian C, von Arnim AG, Johnson CH (2007)
Imaging protein interactions with bioluminescence resonance energy transfer (BRET) in
plant and mammalian cells and tissues. Proc Natl Acad Sci U S A 104:10264–10269
91. Geissler D, Linden S, Liermann K, Wegner KD, Charbonniere LJ, Hildebrandt N (2014)
Lanthanides and quantum dots as Forster resonance energy transfer agents for diagnostics and
cellular imaging. Inorg Chem 53(4):1824–1838
92. Rajapakse HE, Gahlaut N, Mohandessi S, Yu D, Turner JR, Miller LW (2010) Time-resolved
luminescence resonance energy transfer imaging of protein-protein interactions in living
cells. Proc Natl Acad Sci U S A 107:13582–13587
93. Rajapakse HE, Reddy DR, Mohandessi S, Butlin NG, Miller LW (2009) Luminescent
terbium protein labels for time-resolved microscopy and screening. Angew Chem Int Ed
Engl 48:4990–4992
94. Rajapakse HE, Miller LW (2012) Time-resolved luminescence resonance energy transfer
imaging of protein-protein interactions in living cells. Methods Enzymol 505:329–345
95. Geissler D, Charbonniere LJ, Ziessel RF, Butlin NG, Lohmannsroben HG, Hildebrandt N
(2010) Quantum dot biosensors for ultrasensitive multiplexed diagnostics. Angew Chem Int
Ed Engl 49:1396–1401
96. Comps-Agrar L, Kniazeff J, Brock C, Trinquet E, Pin JP (2012) Stability of GABAB receptor
oligomers revealed by dual TR-FRET and drug-induced cell surface targeting. FASEB J
26:3430–3439
97. George SR, Fan T, Xie Z, Tse R, Tam V, Varghese G, O’Dowd BF (2000) Oligomerization of
mu- and delta-opioid receptors. Generation of novel functional properties. J Biol Chem
275:26128–26135
98. Lin H, Trejo J (2013) Transactivation of the PAR1-PAR2 heterodimer by thrombin elicits
beta-arrestin-mediated endosomal signaling. J Biol Chem 288:11203–11215
99. Rocheville M, Lange DC, Kumar U, Sasi R, Patel RC, Patel YC (2000) Subtypes of the
somatostatin receptor assemble as functional homo- and heterodimers. J Biol Chem
275:7862–7869
100. Sartania N, Appelbe S, Pediani JD, Milligan G (2007) Agonist occupancy of a single
monomeric element is sufficient to cause internalization of the dimeric beta2-adrenoceptor.
Cell Signal 19:1928–1938
101. Herrick-Davis K, Weaver BA, Grinde E, Mazurkiewicz JE (2006) Serotonin 5-HT2C recep-
tor homodimer biogenesis in the endoplasmic reticulum: real-time visualization with confo-
cal fluorescence resonance energy transfer. J Biol Chem 281:27109–27116
102. Zou X, Rajendran M, Magda D, Miller LW (2015) Cytoplasmic delivery and selective,
multicomponent labeling with oligoarginine-linked protein tags. Bioconjug Chem
26:460–465
103. Pontier SM, Percherancier Y, Galandrin S, Breit A, Gales C, Bouvier M (2008) Cholesterol-
dependent separation of the beta2-adrenergic receptor from its partners determines signaling
efficacy: insight into nanoscale organization of signal transduction. J Biol Chem
283:24659–24672
104. Kniazeff J, Prezeau L, Rondard P, Pin JP, Goudet C (2011) Dimers and beyond: the
functional puzzles of class C GPCRs. Pharmacol Ther 130:9–25
105. Maurice P, Kamal M, Jockers R (2011) Asymmetry of GPCR oligomers supports their
functional relevance. Trends Pharmacol Sci 32:514–520
106. Warne T, Moukhametzianov R, Baker JG, Nehme R, Edwards PC, Leslie AG, Schertler GF,
Tate CG (2011) The structural basis for agonist and partial agonist action on a beta(1)-
adrenergic receptor. Nature 469:241–244
107. Warne T, Serrano-Vega MJ, Baker JG, Moukhametzianov R, Edwards PC, Henderson R,
Leslie AG, Tate CG, Schertler GF (2008) Structure of a beta1-adrenergic G-protein-coupled
108. Dorsch S, Klotz KN, Engelhardt S, Lohse MJ, Bunemann M (2009) Analysis of receptor
oligomerization by FRAP microscopy. Nat Methods 6:225–230
109. Rybin VO, Xu X, Lisanti MP, Steinberg SF (2000) Differential targeting of beta -adrenergic
receptor subtypes and adenylyl cyclase to cardiomyocyte caveolae. A mechanism to func-
tionally regulate the cAMP signaling pathway. J Biol Chem 275:41447–41457
110. Ilien B, Glasser N, Clamme JP, Didier P, Piemont E, Chinnappan R, Daval SB, Galzi JL,
Mely Y (2009) Pirenzepine promotes the dimerization of muscarinic M1 receptors through a
three-step binding process. J Biol Chem 284:19533–19543
111. Goin JC, Nathanson NM (2006) Quantitative analysis of muscarinic acetylcholine receptor
homo- and heterodimerization in live cells: regulation of receptor down-regulation by
heterodimerization. J Biol Chem 281:5416–5425
112. Zeng FY, Wess J (1999) Identification and molecular characterization of m3 muscarinic
receptor dimers. J Biol Chem 274:19487–19497
113. Grant M, Collier B, Kumar U (2004) Agonist-dependent dissociation of human somatostatin
receptor 2 dimers: a role in receptor trafficking. J Biol Chem 279:36179–36183
114. Briddon SJ, Middleton RJ, Cordeaux Y, Flavin FM, Weinstein JA, George MW, Kellam B,
Hill SJ (2004) Quantitative analysis of the formation and diffusion of A1-adenosine receptor-
antagonist complexes in single living cells. Proc Natl Acad Sci U S A 101:4673–4678
115. Cordeaux Y, Briddon SJ, Alexander SP, Kellam B, Hill SJ (2008) Agonist-occupied A3
adenosine receptors exist within heterogeneous complexes in membrane microdomains of
individual living cells. FASEB J 22:850–860
116. Corriden R, Kilpatrick LE, Kellam B, Briddon SJ, Hill SJ (2014) Kinetic analysis of
antagonist-occupied adenosine-A3 receptors within membrane microdomains of individual
cells provides evidence of receptor dimerization and allosterism. FASEB J 28:4211–4222
117. Briddon SJ, Gandia J, Amaral OB, Ferre S, Lluis C, Franco R, Hill SJ, Ciruela F (2008)
Plasma membrane diffusion of G protein-coupled receptor oligomers. Biochim Biophys Acta
1783:2262–2268
118. Patel RC, Kumar U, Lamb DC, Eid JS, Rocheville M, Grant M, Rani A, Hazlett T, Patel SC,
Gratton E, Patel YC (2002) Ligand binding to somatostatin receptors induces receptor-
specific oligomer formation in live cells. Proc Natl Acad Sci U S A 99:3294–3299
119. Comar WD, Schubert SM, Jastrzebska B, Palczewski K, Smith AW (2014) Time-resolved
fluorescence spectroscopy measures clustering and mobility of a G protein-coupled receptor
opsin in live cell membranes. J Am Chem Soc 136:8342–8349
120. Kasai RS, Suzuki KG, Prossnitz ER, Koyama-Honda I, Nakada C, Fujiwara TK, Kusumi A
(2011) Full characterization of GPCR monomer-dimer dynamic equilibrium by single mol-
ecule imaging. J Cell Biol 192:463–480
121. Moutin E, Compan V, Raynaud F, Clerte C, Bouquier N, Labesse G, Ferguson ML, Fagni L,
Royer CA, Perroy J (2014) The stoichiometry of scaffold complexes in living neurons –
DLC2 functions as a dimerization engine for GKAP. J Cell Sci 127:3451–3462
122. Hern JA, Baig AH, Mashanov GI, Birdsall B, Corrie JE, Lazareno S, Molloy JE, Birdsall NJ
(2010) Formation and dissociation of M1 muscarinic receptor dimers seen by total internal
reflection fluorescence imaging of single molecules. Proc Natl Acad Sci U S A
107:2693–2698
123. Olofsson L, Felekyan S, Doumazane E, Scholler P, Fabre L, Zwier JM, Rondard P, Seidel
CA, Pin JP, Margeat E (2014) Fine tuning of sub-millisecond conformational dynamics
controls metabotropic glutamate receptors agonist efficacy. Nat Commun 5:5206
124. Betzig E, Patterson GH, Sougrat R, Lindwasser OW, Olenych S, Bonifacino JS, Davidson
MW, Lippincott-Schwartz J, Hess HF (2006) Imaging intracellular fluorescent proteins at
nanometer resolution. Science 313:1642–1645
125. Sengupta P, Jovanovic-Talisman T, Lippincott-Schwartz J (2013) Quantifying spatial orga-
nization in point-localization superresolution images using pair correlation analysis. Nat
Protoc 8:345–354
126. Ester M, Kriegel H-P, Sander J, Xu X (1996) A density-based algorithm for discovering
clusters in large spatial databases with noise. The AAAI Press, Menlo Park, CA
127. Scarselli M, Annibale P, Gerace C, Radenovic A (2013) Enlightening G-protein-coupled
receptors on the plasma membrane using super-resolution photoactivated localization
microscopy. Biochem Soc Trans 41:191–196
128. Scarselli M, Annibale P, Radenovic A (2012) Cell type-specific beta2-adrenergic receptor
clusters identified using photoactivated localization microscopy are not lipid raft related, but
depend on actin cytoskeleton integrity. J Biol Chem 287:16768–16780
129. Patowary S, Alvarez-Curto E, Xu TR, Holz JD, Oliver JA, Milligan G, Raicu V (2013) The
muscarinic M3 acetylcholine receptor exists as two differently sized complexes at the plasma
membrane. Biochem J 452:303–312
130. Rondard P, Pin JP (2015) Dynamics and modulation of metabotropic glutamate receptors.
Curr Opin Pharmacol 20:95–101
131. Sakon JJ, Weninger KR (2010) Detecting the conformation of individual proteins in live
cells. Nat Methods 7:203–205
132. Irannejad R, Tomshine JC, Tomshine JR, Chevalier M, Mahoney JP, Steyaert J, Rasmussen
SG, Sunahara RK, El-Samad H, Huang B, von Zastrow M (2013) Conformational biosensors
reveal GPCR signalling from endosomes. Nature 495:534–538
133. Jonas KC, Fanelli F, Huhtaniemi IT, Hanyaloglu AC (2015) Single molecule analysis of
functionally asymmetric G protein-coupled receptor (GPCR) oligomers reveals diverse
spatial and structural assemblies. J Biol Chem 290:3875–3892
134. Benke A, Olivier N, Gunzenhauser J, Manley S (2012) Multicolor single molecule tracking of
stochastically active synthetic dyes. Nano Lett 12:2619–2624
Top Med Chem (2019) 30: 253–284
DOI: 10.1007/7355_2017_32
Published online: 11 February 2018
Modulation of Metabotropic Glutamate

Receptors by Orthosteric, Allosteric,
and Light-Operated Ligands
Cyril Goudet, Xavier Rovira, Philippe Rondard, Jean-Philippe Pin,

Amadeu Llebaria, and Francine Acher
Abstract Glutamate is the major excitatory neurotransmitter of the mammalian central

nervous system (CNS). Its actions are mediated by two broad classes of receptors: ligand-
gated ion channels and G-protein-coupled receptors (GPCRs), named ionotropic and
metabotropic glutamate receptors (mGluRs), respectively. The mGluR family consists of
eight subtypes which are responsible for glutamate neuromodulatory actions throughout
the CNS. These receptors are complex allosteric machines, offering multiple possibilities
to regulate their activity. Their structure is composed of a seven-helix transmembrane
domain, common to all GPCRs, and a large bilobate extracellular domain (ECD) where
glutamate binds. Moreover, mGluRs are constitutive dimers crosslinked by a disulfide
bridge. This chapter will provide an overview of mGluR structure, dynamics, and
pharmacology, describing the different types of pharmacological tools that modulate
mGluRs function, from orthosteric to allosteric molecules, including nanobodies, ions,
and light-operated ligands.
Keywords Glutamate, Neurotransmitter, Receptor, Pharmacology, Optogenetic,

Optopharmacology, Photopharmacology
C. Goudet (*), P. Rondard, and J.-P. Pin

IGF, CNRS, INSERM, Univ. de Montpellier, Montpellier, France
e-mail: cyril.goudet@igf.cnrs.fr
X. Rovira
Molecular Photopharmacology Research Group, The Tissue Repair and Regeneration
Laboratory, University of Vic – Central University of Catalonia, Vic, Spain
A. Llebaria
MCS, Laboratory of Medicinal Chemistry, Institute for Advanced Chemistry of Catalonia
(IQAC-CSIC), Barcelona, Spain
F. Acher
Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, CNRS UMR8601,
Université Paris Descartes, Paris, Cedex 6, France
254 C. Goudet et al.
Contents
1 Class C G-Protein-Coupled Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254
2 Metabotropic Glutamate Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
2.1 Distribution and Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256
2.2 Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256
2.3 Therapeutic Potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
3 Modulation by Orthosteric Ligands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259
4 Modulation by Allosteric Ligands Binding to the 7TM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262
5 Modulation by Cations and Anions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266
6 Modulation by Nanobodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
7 Ligand Control of Heterodimeric mGluRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268
8 Modulation by Light-Operated Ligands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
8.1 Optogenetic Pharmacology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
8.2 Photopharmacology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271
9 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
1 Class C G-Protein-Coupled Receptors
G-protein-coupled receptors constitute a large family of membrane proteins respon-

sible for transduction of various external signals into intracellular responses through
heterotrimeric G proteins. As a result, GPCRs are involved in the regulation of many
physiological or pathological processes and are the target of about a quarter of the
drugs available on the market.
Based on sequence comparison of their heptahelical domain (HD), four main
classes of GPCRs have been defined: class A rhodopsin-like, class B secretin-like,
class C metabotropic glutamate/pheromone, and frizzled receptors. Class C GPCRs
include the eight metabotropic glutamate receptors (named mGlu1 to mGlu8); the
metabotropic receptor for GABA which is composed of two subunits, GABAB1 and
GABAB2; the calcium-sensing (CaS) and CaS-related (GPRC6a) receptor; and the
receptors involved in sweet and umami taste detection (T1R1, T1R2, and T1R3).
Several pheromone receptors are also part of class C; however, although they play an
important role in pheromonal detection in rodents, all but one are pseudogenic in
human. Seven additional orphan class C GPCRs have also been identified in mam-
malian genomes.
Class C GPCRs possess two remarkable features that are important for their regulation
and function. The first structural particularity of most class C receptors is the presence of a
large extracellular domain (ECD), containing a bilobate domain called the Venus flytrap
(VFT) domain where natural ligands bind [1, 2]. This domain is juxtaposed to a core
transmembrane domain composed of seven helices, a feature common to all GPCRs and
responsible of G-protein coupling (Fig. 1). Another specific feature of most class C
GPCRs is their constitutive dimeric nature. Both CaS and mGlu receptors are dimers
linked by a disulfide bridge. mGluRs have long been believed to form exclusively
homodimers, but recent data are suggesting that they may also form heterodimers [3–
6]. GABAB and taste receptors form obligate heterodimers. Sweet and umami taste
receptors function as T1R2-T1R3 and T1R1-T1R3 heterodimers [7]. In GABAB
Modulation of Metabotropic Glutamate Receptors by Orthosteric. . . 255
Fig. 1 Complex structure

of mGluRs offers different
possibilities for modulation
of their activity. Structural
model of a dimeric mGlu
receptor composed of Venus
flytrap (VFT), cysteine-rich
(CRD), and heptahelical
(HD) domains. Different
types of ligands and their
binding site locations are
shown
receptors, the subunit GABAB1 contains the agonist binding site, whereas GABAB2
couples to the G protein and allows the targeting of the heterodimer to the cell surface by
masking a retention signal located in the C terminus of the GABAB1 subunit [8].
2 Metabotropic Glutamate Receptors
Glutamate is the major excitatory neurotransmitter of the mammalian central ner-

vous system. This neurotransmitter exerts its actions through two broad types of
receptors: ionotropic and metabotropic receptors. While ionotropic receptors are
ligand-gated ion channels involved in fast synaptic transmission, metabotropic
receptors belong to the superfamily of G-protein-coupled receptors (GPCRs) and
are responsible for the neuromodulatory effects of glutamate.
Eight genes encoding metabotropic glutamate receptors have been identified in
mammalian genomes. Based on sequence homology, signal transduction, and phar-
macology, they can be subdivided into three groups. Group I mGluRs (mGlu1 and
mGlu5) are mainly coupled to Gq and, as such, activate PLC and generate intracel-
lular calcium signals. Both group II (mGlu2 and mGlu3) and group III (mGlu4,
mGlu6, mGlu7, and mGlu8) mGluRs are mainly coupled to Gi/o and can therefore
inhibit adenylate cyclase and regulate the activity of various ion channels. Several
mGluR splice variants have been described, most of them altering the intracellular
C-terminal tails [9].
2.1 Distribution and Function
The different mGluRs are widely distributed throughout the peripheral and central
nervous system. Group I mGluRs are mostly localized at the post-synapse where they
play an important role in upregulating neuronal excitability and in regulating currents
through ionotropic glutamate receptors. Group II and III mGluRs are mostly presynaptic
receptors and their activation tends to reduce synaptic transmission and neuronal excit-
ability. As autoreceptors, mGluRs are involved in reducing transmission at glutamatergic
synapses, but they are also present in other types of synapses, such as dopaminergic and
GABAergic synapses, where they play the role of heteroreceptors reducing neurotrans-
mitter release. In addition, besides being expressed in neurons, mGluRs have also been
detected in different glial cells (astrocytes, oligodendrocytes, and microglia) which are
active regulators and protectors of the nervous system (see [10] for review). Moreover,
mGluRs are also distributed outside the CNS, for example in the heart, in adrenal glands
or in lymphocytes (see [11] for review).
2.2 Structure
Like most class C GPCRs, mGluRs possess a large ECD composed of a VFT, connected
via a cysteine-rich domain (CRD) to the heptahelical transmembrane domain (HD). They
are mandatory dimers, crosslinked by a disulfide bridge connecting two flexible loops at
the top of the VFT (Fig. 1). Whereas several high-resolution X-ray structures of the
isolated ECD and HD of mGluRs are available, no crystal structure of a full-length
mGluR has been solved yet.
Dimerization is a prerequisite for function of mGluRs. Indeed, when reconstituted
in nanodiscs that reproduce a native-like environment, solely the fraction containing
the dimeric mGluRs was able to activate a purified G protein following stimulation by
glutamate, whereas a monomeric form can be activated by an allosteric molecule
binding on the transmembrane (functional) domain [12]. However, despite its dimeric
nature, the receptor functions asymmetrically with only one of the two subunits that
activates the G protein at a time [13–15]. Contrary to GABAB receptors that form
higher-order complexes [16], only strict dimers of mGluRs have been detected so far,
but this remains to be confirmed with receptors in their native environment [3, 17].
mGluRs were previously believed to exclusively form homodimers. However,
mGluRs can form heterodimers within their classification groups and between
groups II and III in transfected cells [3]. There is evidence for the existence of
mGlu2-4 heterodimers in native tissues based on specific pharmacological signatures
[5, 6]. There is also evidence for the association of mGlu1 and mGlu5 in brain tissues
and potentially for mGlu7 and mGlu8 as well [4, 18]. Further studies are required to
explore the expression and function of mGlu heterodimers.
Crystal structures of the isolated VFT dimers have been solved for most mGluRs
(mGlu1, 2, 3, 5 and 7) [1, 2, 19–21]. Structures revealed that each VFT can adopt two
major states: (1) an “open” conformation in the absence of ligand or in the presence
of competitive antagonists and (2) a “closed” conformation stabilized by agonists
[1, 2, 22, 23]. Of note, the VFT domain also binds endogenous allosteric modulators,
notably extracellular ions, like calcium and chloride, that potentiate receptor activity
[24–29].
The CRD is a short and rigid domain that is stabilized by disulfide bridges, one of
which connects the CRD to the VFT [30]. The CRD is required for the propagation
of the activation from the VFT to the HD. The agonist-induced change of configu-
ration of the VFT dimer brings the CRDs in contact in the active state. The activation
process is then propagated to the HDs, which undergo major rearrangement influenc-
ing their relative position and changing the dimer interface from transmembrane
helices 4–5 (TM 4–5) to TM6 [31].
The HD of mGluRs is responsible for G-protein activation. It contains the binding
site for synthetic allosteric modulators (AM). These allosteric ligands can be either
positive (PAM) or negative allosteric modulators (NAM) if they enhance or inhibit
the agonist-induced activity of the receptor, respectively. These modulators affect
the stability of the active conformation of the receptor. The overall structure of the
HD is conserved between class C and class A GPCRs, despite the low sequence
conservation (<20% identical residues) as revealed by the high-resolution structures
of isolated mGlu1 and mGlu5 HDs [32, 33]. Of note, no endogenous ligand targeting
this domain has been identified until now.
2.3 Therapeutic Potential
Due to their vital role in the regulation of neurotransmission and neuronal excitabil-
ity, mGluRs can be considered as valuable targets for treating neurological disorders.
This is supported by many preclinical studies that have demonstrated their role in
several CNS diseases where it is suggested that mGluRs correct the dysregulation of
glutamatergic signaling or neurological imbalances in non-glutamatergic systems.
Hereafter, we propose a non-exhaustive list of pathologies currently associated with
the different mGluRs in preclinical studies, some of which have led to clinical trials.
So far, however, there is no marketed drug which targets mGluRs.
2.3.1 Group I mGluRs
Defects of mGlu1 are linked to motor impairments [34], and activators of mGlu1 are
considered as potential candidates for the treatment of ataxia associated with a loss of
function or expression mGlu1 [35]. Expression of mGlu1 has been detected in human
malignant melanomas where its oncogenic activity is inhibited by mGlu1 NAMs [36].
mGlu5 is one of the most well-studied receptors in this family. It is considered as a target
of interest for the treatment of anxiety. Blockade of mGlu5 attenuates excitability in brain
structures involved in anxiety such as amygdala. Interestingly, the anxiolytic drug fenobam
developed in the 1970s–1980s has subsequently been found to act as an mGlu5 NAM
[37]. There is also a strong interest in mGlu5 for the symptomatic treatment of Parkinson’s
disease (PD) and related movement disorders. Several clinical trials have been launched to
evaluate the potential of mGlu5 blockade in PD. Mavoglurant (AFQ056) (Fig. 6), an mGlu5
NAM developed by Novartis, displayed significant antikinetic effects without interfering
with L-DOPA in phase 2a and 2b studies. Unfortunately, the program was discontinued
due to lack of efficacy in further studies. Another initial phase 2 study has revealed that
dipraglurant (ADX-48621), an mGlu5 NAM developed by Addex Pharmaceuticals,
displayed significant efficacy in the treatment of PD levodopa-induced dyskinesia. Antag-
onism of mGlu5 is also of interest in migraine and in gastroesophageal reflux disorders
(GERD). Unfortunately, phase I clinical trials with mGlu5 NAMs for migraine or GERD
have been performed but discontinued due to liver toxicity in some volunteers. Although
promising results were obtained in preclinical animal models [38], the use of mavoglurant
to relieve autistic symptoms in patients with fragile X syndrome did not demonstrate
sufficient efficacy.
2.3.2 Group II mGluRs
Group II mGluRs are considered as interesting targets for schizophrenia. Indeed, the
mGlu2/3 agonists LY354740 and LY379268 (Fig. 4) alleviate symptoms induced by
the NMDA receptor antagonist phencyclidine (PCP), an animal model of schizo-
phrenia [39, 40]. Activation of mGlu2/3 receptors by pomaglumetad methionil, the
oral prodrug of LY404039 (an mGlu2/3 receptor agonist) developed by Lilly, has
shown good efficacy on positive and negative symptoms of schizophrenia in an
initial phase 2 clinical trial [41]. Unfortunately the program was discontinued due to
the lack of overall efficacy in subsequent phase 3 trial, although the drug was
efficient in reversing symptoms in early-in-disease patients [42–44]. The develop-
ment of pomaglumetad methionil is now continuing for evaluation in early schizo-
phrenia by Denovo Biopharma. Preclinical studies have also linked mGlu2 to anxiety
and depression. The mGlu2/3 agonist eglumagad (LY354740) from Lilly has shown
efficacy in the treatment of generalized anxiety disorder (GAD), but the phase 3 trial
was discontinued due to some toxicity observed in preclinical studies [45].
Genetic studies revealed the possible involvement of mGlu3 in schizophrenia and
other psychiatric disorders [46–50], as well as cancer development at the periphery
[51, 52].
2.3.3 Group III mGluRs
mGlu4 is an emerging target for the treatment of PD [53]. The antiparkinsonian effect
of mGlu4 activation results from the correction of the imbalance of neurotransmission
among the basal ganglia circuitry, as shown with the mGlu4 PAM PHCCC [54]
(Fig. 6) or with mGlu4 agonists [55]. Very recently (fall 2017), a phase 2 clinical
trial has been initiated by Prexton therapeutics to evaluate the efficacy and safety of
the mGlu4 PAM Foliglurax (PXT002331, Fig. 6) [56] in PD patients treated with
levodopa, experiencing end-of-dose wearing off and levodopa-induced dyskinesia. In
addition, preclinical studies suggest that mGlu4 could be a potential therapeutic target
in anxiety and depression [57], inflammatory and neuropathic pain [58–61], schizo-
phrenia [62], neuroinflammation [63], and autism [64].
mGlu6 is expressed mainly in the retina where it is responsible for the ON-bipolar
neuronal transmission [65]. Genetic mutations of mGlu6 have been linked to con-
genital stationary night blindness [66].
mGlu7 could be an attractive therapeutic target for several CNS diseases, notably
anxiety, depression [67], or epilepsy [68]. However, until recently, preclinical studies
have been difficult to perform due to the lack of selective and potent pharmacology for
this receptor. The recent discovery of mGlu7 NAMs such as MMPIP, ADX71743 or
XAP044, or the dual mGlu7/8 PAM VU6005649 should facilitate the discovery of the
particular role of this receptor (Fig. 6). Of note AMN082, an mGlu7 ago-PAM [69],
has attracted much interest but yields contradictory results in vivo that should be
interpreted with precaution as AMN082 is rapidly metabolized into a monoamine
transporter inhibitor [70].
mGlu8 is one of the least studied receptors of the mGluR family due to the lack of
selective pharmacological tools that have hampered the preclinical studies. mGlu8
may be of interest in anxiety since mGlu8 knockout mice exhibit higher levels of
anxiety [71]. mGlu8 has also been shown to regulate thermal and mechanical pain
perception in neuropathic pain [72].
3 Modulation by Orthosteric Ligands
Orthosteric ligands act in the same binding pocket as the endogenous ligand. They
are also referred to as “competitive” ligands, since they are competing with the
endogenous ligand for the same site. They can either be agonists or antagonists.
In mGluRs, glutamate and orthosteric agonists bind in the cleft between the two
lobes of the VFT, stabilizing its close state, a first step in the activation process
[73]. The closed VFTs are more prone to associate differently, leading to a change in
their relative orientation such that the dimer of ECD moves from a “resting” (R) to an
active (A) state through a rotation of 70 . On the other hand, competitive antagonists
prevent the full closure of the VFT [1, 2, 23, 73]. Based on crystal structures, the main
conformations that define the inactive and active states of the mGluR are the resting
state Roo where both VFTs are open and the active states Aco or Acc where one or
both VFTs are closed, respectively. Indeed, closure of one VFT to reach the Aco form
is sufficient to generate a signal, although the closure of both VFTs to reach the Acc
state is required for full activity [74]. The two lobes (lower lobes linked to the CRD
domain) are distant in the resting state and become closer in the active state [1, 2].
The L-glutamate binding site is highly conserved between homologs, making it
very difficult to identify compounds acting selectively on mGlu receptor subtypes
[75, 76]. Residues in direct interactions with the amino acid moiety of glutamate are
Fig. 2 Glutamate binding site. X-ray structure of glutamate bound to mGlu1 VFT domain (PDB 1D
1EWK). Conserved and selective residues of the glutamate binding site are highlighted in similar
colors as in Fig. 3
fully conserved [75] as well as two basic residues (Arg and Lys) (Figs. 2 and 3) from
lobe 1 that bind the distal carboxylate of glutamate [22]. In addition, glutamate adopts
a similar conformation when binding to all mGlu subtypes [77]. Consequently
L-CCG-I, a partly constrained glutamate analogue, activates all mGlu receptors
with increased potency [78]. Moreover, LY341495, an L-CCG-I derivative, is the
most popular mGlu competitive antagonist for all subtypes yet with higher potency at
mGlu2/3 receptors [79].
In spite of these common features, several group selective residues at the binding
sites have allowed early identification of group I, group II, and group III selective
ligands. Among them the most popular agonists are 3,5-DHPG and quisqualic acid
for group I, DCG-IV [78] and LY354740 [80] and analogues for group II, and
L-AP4 and ACPT-I [81] for group III (Fig. 4) (see [82] for review).
A few molecules deriving from group selective agonists have gained subtype-
specific properties due to interaction with selective residues adjacent to the glutamate
binding site. Such ligands were found for mGlu2 (LY541850 [20] and LY2812223
[19]) and mGlu4 (LSP4-2022 [83]) (Fig. 5). Docking studies of LSP4-2022 and
analogues revealed that this molecule extends out of the mGlu4 glutamate binding
site. Further modeling investigations showed that two pockets may be reached by
these compounds taking the place of chloride ions that are binding to these sites in the
endogenous protein [28, 83–85] (Fig. 7). These ions were demonstrated to be strong
PAMs of mGluRs [28, 87]. Therefore, it also implies that these agonists, which target
simultaneously an orthosteric and an allosteric binding site on the same receptor VFT,
constitute a new class of bitopic ligands. This particular binding offers new possibil-
ities to design subtype-specific ligands as the topology of this new pocket is less
conserved than the agonist-binding pocket, as exemplified by the first selective mGlu4
orthosteric agonist LSP4-2022 [83].
Fig. 3 Sequence alignment. mGluR VFT sequence alignment. Conserved and selective residues of
the glutamate binding site are highlighted
Fig. 4 Structures of several classical orthosteric mGluR ligands
Fig. 5 Structure of subtype selective agonists of mGluRs. LY41850 and LY2812223 selectively
activate mGlu2 [19, 20] whereas LSP4-2022 is selective of mGlu4 [83]
4 Modulation by Allosteric Ligands Binding to the 7TM
Allosteric modulators are ligands that regulate the activity of a receptor from a binding
site distinct from the orthosteric site where the endogenous ligands bind. Accordingly,
allosteric modulators have specific properties different from those of orthosteric
ligands.
Allosteric modulators can either inhibit or potentiate the activation of the receptors. In
consequence, they are divided in two main categories, the negative and positive allosteric
modulators (NAM and PAMs). In addition, several neutral (also known as silent)
allosteric modulators have been described, i.e., ligands which bind to an allosteric site
without modifying receptor activity [88]. NAMs act as noncompetitive antagonists and
can present inverse agonist properties, meaning that they can inhibit the receptor consti-
tutive activity. In contrast, PAMs potentiate the activity of orthosteric agonists by
enhancing either their potency or efficacy or both. In some cases, PAMs can directly
activate the receptor and are referred to as allosteric agonist or ago-PAM, although such
activity is usually partial. When devoid of agonist activity by themselves, they are
referred to as “pure” PAMs. Of interest, these compounds should present relatively
fewer side effects and lead to less desensitization than agonists. They are, therefore,
very promising as potential therapeutic drugs. Moreover, while the site of glutamate has
been subjected to intense pressure during evolution and is thus highly conserved between
homologs, the binding pocket of allosteric modulators is more variable allowing the
discovery of subtype selective ligands of mGluRs. For these reasons, the identification of
new allosteric modulators is a significant research focus by pharmaceutical companies.
For mGluRs, the first allosteric ligands that have been described were the mGlu1
NAMs CPCCOEt [89, 90] and BAY36-7620 [91] and the mGlu5 NAM MPEP
[92, 93] (Fig. 6). These noncompetitive antagonists have a structure which differs
from that of orthosteric agonists and antagonists. They were soon found to bind in
the HD of the receptor [91, 93]. Shortly afterward, the first mGlu1 PAMs was
reported [94]. These pioneering studies have opened avenues for the discovery of
allosteric ligands for most mGluRs. Of note, no endogenous PAM or NAM binding
in the HD of mGluRs have been described so far.
The high-resolution X-ray structures of the isolated HD of mGlu1 and mGlu5
have been solved in their inactive state in complex with NAMs (Fig. 7), bringing
much information on the binding mode of the allosteric modulators. The molecule
FITM was co-crystalized in mGlu1 [32] and mavoglurant or fragments that led to the
design of HTL14242 in mGlu5 [32, 33, 86]. First, these crystals confirmed that the
overall structure of the HD is conserved between mGluRs and class A GPCRs
despite the lack of sequence conservation (<15% identical residues). Some differ-
ences are also observed between class C and other classes, such as distinct distribu-
tion patterns of proline-induced kinks in the helical backbone and a more tightly
restricted ligand binding cavity between the TMs. These structures also revealed the
binding pocket of NAM in mGluRs HD. The two NAMs bind in a cavity located
centrally in the HD. The binding pocket for FITM is located in the upper part of the
mGlu1 HD and partially overlaps with the orthosteric binding sites of many class A
GPCRs. The narrow pocket is defined by residues on TM 2, 3, 5, 6, and 7 and is
partially surmounted with ECL2 [33]. Mavoglurant binds deeper in the mGlu5 HD,
in a pocket delimited by the same TMs than FITM in mGlu1 but at 8 Å from the
receptor surface [32] (Fig. 7).
Interestingly, the existence of these two distinct binding pockets confirmed that
allosteric modulators can bind in multiple sites in mGluRs HD [32, 33]. Most studies
on the binding mode of allosteric modulators have been performed on mGlu5 (see [95] for
review) and to a lesser extent on mGlu1 [94], mGlu2 [96, 97], and mGlu4 [98]. The
existence of multiple allosteric sites in the HD of mGluRs has been proposed, notably in
mGlu5 [95, 99] and mGlu4 [98]. For example, the two mGlu5 PAMs CPPHA and
VU0357121 do not displace MPEP, contrary to other mGlu5 PAMs such as VU29
[100, 101]. Also, the mGlu5 PAM DFB is suggested to occupy an overlapping, but
distinct, binding site to MPEP in mGlu5 HD [102]. On mGlu4, the ago-PAM VU0155041
and the PAM PHCCC are not competing for the same site [103]. Accordingly, two
partially overlapping binding pockets have been identified in mGlu4 HD [98]. Interest-
ingly, it appears that the intrinsic efficacy and cooperativity of mGlu4 PAMs are
correlated with their binding mode. A first shallow site is analogous to the pocket of
natural agonists of class A GPCRs and is linked to PAM intrinsic allosteric agonism. A
second site is located more deeply within the HD, pointing toward a site topographically
homologous to the Na + binding pocket of class A GPCRs and is occupied by PAMs
Fig. 6 Structure of several positive and negative allosteric modulators of mGluRs
exhibiting the strongest cooperativity. Interestingly, this deep site also corresponds to that
of mavoglurant in the crystal structure of the mGlu5 HD [32].
PAMs would facilitate receptor activation by stabilizing the active conformation of the
HD. From the recent crystal structures and the previous structure-function studies, it has
Fig. 7 Superposition of all allosteric ligands crystalized in complex with the transmembrane
domains of mGlu1 (violet) and mGlu5 (brown). The depth of the ligand binding pocket and the
amino acids involved in the interaction are shown. The structure of mGlu5 (4OO9) is shown as a
reference for the binding site location of ligands [32, 33, 86]
been hypothesized that similar conformational changes would occur in class A and C
GPCRs. Accordingly, when deleted from their VFT and CRD, headless mGluRs can be
directly activated or inhibited by PAMs and NAMs acting at the level of their 7TM
[104, 105], similarly to agonist and antagonist in class A GPCRs.
As already mentioned, pure PAMs (that do not directly activate the receptor but
facilitate its activation by glutamate) are considered valuable drug-like compounds
with therapeutic potential since the adverse effects that may be caused by treatment
with agonists, induced by long-lasting activation and desensitization, should be
lowered. On the other hand, PAMs with intrinsic efficacy (with partial agonist
activity) (ago-PAMs) may lead to side effects since their activity is not dependent
solely on endogenous glutamate release. This has recently been exemplified in a
study comparing the therapeutic potential of pure vs ago mGlu5 PAMs in schizo-
phrenia, which revealed that both types of PAMs reduce symptoms in preclinical
models but that, contrary to pure PAMs, ago-PAMs induce epileptic activity [106].
Of interest, by stabilizing particular conformations of the HD, it can be speculated
that PAMs or NAMs could modulate a specific signaling pathway while agonists can
activate several pathways, a concept known as functional selectivity or bias signaling
[107]. Recently, a biased mGlu5 PAM (VU0409551) has been described [108]. This
PAM enhances mGlu5 coupling to Gαq-mediated signaling but not its modulation of
NMDAR currents. In vivo, this PAM induces robust antipsychotic-like and cognition-
enhancing activity, suggesting that modulation of NMDAR currents is not critical for
efficacy in these animal models of schizophrenia, as previously thought. Biased
mGluRs AM are promising pharmacological tools that may open the way to modu-
lation for specific signaling cascades [107].
5 Modulation by Cations and Anions
Ion modulation of mGluRs has previously been reported, notably to calcium (Ca2+),
gadolinium (Gd3+), and chloride (Cl) [2, 24–26, 109–111]].
Cl sensitivity has been reported for most mGluRs [24, 25, 28, 29, 109, 111]. Extra-
cellular Cl ions favor the agonist-induced active conformation of mGluRs. All mGluRs
are positively modulated by Cl ion, but they display different sensitivities – mGlu3 and
mGlu4 being the most sensitive and mGlu2 the least [28, 87]. Recent crystal structure of
mGlu3 VFT showed several halide binding sites [19]. The Cl sensitivity appears to be
carried mainly by two sites localized in mGluRs VFT (Fig. 8), with the remarkable
exception of mGlu2 that possesses only one functional site. A first site is located within
lobe 1, in a binding pocket adjacent to the glutamate binding site. Interestingly, this site is
well conserved not only in mGluRs but also in other members of the LIVBP-like family.
It corresponds to the Cl binding site of the structurally closely related natriuretic peptide
receptor, NPR-A [112]. Of note, this site has previously been described as the binding
pocket for the carboxylate moiety of the mGlu4 selective agonist LSP4-2022, and the
putative presence of a chloride ion was considered [83, 84]. The second site is created by
the interface of both VFT lobes, when the receptor is in a closed active conformation and
shows more divergence within the mGluR family. The key residue for Cl binding appears
to be a conserved serine in all mGluRs (in position 166 in mGlu1a and position 152 in
mGlu3), except in mGlu2 where it is replaced by an aspartate [28, 84, 87]. Cl binding at
Fig. 8 Binding sites of chloride ions in the extracellular domain of mGluRs. All mGluRs are
positively modulated by extracellular Cl anion which favor the agonist-induced active conforma-
tion. The Cl sensitivity appears to be carried mainly by two sites localized in mGluRs VFT, except
in mGlu2 which possesses only one functional site. A first site is located within lobe 1, in a well-
conserved binding pocket adjacent to glutamate binding site. The second site is located at the
interface of both VFT lobes, when the receptor is in a closed active conformation and shows more
divergence within the mGluR family. Localization of the two Cl binding sites in a 3D homology
model of the mGlu4 VFT (adapted from [28])
this site should enhance the stability of the agonist-induced closed active state of the VFT,
consistent with PAM activity.
Most mGluRs are positively modulated by Ca2+ ion [27, 29, 110, 113]. The
binding site of Ca2+ was proposed to be located close to the entrance of the VFT
[110]. The conserved serine residues in position 166 in mGlu1a and position 152 in
mGlu3 were also previously reported to be involved in Ca2+ activity [27]. However,
no Ca2+ ion was found in crystal structures at this location. Rather it appears to be
involved in Cl sensing.
In addition, Gd3+ has been reported to be a potentiator of glutamate action [27, 114,
115]. Gd3+ binding was observed in the crystal structure of the mGlu1 VFT dimer,
where both VFTs are closed (conformation Acc) [2]. Gd3+ binds at the interface
between the two lobes of the VFT dimer in the Acc conformation and neutralizes the
negative charges that would face each other in the absence of the ion [2], hence
promoting activation of the receptor.
Exploiting ion-binding pockets may yield to the development of innovative regulators
of mGluR activity. A first example is given by bitopic agonists, such as the mGlu4
selective agonist LSP4-2022 [83], which bind concomitantly to the glutamate binding
pocket and to one of the Cl allosteric binding site.
6 Modulation by Nanobodies
Monoclonal antibodies (mAbs) represent an alternative approach to target mGluRs

and control their activity [116, 117]. Unfortunately, development of antibody-based
therapies for targets in the CNS has lagged behind small molecule drugs due to the
complex architecture and poor blood-brain-barrier (BBB) permeability of mAbs.
Consequently, many attempts have been made to minimize the size of mAbs and
generate smaller antigen-binding antibody fragments. Particular antibodies found in
the Camelidae family (llama, dromedary, Bactrian camel) do not have a light chain
[118] and have been developed as single-domain antibodies by protein engineering
called nanobodies [119, 120]. These 13 kDa domains generate affinities in the low
nanomolar range, and they can easily be mass-produced in bacteria. Due to their
small antigen binding site, nanobodies can penetrate into cavities on the antigen
surface, and such epitopes are usually inaccessible to conventional antibodies.
Nanobodies could then offer new possibilities to modulate GPCR activities.
Llama nanobodies that specifically recognize mGlu2 receptors, among the eight
subtypes of mGluR subunits, have recently been developed [121]. Among these
nanobodies, DN10 and 13 act as positive allosteric modulators (PAM) on homodimeric
mGlu2, while DN10 displays a significant partial agonist activity. DN10 and DN13 had
no effect on mGlu2-3 and mGlu2-4 heterodimers. These PAMs enhance the inhibitory
action of the orthosteric mGlu2/mGlu3 agonist, DCG-IV, at mossy fiber terminals in the
CA3 region of hippocampal slices. DN13 also impairs contextual fear memory when
injected in the CA3 region of hippocampal region. Finally, we have also mapped the
binding site of these nanobodies by modeling and swapping residues between mGlu2 and
mGlu3 and found they bind at the interface between the two VFTs in the mGlu2
homodimer, then stabilizing a site that is formed upon activation of the receptor.
7 Ligand Control of Heterodimeric mGluRs
With the recent evidence of the existence of mGlu heterodimers, a complex pharmacology
is expected due to the presence of two distinct orthosteric sites and two distinct allosteric
sites, each of these sites being allosterically interconnected. Previous studies with mGlu5
receptors revealed a positive cooperativity between both orthosteric sites [74], as illustrated
by the increased affinity of a mutated subunit by the closed state of the associated subunit. In
addition, the closure of a single VFT leads to a partial G-protein activation [74]. Accord-
ingly, specific agonist binding in one subunit in an mGlu heterodimer is expected to
generate a partial response and to increase the potency of an agonist specifically acting at
the associated VFT. This was recently validated for the mGlu2-4 heterodimer, for which
mGlu2 or mGlu4 selective agonists only lead to partial activity of the receptor, and mGlu2
agonist potency was largely increased in the presence of low concentration of the mGlu4
agonist L-AP4 [5].
The action of allosteric modulators is likely to be even more complex. Indeed, in an
mGlu 7TM dimer, only one monomer reaches a fully active state at a time [13, 14], while
the other monomer also likely reaches a specific conformation though not able to activate
G proteins directly, but potentiating the activity of the associated 7TM. Indeed, in an
mGlu2-42-4 heterodimer, G-protein coupling occurs exclusively through the mGlu4 7TM,
while a conformational change in the mGlu2 7TM is required for an efficient G-protein
activation. Under such conditions, the effect of PAMs and NAMs are difficult to predict
and may easily lead to ligand-specific properties at mGlu heterodimers. Indeed, in an
mGlu2-4 heterodimer, either an mGlu2 PAM or an mGlu4 NAM, reorients the G-protein
coupling to the mGlu2 7TM, and an mGlu2 NAM largely diminishes the G-protein
coupling efficacy of the heterodimer [122]. Such complex allosteric interactions between
the 7TMs in an mGlu heterodimer likely explain the differential effect of PAMs. For
example, whereas the mGlu2 PAMs LY487379 and BINA, as well as the mGlu4 PAMs
VU0415374, VU0418506, and PHCCC, display no or low PAM activity at mGlu2-4
heterodimers, the mGlu4 PAM VU0155041 retains its full activity at this heterodimer
[5, 6, 123]. Due to the allosteric interaction between the 7TMs within such heterodimeric
receptors, positive or negative interactions between allosteric modulators are also
expected. A first illustration is the large potentiating effect observed on the mGlu2-4
heterodimer upon application of both the mGlu4 PAM VU0415374 and the mGlu2 PAM
BINA, both being inactive when applied alone [5] (Fig. 9). Such complex pharmacolog-
ical properties offer great opportunities to specifically control mGlu heterodimer activity,
and this has already been used to bring forward evidence of the existence of mGlu2-4 in
the brain [5, 6].
Fig. 9 Heterodimeric mGluRs. Studies with heterologous cells revealed the possible existence of
16 heterodimeric mGluRs [3]. Based on specific pharmacological properties, mGlu2-4 heterodimers
have been detected in the terminals of the lateral perforant path on the granule neurons in the dentate
gyrus of the hippocampus (left). Molecular studies revealed that glutamate binding in both VFTs of
the mGlu2-4 heterodimer leads to signaling via activation of the mGlu4 7TM. Although not directly
involved in coupling to G proteins, the mGlu2 7TM increases coupling efficacy (right) [5]
8 Modulation by Light-Operated Ligands
Optical control of mGluRs can be achieved via two techniques: optogenetic pharma-
cology based on tethered light-operated ligands [124] and photopharmacology (also
known as optopharmacology) which is based on diffusible light-operated ligands
[125, 126]. The aim of both techniques is to control the target with an improved spatial
and temporal resolution via the use of light.
8.1 Optogenetic Pharmacology
Optogenetic pharmacology is based on photoswitchable tethered ligands (PTLs). These

ligands bind covalently to genetically modified proteins and, once attached to their target,
enable its photoactivation or photoantagonism (Fig. 10).
Different light-agonized and light-antagonized “LimGluRs” have been designed
by Isacoff’s and Trauner’s lab. The first generation of LimGluRs was based on PTLs
specifically developed for mGluRs, named MAGs because they contain a maleimide
at one end for cysteine attachment, a photoisomerizable azobenzene linker, and
glutamate as the ligand at the other end. These molecules bind covalently to genet-
ically engineered mGluRs that possess geometrically appropriate cysteine-attachment
points. Once attached, these ligands mimic or block the action of glutamate under the
control of light. Using this approach, LimGluR2, LimGluR3, and LimGluR6 were
designed. The system works in heterologous cells, in rodent brain slices, and in vivo in
Fig. 10 Principle of optogenetic pharmacology on mGluRs. Scheme showing the optogenetic

pharmacology strategy used to control mGlu receptor activation [127]. A photoisomerizable ligand
is covalently bound to a mutated amino acid (yellow star) through a reactive group (yellow square).
The light control of the receptor activity is exerted by the photoisomerization of an azobenzene
group (violet square) and the binding of the glutamate moiety (blue ellipse) in the binding site
zebrafish [127]. Later, modified MAGs have been developed to enable activation of
mGluRs by 2 photons to improve the spatiotemporal resolution [128].
A second generation of light-controlled mGluRs has been developed based on
photoswitchable orthogonal remotely tethered ligands (PORTL) and the SNAP-tag
technology [129]. This time, the receptor is genetically modified to contain a SNAP-
tag at the N-terminus. The photoswitchable ligands are composed of a glutamate moiety,
followed by a long flexible linker containing an azobenzene and a benzylguanine that will
anchor the PORTL to the SNAP. This strategy has been applied to mGlu2 permitting the
photoactivation of mGlu2 and the control of excitability in heterologous cells or
transfected neurons [130]. The same principle can be applied to CLIP-tagged receptors
[131]. Since SNAP-tag and CLIP-tag possess orthogonal substrate specificities, SNAP-
and CLIP-tagged proteins can be labeled simultaneously and specifically with different
molecular probes in living cells. This has proven to be a very useful approach to analyze
cell surface protein complexes and notably led to the discovery of specific heterodimeric
mGluRs [3]. Combining SNAP- and CLIP-tagged receptors and specific PORTL, Levitz
and colleagues have created a family of light-gated group II/III mGluRs [132], allowing
the multiplexed orthogonal optical control within homo or heterodimers. Light-controlled
mGluRs can be useful optogenetic tools to understand the activation mechanisms of
mGluRs [133] or to study synaptic activity of neural circuits with high spatiotemporal
resolution and pharmacological specificity.
8.2 Photopharmacology
Photopharmacology is a novel technique to control the function of a given target

through light. Contrary to optogenetics that uses exogenous expression of light-
sensitive channels to take control of specific cells, the control of targets is achieved
through the use of small diffusible, drug-like, photo-regulated ligands. Two types of
non-tethered photo-regulated drugs have been developed for photopharmacology:
photo-caged ligands (Fig. 11) and photoswitchable ligands (Fig. 12).
One of the main interests of photopharmacology resides in the ability to target
endogenous receptors in their native environment. Indeed, this technique doesn’t
require exogenous viral expression of light-activatable proteins as with optogenetics.
Moreover, thanks to the use of optic fibers coupled to a light source, spatial and
temporal control of compound activity is improved as compared to conventional
pharmacological approaches.
The limitation for this technique was believed to be the local delivery of drug and
light in vivo. Although easy on isolated cells or small model organisms, such as
xenopus tadpoles or zebrafish larvae, it was thought to be more complicated in
rodent models. However, two pioneering studies have recently demonstrated the
Fig. 11 Caged ligands of mGluRs. Schematic showing the uncaging or photolysis strategy
developed for mGlu5. JF-NP-26 is an inactive photo-caged derivative of the mGlu5 NAM
raseglurant [134]. Illumination of JF-NP-26 with violet light induces a photochemical reaction
prompting the dissociation of the active ligand raseglurant and the coumarin DEACM and thus the
blockade of mGlu5 activity
Fig. 12 Photochromic ligands of mGluRs. Schematic showing the photopharmacological approach

developed for mGlu5. The diffusible ligand Alloswitch-1, a photochromic negative allosteric mod-
ulator (NAM) of mGlu5 [135], can be reversibly photoisomerized to the cis configuration (inactive)
upon illumination with a violet light, whereas it relaxes back to the trans configuration (active) in dark
or, in a faster manner, by the use of green light. This approach has the advantage that no modification
of the receptor is required to photo-control the receptor activity since the azobenzene is integrated
within the active moiety
feasibility of controlling photoswitchable [61] or photo-caged drugs [134] in the

brain of freely moving mice.
8.2.1 Photo-Caged Ligands
Caged ligands possess a photolabile protecting group that is removed following

illumination, enabling the uncaged ligand to bind to its receptor (Fig. 11). Therefore,
these ligands are inactive photo-caged ligands that can be turned on by light. They
are also referred to as photoactivatable ligands. They enable the precise control of the
onset of drug activity as well as its location.
First applied to ATP [136], the caged strategy has been adapted to different
neurotransmitters, including glutamate. Caged glutamate improved the spatial reso-
lution of neuronal circuit mapping [137]. It has also been used to study the function
of mGluRs [138]. However, the use of caged glutamate is somehow limited due to
the lack of subtype selectivity, leading to the development of iGluR and mGluR
selective compounds.
The first mGluR subtype selective caged compound is JF-NP-26, an inactive photo-
caged derivative of the mGlu5 NAM raseglurant [134] (Fig. 11). Violet light illumination
of JF-NP-26 induces a photochemical reaction prompting the active-drug’s release,
which effectively controls mGlu5 activity in HEK293 cells expressing the receptor.
Systemic administration in mice followed by local illumination in peripheral tissues or
in the thalamus induced JF-NP-26-mediated light-dependent analgesia in inflammatory
or neuropathic pain models.
8.2.2 Photoswitchable Ligands
These ligands possess an azobenzene moiety which can reversibly photoisomerize,

such that these molecules can be rapidly and reversibly turned on and off with a
specific light (Fig. 12). In the dark or under white light, the azobenzene moiety is in a
trans configuration while it reaches the cis configuration upon illumination at the
adequate wavelength (usually in the ultraviolet range). Relaxation to the thermody-
namically more stable trans-isomer can be induced by irradiation or by thermal
relaxation. Azobenzene changes geometry during photoisomerization, inducing a
change in the conformation of the ligand, when present in an active molecule,
drastically affecting its affinity for the targeted receptor.
In the last few years, photoswitchable ligands have been developed for various
receptors and ion channels [135, 139–143]. The first allosteric photoswitchable ligand
targeting a GPCR is Alloswitch-1, an mGlu5 NAM [135] (Fig. 12). This ligand is a
derivative of VU0415374, an allosteric ligand of mGlu4 having high chemical and
structural homology with the scaffold present in azobenzene [144]. Based on a design
named azologization, an azobenzene was inserted in the core of the ligand in order to
minimally modify the steric occupancy, binding determinants and physicochemical
properties of the parent compounds. Illumination by green or violet light stabilizes
either the trans or the cis configuration of the ligand that corresponds to high and low
pharmacological activity, respectively, on heterologous or native cells expressing the
mGlu5 receptor. In vivo, Alloswitch-1 allows light-dependent control of the motility
of Xenopus laevi tadpoles.
Optogluram is a photoswitchable mGlu4 PAM [61] which allows for selective,
reversible, and repeated optical manipulation of mGlu4 activity with light. This ligand
is also a derivative of VU0415374 but with the azobenzene inserted in a different position
than in Alloswitch-1. Enhancement of mGlu4 activity by optogluram observed in the
trans-form was reduced upon isomerization to the cis configuration after irradiation with
violet light. Using stereotaxically implanted hybrid optic-fluid cannula, Zussy and
colleagues have injected optogluram and controlled activity of endogenous mGlu4 with
light in a specific area of the brain of freely behaving mice. This study revealed that it is
possible to regulate persistent pain-related symptoms in a temporally and spatially
restricted manner taking control of amygdala mGlu4 receptors by light. To our knowl-
edge, this is the first work to establish that photopharmacology with a small diffusible
drug-like photoswitchable ligand can be used in vivo to regulate behavior in a disease
model.
Since ultraviolet light could be potentially damaging to irradiated tissues, it is
interesting to design ligands that can photoisomerize at red-shifted wavelengths.
Recently, OptoGluNAM4.1, a blue light-sensitive mGlu4 photoswitchable NAM,
has been described [145]. This compound is active both in vitro and in vivo, being
able to block the analgesic activity of an mGlu4 receptor agonist in a mouse model of
chronic pain and to photo-control the mobility of zebrafish larvae.
More recently, a series of photoswitchable mGlu5 NAMs based on the phenyl-
azopyridine scaffold have been generated. Most of the trans-isomers of this series are
active both in vitro, inhibiting mGlu5 function in heterologous cells, and in vivo,
photo-controlling zebrafish motility. However, different photoisomerization proper-
ties are observed in this structure-activity relationship study with a robust translation
from the photoswitchable properties observed in vitro and in vivo. Notably,
depending on the compound, the optimal wavelength of illumination is between
360 and 500 nm.
Photopharmacology and light-operated drugs constitute powerful tools to manip-
ulate and explore the function and therapeutic potential of endogenous receptors in
living animals. Dedicated to fundamental research to date, the small, drug-like,
diffusible photoswitchable molecules used in photopharmacology could be amena-
ble to drug development in the future.
9 Conclusion
These past few years have seen several breakthroughs in the field of mGluRs. First,
more than 10 years after the resolution of the first crystal structures of the mGluR
extracellular domain, we have only recently seen the advent of the structures of their
transmembrane domains. These structures are a gold mine for the deep understand-
ing of drug mechanisms of action and the design of new ligands. The next achieve-
ment will probably be the resolution of the full-length receptor structure. The level of
complexity of mGluR physiology has increased with another key finding of the last
years: mGluRs that were long believed to form exclusively homodimers can also
assemble into heterodimers, multiplying the receptor combinations and their subse-
quent signaling and modulatory mechanisms. The discovery of particular pharma-
cological signatures and selective ligands for these heterodimers will be key to
understand their function. Fortunately, researchers can count on an increasing
number of pharmacological tools to investigate the physiology and pathology of
mGluRs, from orthosteric to allosteric ligands, including nanobodies, ions, and light-
operated ligands. Last, but not least, despite being considered valuable targets for
treating neurological disorders and the subsequent efforts of pharmaceutical indus-
try, there is no marketed drug targeting mGluRs so far. However, clinical trials are
still ongoing, some targeting unexplored subtypes, and will hopefully validate the
therapeutic potential of mGluRs anticipated from preclinical models. In conclusion,
there are still many exciting challenges ahead for fundamental and clinical research
on mGluRs.
Acknowledgments The authors wish to thank Dr. Guillaume Lebon and Dr. Ebba L. Lagerqvist for
critical reading of the manuscript. We acknowledge financial support from the Agence Nationale de la
Recherche (ANR-12-NEUR-0003 and ANR-13-BSV1-006), the ERANET Neuron LIGHTPAIN pro-
ject, the Fondation Recherche Médicale (FRM team DEQ20130326522), the Centre National de la
Recherche Scientifique, the Spanish Ministry of Economy, Industry and Competitiveness (SAF2015-
74132-JIN), and the Catalan government.
References
1. Kunishima N, Shimada Y, Tsuji Y, Sato T, Yamamoto M, Kumasaka T, Nakanishi S,

Jingami H, Morikawa K (2000) Structural basis of glutamate recognition by a dimeric
metabotropic glutamate receptor. Nature 407:971–977
2. Tsuchiya D, Kunishima N, Kamiya N, Jingami H, Morikawa K (2002) Structural views of the
ligand-binding cores of a metabotropic glutamate receptor complexed with an antagonist and
both glutamate and Gd3+. Proc Natl Acad Sci U S A 99:2660–2665
3. Doumazane E, Scholler P, Zwier JM, Eric T, Rondard P, Pin JP (2011) A new approach to
analyze cell surface protein complexes reveals specific heterodimeric metabotropic glutamate
receptors. FASEB J 25:66–77
4. Ferraguti F, Klausberger T, Cobden P, Baude A, Roberts JD, Szucs P, Kinoshita A,
Shigemoto R, Somogyi P, Dalezios Y (2005) Metabotropic glutamate receptor 8-expressing
nerve terminals target subsets of GABAergic neurons in the hippocampus. J Neurosci
25:10520–10536
5. Moreno Delgado D, Moller TC, Ster J, Giraldo J, Maurel D, Rovira X, Scholler P, Zwier JM,
Perroy J, Durroux T, Trinquet E, Prezeau L, Rondard P, Pin JP (2017) Pharmacological
evidence for a metabotropic glutamate receptor heterodimer in neuronal cells. eLife 6. pii:
e25233
6. Yin S, Noetzel MJ, Johnson KA, Zamorano R, Jalan-Sakrikar N, Gregory KJ, Conn PJ,
Niswender CM (2014) Selective actions of novel allosteric modulators reveal functional
heteromers of metabotropic glutamate receptors in the CNS. J Neurosci 34:79–94
7. Servant G, Tachdjian C, Li X, Karanewsky DS (2011) The sweet taste of true synergy: positive
allosteric modulation of the human sweet taste receptor. Trends Pharmacol Sci 32:631–636
8. Pin JP, Bettler B (2016) Organization and functions of mGlu and GABAB receptor complexes.
Nature 540:60–68
9. Pin JP, Galvez T, Prezeau L (2003) Evolution, structure, and activation mechanism of family
3/C G-protein-coupled receptors. Pharmacol Ther 98:325–354
10. Nicoletti F, Bockaert J, Collingridge GL, Conn PJ, Ferraguti F, Schoepp DD, Wroblewski JT,
Pin JP (2011) Metabotropic glutamate receptors: from the workbench to the bedside. Neuro-
pharmacology 60:1017–1041
11. Julio-Pieper M, Flor PJ, Dinan TG, Cryan JF (2011) Exciting times beyond the brain: metabotropic
glutamate receptors in peripheral and non-neural tissues. Pharmacol Rev 63:35–58
12. El Moustaine D, Granier S, Doumazane E, Scholler P, Rahmeh R, Bron P, Mouillac B, Baneres
JL, Rondard P, Pin JP (2012) Distinct roles of metabotropic glutamate receptor dimerization in
agonist activation and G-protein coupling. Proc Natl Acad Sci U S A 109:16342–16347
13. Goudet C, Kniazeff J, Hlavackova V, Malhaire F, Maurel D, Acher F, Blahos J, Prezeau L, Pin
JP (2005) Asymmetric functioning of dimeric metabotropic glutamate receptors disclosed by
positive allosteric modulators. J Biol Chem 280:24380–24385
14. Hlavackova V, Goudet C, Kniazeff J, Zikova A, Maurel D, Vol C, Trojanova J, Prezeau L, Pin
JP, Blahos J (2005) Evidence for a single heptahelical domain being turned on upon activation
of a dimeric GPCR. EMBO J 24:499–509
15. Liu J, Zhang Z, Moreno-Delgada D, Dalton J, Rovira X, Trapero A, Goudet C, Llebaria A,
Giraldo J, Yuan Q, Rondard P, Huang S, Liu J, Pin J-P (2017) Allosteric control of an
asymmetric transduction in a G protein-coupled receptor heterodimer. eLife 6:e26985
16. Comps-Agrar L, Kniazeff J, Norskov-Lauritsen L, Maurel D, Gassmann M, Gregor N,
Prezeau L, Bettler B, Durroux T, Trinquet E, Pin JP (2011) The oligomeric state sets GABA
(B) receptor signalling efficacy. EMBO J 30:2336–2349
17. Maurel D, Comps-Agrar L, Brock C, Rives ML, Bourrier E, Ayoub MA, Bazin H, Tinel N,
Durroux T, Prezeau L, Trinquet E, Pin JP (2008) Cell-surface protein-protein interaction
analysis with time-resolved FRET and snap-tag technologies: application to GPCR oligomer-
ization. Nat Methods 5:561–567
18. Pandya NJ, Klaassen RV, van der Schors RC, Slotman JA, Houtsmuller A, Smit AB, Li KW
(2016) Group 1 metabotropic glutamate receptors 1 and 5 form a protein complex in mouse
hippocampus and cortex. Proteomics 16:2698–2705
19. Monn JA, Prieto L, Taboada L, Hao J, Reinhard MR, Henry SS, Beadle CD, Walton L, Man T,
Rudyk H, Clark B, Tupper D, Baker SR, Lamas C, Montero C, Marcos A, Blanco J, Bures M,
Clawson DK, Atwell S et al (2015) Synthesis and pharmacological characterization of
C4-(thiotriazolyl)-substituted-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylates. Identification
of (1R,2S,4R,5R,6R)-2-amino-4-(1H-1,2,4-triazol-3-ylsulfanyl)bicyclo[3.1.0]hexane-2,
6-dicarboxylic acid (LY2812223), a highly potent, functionally selective mGlu2 receptor
agonist. J Med Chem 58:7526–7548
20. Monn JA, Prieto L, Taboada L, Pedregal C, Hao J, Reinhard MR, Henry SS, Goldsmith PJ,
Beadle CD, Walton L, Man T, Rudyk H, Clark B, Tupper D, Baker SR, Lamas C, Montero C,
Marcos A, Blanco J, Bures M et al (2015) Synthesis and pharmacological characterization of
C4-disubstituted analogs of 1S,2S,5R,6S-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate:
identification of a potent, selective metabotropic glutamate receptor agonist and determination
of agonist-bound human mGlu2 and mGlu3 amino terminal domain structures. J Med Chem
58:1776–1794
21. Muto T, Tsuchiya D, Morikawa K, Jingami H (2007) Structures of the extracellular regions of
the group II/III metabotropic glutamate receptors. Proc Natl Acad Sci U S A 104:3759–3764
22. Bertrand HO, Bessis AS, Pin JP, Acher FC (2002) Common and selective molecular determi-
nants involved in metabotopic glutamate receptor agonist activity. J Med Chem 45:3171–3183
23. Bessis AS, Bertrand HO, Galvez T, De Colle C, Pin JP, Acher F (2000) Three-dimensional
model of the extracellular domain of the type 4a metabotropic glutamate receptor: new insights
into the activation process. Protein Sci 9:2200–2209
24. DiRaddo JO, Miller EJ, Hathaway HA, Grajkowska E, Wroblewska B, Wolfe BB, Liotta DC,
Wroblewski JT (2014) A real-time method for measuring cAMP production modulated by
Galphai/o-coupled metabotropic glutamate receptors. J Pharmacol Exp Ther 349:373–382
25. Jiang JY, Nagaraju M, Meyer RC, Zhang L, Hamelberg D, Hall RA, Brown EM, Conn PJ,
Yang JJ (2014) Extracellular calcium modulates actions of orthosteric and allosteric ligands on
metabotropic glutamate receptor 1alpha. J Biol Chem 289:1649–1661
26. Kuang D, Hampson DR (2006) Ion dependence of ligand binding to metabotropic glutamate
receptors. Biochem Biophys Res Commun 345:1–6
27. Kubo Y, Miyashita T, Murata Y (1998) Structural basis for a Ca2+-sensing function of the
metabotropic glutamate receptors. Science 279:1722–1725
28. Tora AS, Rovira X, Dione I, Bertrand HO, Brabet I, De Koninck Y, Doyon N, Pin JP, Acher F,
Goudet C (2015) Allosteric modulation of metabotropic glutamate receptors by chloride ions.
FASEB J 29:4174–4188
29. Vafabakhsh R, Levitz J, Isacoff EY (2015) Conformational dynamics of a class C G-protein-

30. Rondard P, Liu J, Huang S, Malhaire F, Vol C, Pinault A, Labesse G, Pin JP (2006) Coupling
of agonist binding to effector domain activation in metabotropic glutamate-like receptors. J
Biol Chem 281:24653–24661
31. Xue L, Rovira X, Scholler P, Zhao H, Liu J, Pin JP, Rondard P (2015) Major ligand-induced
rearrangement of the heptahelical domain interface in a GPCR dimer. Nat Chem Biol 11:134–140
32. Dore AS, Okrasa K, Patel JC, Serrano-Vega M, Bennett K, Cooke RM, Errey JC, Jazayeri A,
Khan S, Tehan B, Weir M, Wiggin GR, Marshall FH (2014) Structure of class C GPCR
metabotropic glutamate receptor 5 transmembrane domain. Nature 511:557–562
33. Wu H, Wang C, Gregory KJ, Han GW, Cho HP, Xia Y, Niswender CM, Katritch V, Meiler J,
Cherezov V, Conn PJ, Stevens RC (2014) Structure of a class C GPCR metabotropic glutamate
receptor 1 bound to an allosteric modulator. Science 344:58–64
34. Conquet F, Bashir ZI, Davies CH, Daniel H, Ferraguti F, Bordi F, Franz-Bacon K, Reggiani A,
Matarese V, Conde F et al (1994) Motor deficit and impairment of synaptic plasticity in mice
lacking mGluR1. Nature 372:237–243
35. Notartomaso S, Zappulla C, Biagioni F, Cannella M, Bucci D, Mascio G, Scarselli P, Fazio F,
Weisz F, Lionetto L, Simmaco M, Gradini R, Battaglia G, Signore M, Puliti A, Nicoletti F
(2013) Pharmacological enhancement of mGlu1 metabotropic glutamate receptors causes a
prolonged symptomatic benefit in a mouse model of spinocerebellar ataxia type 1. Mol Brain
6:48
36. Pollock PM, Cohen-Solal K, Sood R, Namkoong J, Martino JJ, Koganti A, Zhu H, Robbins C,
Makalowska I, Shin SS, Marin Y, Roberts KG, Yudt LM, Chen A, Cheng J, Incao A, Pinkett
HW, Graham CL, Dunn K, Crespo-Carbone SM et al (2003) Melanoma mouse model
implicates metabotropic glutamate signaling in melanocytic neoplasia. Nat Genet 34:108–112
37. Porter RH, Jaeschke G, Spooren W, Ballard TM, Buttelmann B, Kolczewski S, Peters JU,
Prinssen E, Wichmann J, Vieira E, Muhlemann A, Gatti S, Mutel V, Malherbe P (2005)
Fenobam: a clinically validated nonbenzodiazepine anxiolytic is a potent, selective, and
noncompetitive mGlu5 receptor antagonist with inverse agonist activity. J Pharmacol Exp
Ther 315:711–721
38. Rojas DC (2014) The role of glutamate and its receptors in autism and the use of glutamate
receptor antagonists in treatment. J Neural Transm (Vienna) 121:891–905
39. Cartmell J, Monn JA, Schoepp DD (1999) The metabotropic glutamate 2/3 receptor agonists
LY354740 and LY379268 selectively attenuate phencyclidine versus d-amphetamine motor
behaviors in rats. J Pharmacol Exp Ther 291:161–170
40. Moghaddam B, Adams BW (1998) Reversal of phencyclidine effects by a group II metabotropic
glutamate receptor agonist in rats. Science 281:1349–1352
41. Patil ST, Zhang L, Martenyi F, Lowe SL, Jackson KA, Andreev BV, Avedisova AS,
Bardenstein LM, Gurovich IY, Morozova MA, Mosolov SN, Neznanov NG, Reznik AM,
Smulevich AB, Tochilov VA, Johnson BG, Monn JA, Schoepp DD (2007) Activation of
mGlu2/3 receptors as a new approach to treat schizophrenia: a randomized phase 2 clinical
trial. Nat Med 13:1102–1107
42. Adams DH, Kinon BJ, Baygani S, Millen BA, Velona I, Kollack-Walker S, Walling DP
(2013) A long-term, phase 2, multicenter, randomized, open-label, comparative safety study of
pomaglumetad methionil (LY2140023 monohydrate) versus atypical antipsychotic standard of
care in patients with schizophrenia. BMC Psychiatry 13:143
43. Kinon BJ, Zhang L, Millen BA, Osuntokun OO, Williams JE, Kollack-Walker S, Jackson K,
Kryzhanovskaya L, Jarkova N (2011) A multicenter, inpatient, phase 2, double-blind, placebo-
controlled dose-ranging study of LY2140023 monohydrate in patients with DSM-IV schizo-
phrenia. J Clin Psychopharmacol 31:349–355
44. Stauffer VL, Millen BA, Andersen S, Kinon BJ, Lagrandeur L, Lindenmayer JP, Gomez JC
(2013) Pomaglumetad methionil: no significant difference as an adjunctive treatment for
patients with prominent negative symptoms of schizophrenia compared to placebo. Schizophr

Res 150:434–441
45. Dunayevich E, Erickson J, Levine L, Landbloom R, Schoepp DD, Tollefson GD (2008)
Efficacy and tolerability of an mGlu2/3 agonist in the treatment of generalized anxiety
disorder. Neuropsychopharmacology 33:1603–1610
46. Consortium SWGotPG (2014) Biological insights from 108 schizophrenia-associated genetic
loci. Nature 511:421–427
47. De Filippis B, Lyon L, Taylor A, Lane T, Burnet PW, Harrison PJ, Bannerman DM (2015)
The role of group II metabotropic glutamate receptors in cognition and anxiety: comparative
studies in GRM2(/), GRM3(/) and GRM2/3(/) knockout mice. Neuropharmacol-
ogy 89:19–32
48. Egan MF, Straub RE, Goldberg TE, Yakub I, Callicott JH, Hariri AR, Mattay VS, Bertolino A,
Hyde TM, Shannon-Weickert C, Akil M, Crook J, Vakkalanka RK, Balkissoon R, Gibbs RA,
Kleinman JE, Weinberger DR (2004) Variation in GRM3 affects cognition, prefrontal gluta-
mate, and risk for schizophrenia. Proc Natl Acad Sci U S A 101:12604–12609
49. Harrison PJ, Lyon L, Sartorius LJ, Burnet PW, Lane TA (2008) The group II metabotropic
glutamate receptor 3 (mGluR3, mGlu3, GRM3): expression, function and involvement in
schizophrenia. J Psychopharmacol 22:308–322
50. O'Brien NL, Way MJ, Kandaswamy R, Fiorentino A, Sharp SI, Quadri G, Alex J, Anjorin A,
Ball D, Cherian R, Dar K, Gormez A, Guerrini I, Heydtmann M, Hillman A, Lankappa S,
Lydall G, O'Kane A, Patel S, Quested D et al (2014) The functional GRM3 Kozak sequence
variant rs148754219 affects the risk of schizophrenia and alcohol dependence as well as
bipolar disorder. Psychiatr Genet 24:277–278
51. Kunz M (2014) Oncogenes in melanoma: an update. Eur J Cell Biol 93:1–10
52. Yi H, Geng L, Black A, Talmon G, Berim L, Wang J (2017) The miR-487b-3p/GRM3/
TGFbeta signaling axis is an important regulator of colon cancer tumorigenesis. Oncogene 36
(24):3477–3489
53. Amalric M, Lopez S, Goudet C, Fisone G, Battaglia G, Nicoletti F, Pin JP, Acher FC (2013)
Group III and subtype 4 metabotropic glutamate receptor agonists: discovery and pathophys-
iological applications in Parkinson’s disease. Neuropharmacology 66:53–64
54. Marino MJ, Williams DL Jr, O’Brien JA, Valenti O, McDonald TP, Clements MK, Wang R,
DiLella AG, Hess JF, Kinney GG, Conn PJ (2003) Allosteric modulation of group III
metabotropic glutamate receptor 4: a potential approach to Parkinson’s disease treatment.
55. Beurrier C, Lopez S, Revy D, Selvam C, Goudet C, Lherondel M, Gubellini P, Kerkerian-
LeGoff L, Acher F, Pin JP, Amalric M (2009) Electrophysiological and behavioral evidence
that modulation of metabotropic glutamate receptor 4 with a new agonist reverses experimen-
tal parkinsonism. FASEB J 23:3619–3628
56. Charvin D, Pomel V, Ortiz M, Frauli M, Scheffler S, Steinberg E, Baron L, Deshons L,
Rudigier R, Thiarc D, Morice C, Manteau B, Mayer S, Graham D, Giethlen B, Brugger N,
Hedou G, Conquet F, Schann S (2017) Discovery, structure-activity relationship and anti-
parkinsonian effect of a potent and brain-penetrant chemical series of positive allosteric
modulators of metabotropic glutamate receptor 4. J Med Chem 60(20):8515–8537
57. Kalinichev M, Le Poul E, Bolea C, Girard F, Campo B, Fonsi M, Royer-Urios I, Browne SE,
Uslaner JM, Davis MJ, Raber J, Duvoisin R, Bate ST, Reynolds IJ, Poli S, Celanire S (2014)
Characterization of the novel positive allosteric modulator of the metabotropic glutamate
receptor 4 ADX88178 in rodent models of neuropsychiatric disorders. J Pharmacol Exp
Ther 350:495–505
58. Goudet C, Chapuy E, Alloui A, Acher F, Pin JP, Eschalier A (2008) Group III metabotropic
glutamate receptors inhibit hyperalgesia in animal models of inflammation and neuropathic
pain. Pain 137:112–124
59. Vilar B, Busserolles J, Ling B, Laffray S, Ulmann L, Malhaire F, Chapuy E, Aissouni Y,
Etienne M, Bourinet E, Acher F, Pin JP, Eschalier A, Goudet C (2013) Alleviating pain
hypersensitivity through activation of type 4 metabotropic glutamate receptor. J Neurosci

33:18951–18965
60. Wang H, Jiang W, Yang R, Li Y (2011) Spinal metabotropic glutamate receptor 4 is involved
in neuropathic pain. Neuroreport 22:244–248
61. Zussy C, Gomez-Santacana X, Rovira X, De Bundel D, Ferrazzo S, Bosch D, Asede D,
Malhaire F, Acher F, Giraldo J, Valjent E, Ehrlich I, Ferraguti F, Pin JP, Llebaria A, Goudet C
(2016) Dynamic modulation of inflammatory pain-related affective and sensory symptoms by
optical control of amygdala metabotropic glutamate receptor 4. Mol Psychiatry. https://doi.
org/10.1038/mp.2016.223
62. Wieronska JM, Stachowicz K, Acher F, Lech T, Pilc A (2012) Opposing efficacy of group III
mGlu receptor activators, LSP1-2111 and AMN082, in animal models of positive symptoms
of schizophrenia. Psychopharmacology 220:481–494
63. Fallarino F, Volpi C, Fazio F, Notartomaso S, Vacca C, Busceti C, Bicciato S, Battaglia G,
Bruno V, Puccetti P, Fioretti MC, Nicoletti F, Grohmann U, Di Marco R (2010) Metabotropic
glutamate receptor-4 modulates adaptive immunity and restrains neuroinflammation. Nat Med
16:897–902
64. Becker JA, Clesse D, Spiegelhalter C, Schwab Y, Le Merrer J, Kieffer BL (2014) Autistic-like
syndrome in mu opioid receptor null mice is relieved by facilitated mGluR4 activity.
Neuropsychopharmacology 39(9):2049–2060
65. Vardi N, Duvoisin R, Wu G, Sterling P (2000) Localization of mGluR6 to dendrites of ON
bipolar cells in primate retina. J Comp Neurol 423:402–412
66. Zeitz C, Forster U, Neidhardt J, Feil S, Kalin S, Leifert D, Flor PJ, Berger W (2007) Night
blindness-associated mutations in the ligand-binding, cysteine-rich, and intracellular domains
of the metabotropic glutamate receptor 6 abolish protein trafficking. Hum Mutat 28:771–780
67. O’Connor RM, Finger BC, Flor PJ, Cryan JF (2010) Metabotropic glutamate receptor 7: at the
interface of cognition and emotion. Eur J Pharmacol 639:123–131
68. Bertaso F, Zhang C, Scheschonka A, de Bock F, Fontanaud P, Marin P, Huganir RL, Betz H,
Bockaert J, Fagni L, Lerner-Natoli M (2008) PICK1 uncoupling from mGluR7a causes
absence-like seizures. Nat Neurosci 11:940–948
69. Mitsukawa K, Yamamoto R, Ofner S, Nozulak J, Pescott O, Lukic S, Stoehr N, Mombereau C,
Kuhn R, McAllister KH, van der Putten H, Cryan JF, Flor PJ (2005) A selective metabotropic
glutamate receptor 7 agonist: activation of receptor signaling via an allosteric site modulates
stress parameters in vivo. Proc Natl Acad Sci U S A 102:18712–18717
70. Sukoff Rizzo SJ, Leonard SK, Gilbert A, Dollings P, Smith DL, Zhang MY, Di L, Platt BJ,
Neal S, Dwyer JM, Bender CN, Zhang J, Lock T, Kowal D, Kramer A, Randall A, Huselton C,
Vishwanathan K, Tse SY, Butera J et al (2011) The metabotropic glutamate receptor 7 allosteric
modulator AMN082: a monoaminergic agent in disguise? J Pharmacol Exp Ther 338:345–352
71. Fendt M, Imobersteg S, Peterlik D, Chaperon F, Mattes C, Wittmann C, Olpe HR,
Mosbacher J, Vranesic I, van der Putten H, McAllister KH, Flor PJ, Gee CE (2013) Differ-
ential roles of mGlu(7) and mGlu(8) in amygdala-dependent behavior and physiology. Neu-
ropharmacology 72:215–223
72. Rossi F, Marabese I, De Chiaro M, Boccella S, Luongo L, Guida F, De Gregorio D,
Giordano C, de Novellis V, Palazzo E, Maione S (2014) Dorsal striatum metabotropic
glutamate receptor 8 affects nocifensive responses and rostral ventromedial medulla cell
activity in neuropathic pain conditions. J Neurophysiol 111:2196–2209
73. Bessis AS, Rondard P, Gaven F, Brabet I, Triballeau N, Prezeau L, Acher F, Pin JP (2002)
Closure of the Venus flytrap module of mGlu8 receptor and the activation process: insights from
mutations converting antagonists into agonists. Proc Natl Acad Sci U S A 99:11097–11102
74. Kniazeff J, Bessis AS, Maurel D, Ansanay H, Prezeau L, Pin JP (2004) Closed state of both
binding domains of homodimeric mGlu receptors is required for full activity. Nat Struct Mol
Biol 11:706–713
75. Acher FC, Bertrand HO (2005) Amino acid recognition by Venus flytrap domains is encoded
in an 8-residue motif. Biopolymers 80:357–366
76. Parmentier ML, Galvez T, Acher F, Peyre B, Pellicciari R, Grau Y, Bockaert J, Pin JP (2000)
Conservation of the ligand recognition site of metabotropic glutamate receptors during
evolution. Neuropharmacology 39:1119–1131
77. Bessis AS, Jullian N, Coudert E, Pin JP, Acher F (1999) Extended glutamate activates
metabotropic receptor types 1, 2 and 4: selective features at mGluR4 binding site. Neurophar-
macology 38:1543–1551
78. Brabet I, Parmentier ML, De Colle C, Bockaert J, Acher F, Pin JP (1998) Comparative effect
of L-CCG-I, DCG-IV and gamma-carboxy-L-glutamate on all cloned metabotropic glutamate
receptor subtypes. Neuropharmacology 37:1043–1051
79. Kingston AE, Ornstein PL, Wright RA, Johnson BG, Mayne NG, Burnett JP, Belagaje R,
Wu S, Schoepp DD (1998) LY341495 is a nanomolar potent and selective antagonist of group
II metabotropic glutamate receptors. Neuropharmacology 37:1–12
80. Monn JA, Valli MJ, Massey SM, Wright RA, Salhoff CR, Johnson BG, Howe T, Alt CA,
Rhodes GA, Robey RL, Griffey KR, Tizzano JP, Kallman MJ, Helton DR, Schoepp DD (1997)
Design, synthesis, and pharmacological characterization of (+)-2-aminobicyclo[3.1.0]hexane-
2,6-dicarboxylic acid (LY354740): a potent, selective, and orally active group 2 metabotropic
glutamate receptor agonist possessing anticonvulsant and anxiolytic properties. J Med Chem
40:528–537
81. Acher F, Tellier F, Brabet I, Fagni L, Azerad R, Pin J-P (1997) Synthesis and pharmacological
characterization of aminocyclopentane tricarboxylic acids (ACPT): new tools to discriminate
between metabotropic glutamate receptor subtypes. J Med Chem 40:3119–3129
82. Pin JP, Acher F (2002) The metabotropic glutamate receptors: structure, activation mechanism
and pharmacology. Curr Drug Targets CNS Neurol Disord 1:297–317
83. Goudet C, Vilar B, Courtiol T, Deltheil T, Bessiron T, Brabet I, Oueslati N, Rigault D,
Bertrand HO, McLean H, Daniel H, Amalric M, Acher F, Pin JP (2012) A novel selective
metabotropic glutamate receptor 4 agonist reveals new possibilities for developing subtype
selective ligands with therapeutic potential. FASEB J 26:1682–1693
84. Acher FC, Selvam C, Pin JP, Goudet C, Bertrand HO (2011) A critical pocket close to the
glutamate binding site of mGlu receptors opens new possibilities for agonist design. Neuro-
pharmacology 60:102–107
85. Selvam C, Oueslati N, Lemasson IA, Brabet I, Rigault D, Courtiol T, Cesarini S, Triballeau N,
Bertrand HO, Goudet C, Pin JP, Acher FC (2010) A virtual screening hit reveals new possibilities
for developing group III metabotropic glutamate receptor agonists. J Med Chem 53:2797–2813
86. Christopher JA, Aves SJ, Bennett KA, Dore AS, Errey JC, Jazayeri A, Marshall FH, Okrasa K,
Serrano-Vega MJ, Tehan BG, Wiggin GR, Congreve M (2015) Fragment and structure-based
drug discovery for a class C GPCR: discovery of the mGlu5 negative allosteric modulator
HTL14242 (3-chloro-5-[6-(5-fluoropyridin-2-yl)pyrimidin-4-yl]benzonitrile). J Med Chem
58:6653–6664
87. DiRaddo JO, Miller EJ, Bowman-Dalley C, Wroblewska B, Javidnia M, Grajkowska E, Wolfe
BB, Liotta DC, Wroblewski JT (2015) Chloride is an agonist of group II and III metabotropic
glutamate receptors. Mol Pharmacol 88:450–459
88. Christopoulos A, Changeux JP, Catterall WA, Fabbro D, Burris TP, Cidlowski JA, Olsen RW,
Peters JA, Neubig RR, Pin JP, Sexton PM, Kenakin TP, Ehlert FJ, Spedding M, Langmead CJ
(2014) International Union of Basic and Clinical Pharmacology. XC. Multisite pharmacology:
recommendations for the nomenclature of receptor allosterism and allosteric ligands. Pharmacol
Rev 66:918–947
89. Hermans E, Nahorski SR, Challiss RA (1998) Reversible and non-competitive antagonist
profile of CPCCOEt at the human type 1alpha metabotropic glutamate receptor. Neurophar-
macology 37:1645–1647
90. Litschig S, Gasparini F, Rueegg D, Stoehr N, Flor PJ, Vranesic I, Prezeau L, Pin JP,
Thomsen C, Kuhn R (1999) CPCCOEt, a noncompetitive metabotropic glutamate receptor
1 antagonist, inhibits receptor signaling without affecting glutamate binding. Mol Pharmacol
55:453–461
91. Carroll FY, Stolle A, Beart PM, Voerste A, Brabet I, Mauler F, Joly C, Antonicek H,
Bockaert J, Muller T, Pin JP, Prezeau L (2001) BAY36-7620: a potent non-competitive
mGlu1 receptor antagonist with inverse agonist activity. Mol Pharmacol 59:965–973
92. Gasparini F, Lingenhohl K, Stoehr N, Flor PJ, Heinrich M, Vranesic I, Biollaz M, Allgeier H,
Heckendorn R, Urwyler S, Varney MA, Johnson EC, Hess SD, Rao SP, Sacaan AI, Santori EM,
Velicelebi G, Kuhn R (1999) 2-Methyl-6-(phenylethynyl)-pyridine (MPEP), a potent, selective
and systemically active mGlu5 receptor antagonist. Neuropharmacology 38:1493–1503
93. Pagano A, Ruegg D, Litschig S, Stoehr N, Stierlin C, Heinrich M, Floersheim P, Prezeau L,
Carroll F, Pin JP, Cambria A, Vranesic I, Flor PJ, Gasparini F, Kuhn R (2000) The non-competitive
antagonists 2-methyl-6-(phenylethynyl)pyridine and 7-hydroxyiminocyclopropan[b]chromen-1a-
carboxylic acid ethyl ester interact with overlapping binding pockets in the transmembrane region of
group I metabotropic glutamate receptors. J Biol Chem 275:33750–33758
94. Knoflach F, Mutel V, Jolidon S, Kew JN, Malherbe P, Vieira E, Wichmann J, Kemp JA (2001)
Positive allosteric modulators of metabotropic glutamate 1 receptor: characterization, mecha-
nism of action, and binding site. Proc Natl Acad Sci U S A 98:13402–13407
95. Molck C, Harpsoe K, Gloriam DE, Mathiesen JM, Nielsen SM, Brauner-Osborne H (2014)
mGluR5: exploration of orthosteric and allosteric ligand binding pockets and their applications
to drug discovery. Neurochem Res 39(10):1862–1875
96. Lundstrom L, Bissantz C, Beck J, Wettstein JG, Woltering TJ, Wichmann J, Gatti S (2011)
Structural determinants of allosteric antagonism at metabotropic glutamate receptor 2: mech-
anistic studies with new potent negative allosteric modulators. Br J Pharmacol 164:521–537
97. Schaffhauser H, Rowe BA, Morales S, Chavez-Noriega LE, Yin R, Jachec C, Rao SP, Bain G,
Pinkerton AB, Vernier JM, Bristow LJ, Varney MA, Daggett LP (2003) Pharmacological
characterization and identification of amino acids involved in the positive modulation of
metabotropic glutamate receptor subtype 2. Mol Pharmacol 64:798–810
98. Rovira X, Malhaire F, Scholler P, Rodrigo J, Gonzalez-Bulnes P, Llebaria A, Pin JP, Giraldo J,
Goudet C (2015) Overlapping binding sites drive allosteric agonism and positive cooperativity
in type 4 metabotropic glutamate receptors. FASEB J 29:116–130
99. Dalton JA, Gomez-Santacana X, Llebaria A, Giraldo J (2014) Computational analysis of
negative and positive allosteric modulator binding and function in metabotropic glutamate
receptor 5 (in)activation. J Chem Inf Model 54:1476–1487
100. Chen Y, Goudet C, Pin JP, Conn PJ (2008) N-{4-Chloro-2-[(1,3-dioxo-1,3-dihydro-2H-
isoindol-2-yl)methyl]phenyl}-2-hy droxybenzamide (CPPHA) acts through a novel site as a
positive allosteric modulator of group 1 metabotropic glutamate receptors. Mol Pharmacol
73:909–918
101. Hammond AS, Rodriguez AL, Townsend SD, Niswender CM, Gregory KJ, Lindsley CW,
Conn PJ (2010) Discovery of a novel chemical class of mGlu(5) allosteric ligands with distinct
modes of pharmacology. ACS Chem Neurosci 1:702–716
102. Muhlemann A, Ward NA, Kratochwil N, Diener C, Fischer C, Stucki A, Jaeschke G,
Malherbe P, Porter RH (2006) Determination of key amino acids implicated in the actions
of allosteric modulation by 3,30 -difluorobenzaldazine on rat mGlu5 receptors. Eur J Pharmacol
529:95–104
103. Niswender CM, Johnson KA, Weaver CD, Jones CK, Xiang Z, Luo Q, Rodriguez AL, Marlo JE, de
Paulis T, Thompson AD, Days EL, Nalywajko T, Austin CA, Williams MB, Ayala JE, Williams R,
Lindsley CW, Conn PJ (2008) Discovery, characterization, and antiparkinsonian effect of novel
positive allosteric modulators of metabotropic glutamate receptor 4. Mol Pharmacol 74:1345–1358
104. Binet V, Brajon C, Le Corre L, Acher F, Pin JP, Prezeau L (2004) The heptahelical domain of
GABA(B2) is activated directly by CGP7930, a positive allosteric modulator of the GABA
(B) receptor. J Biol Chem 279:29085–29091
105. Goudet C, Gaven F, Kniazeff J, Vol C, Liu J, Cohen-Gonsaud M, Acher F, Prezeau L, Pin JP
(2004) Heptahelical domain of metabotropic glutamate receptor 5 behaves like rhodopsin-like
receptors. Proc Natl Acad Sci U S A 101:378–383
106. Rook JM, Noetzel MJ, Pouliot WA, Bridges TM, Vinson PN, Cho HP, Zhou Y, Gogliotti RD,
Manka JT, Gregory KJ, Stauffer SR, Dudek FE, Xiang Z, Niswender CM, Daniels JS, Jones
CK, Lindsley CW, Conn PJ (2013) Unique signaling profiles of positive allosteric modulators
of metabotropic glutamate receptor subtype 5 determine differences in in vivo activity. Biol
Psychiatry 73:501–509
107. Kenakin T (2017) Signaling bias in drug discovery. Expert Opin Drug Discovery 12:321–333
108. Rook JM, Xiang Z, Lv X, Ghoshal A, Dickerson JW, Bridges TM, Johnson KA, Foster DJ,
Gregory KJ, Vinson PN, Thompson AD, Byun N, Collier RL, Bubser M, Nedelcovych MT,
Gould RW, Stauffer SR, Daniels JS, Niswender CM, Lavreysen H et al (2015) Biased mGlu5-
positive allosteric modulators provide in vivo efficacy without potentiating mGlu5 modulation
of NMDAR currents. Neuron 86:1029–1040
109. Eriksen L, Thomsen C (1995) [3H]-L-2-amino-4-phosphonobutyrate labels a metabotropic
glutamate receptor, mGluR4a. Br J Pharmacol 116:3279–3287
110. Jiang Y, Huang Y, Wong HC, Zhou Y, Wang X, Yang J, Hall RA, Brown EM, Yang JJ (2010)
Elucidation of a novel extracellular calcium-binding site on metabotropic glutamate receptor 1
{alpha} (mGluR1{alpha}) that controls receptor activation. J Biol Chem 285:33463–33474
111. Wright RA, Arnold MB, Wheeler WJ, Ornstein PL, Schoepp DD (2000) Binding of [3H]
(2S,10 S,20 S)-2-(9-xanthylmethyl)-2-(20 -carboxycyclopropyl) glycine ([3H]LY341495) to cell
membranes expressing recombinant human group III metabotropic glutamate receptor sub-
types. Naunyn Schmiedeberg’s Arch Pharmacol 362:546–554
112. Ogawa H, Qiu Y, Philo JS, Arakawa T, Ogata CM, Misono KS (2010) Reversibly bound
chloride in the atrial natriuretic peptide receptor hormone-binding domain: possible allosteric
regulation and a conserved structural motif for the chloride-binding site. Protein Sci 19:544–557
113. Tabata T, Aiba A, Kano M (2002) Extracellular calcium controls the dynamic range of neuronal
metabotropic glutamate receptor responses. Mol Cell Neurosci 20:56–68
114. Miyashita T, Kubo Y (2000) Extracellular Ca2+ sensitivity of mGluR1alpha associated with
persistent glutamate response in transfected CHO cells. Receptors Channels 7:25–40
115. Saunders R, Nahorski SR, Challiss RA (1998) A modulatory effect of extracellular Ca2+ on type
1alpha metabotropic glutamate receptor-mediated signalling. Neuropharmacology 37:273–276
116. Ullmer C, Zoffmann S, Bohrmann B, Matile H, Lindemann L, Flor P, Malherbe P (2012)
Functional monoclonal antibody acts as a biased agonist by inducing internalization of metabotropic
glutamate receptor 7. Br J Pharmacol 167:1448–1466
117. Webster CI, Caram-Salas N, Haqqani AS, Thom G, Brown L, Rennie K, Yogi A, Costain W,
Brunette E, Stanimirovic D (2016) Brain penetration, target engagement, and disposition of the
blood-brain barrier-crossing bispecific antibody antagonist of metabotropic glutamate receptor
1. FASEB J 30(5):1927–1940
118. Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB,
Bendahman N, Hamers R (1993) Naturally occurring antibodies devoid of light chains. Nature
363:446–448
119. Mujic-Delic A, de Wit RH, Verkaar F, Smit MJ (2014) GPCR-targeting nanobodies: attractive
research tools, diagnostics, and therapeutics. Trends Pharmacol Sci 35:247–255
120. Muyldermans S (2013) Nanobodies: natural single-domain antibodies. Annu Rev Biochem
82:775–797
121. Scholler P, Nevoltris D, de Bundel D, Bossi S, Moreno-Delgado D, Rovira X, Moller TC, El
Moustaine D, Mathieu M, Blanc E et al (2017) Allosteric nanobodies uncover a role of
hippocampal mGlu2 receptor homodimers in contextual fear consolidation. Nat Commun
8:1967
122. Liu J, Zhang Z, Moreno-Delgado D, Dalton JA, Rovira X, Trapero A, Goudet C, Llebaria A,
Giraldo J, Yuan Q, Rondard P, Huang S, Pin JP (2017) Allosteric control of an asymmetric
transduction in a G protein-coupled receptor heterodimer. eLife 6. pii: e26985
123. Niswender CM, Jones CK, Lin X, Bubser M, Thompson Gray A, Blobaum AL, Engers DW,
Rodriguez AL, Loch MT, Daniels JS, Lindsley CW, Hopkins CR, Javitch JA, Conn PJ (2016)
Development and antiparkinsonian activity of VU0418506, a selective positive allosteric modulator
of metabotropic glutamate receptor 4 homomers without activity at mGlu2/4 heteromers. ACS

Chem Neurosci 7:1201–1211
124. Kramer RH, Mourot A, Adesnik H (2013) Optogenetic pharmacology for control of native
neuronal signaling proteins. Nat Neurosci 16:816–823
125. Broichhagen J, Frank JA, Trauner D (2015) A roadmap to success in photopharmacology. Acc
Chem Res 48:1947–1960
126. Velema WA, Szymanski W, Feringa BL (2014) Photopharmacology: beyond proof of princi-
ple. J Am Chem Soc 136:2178–2191
127. Levitz J, Pantoja C, Gaub B, Janovjak H, Reiner A, Hoagland A, Schoppik D, Kane B,
Stawski P, Schier AF, Trauner D, Isacoff EY (2013) Optical control of metabotropic glutamate
receptors. Nat Neurosci 16:507–516
128. Carroll EC, Berlin S, Levitz J, Kienzler MA, Yuan Z, Madsen D, Larsen DS, Isacoff EY
(2015) Two-photon brightness of azobenzene photoswitches designed for glutamate receptor
optogenetics. Proc Natl Acad Sci U S A 112:E776–E785
129. Keppler A, Gendreizig S, Gronemeyer T, Pick H, Vogel H, Johnsson K (2003) A general
method for the covalent labeling of fusion proteins with small molecules in vivo. Nat Biotechnol
21:86–89
130. Broichhagen J, Damijonaitis A, Levitz J, Sokol KR, Leippe P, Konrad D, Isacoff EY, Trauner
D (2015) Orthogonal optical control of a G protein-coupled receptor with a SNAP-tethered
photochromic ligand. ACS Cent Sci 1:383–393
131. Gautier A, Juillerat A, Heinis C, Correa IR Jr, Kindermann M, Beaufils F, Johnsson K (2008)
An engineered protein tag for multiprotein labeling in living cells. Chem Biol 15:128–136
132. Levitz J, Broichhagen J, Leippe P, Konrad D, Trauner D, Isacoff EY (2017) Dual optical
control and mechanistic insights into photoswitchable group II and III metabotropic glutamate
receptors. Proc Natl Acad Sci U S A 114:E3546–E3554
133. Levitz J, Habrian C, Bharill S, Fu Z, Vafabakhsh R, Isacoff EY (2016) Mechanism of
assembly and cooperativity of homomeric and heteromeric metabotropic glutamate receptors.
Neuron 92(1):143–159
134. Font J, Lopez-Cano M, Notartomaso S, Scarselli P, Di Pietro P, Bresoli-Obach R, Battaglia G,
Malhaire F, Rovira X, Catena J, Giraldo J, Pin JP, Fernandez-Duenas V, Goudet C, Nonell S,
Nicoletti F, Llebaria A, Ciruela F (2017) Optical control of pain in vivo with a photoactive
mGlu5 receptor negative allosteric modulator. eLife 6. pii: e23545
135. Pittolo S, Gomez-Santacana X, Eckelt K, Rovira X, Dalton J, Goudet C, Pin JP, Llobet A,
Giraldo J, Llebaria A, Gorostiza P (2014) An allosteric modulator to control endogenous G
protein-coupled receptors with light. Nat Chem Biol 10:813–815
136. Kaplan JH, Forbush B 3rd, Hoffman JF (1978) Rapid photolytic release of adenosine
50 -triphosphate from a protected analogue: utilization by the Na:K pump of human red
blood cell ghosts. Biochemistry 17:1929–1935
137. Callaway EM, Katz LC (1993) Photostimulation using caged glutamate reveals functional
circuitry in living brain slices. Proc Natl Acad Sci U S A 90:7661–7665
138. Crawford JH, Wootton JF, Seabrook GR, Scott RH (1997) Activation of Ca2+-dependent currents
in dorsal root ganglion neurons by metabotropic glutamate receptors and cyclic ADP-ribose pre-
cursors. J Neurophysiol 77:2573–2584
139. Bahamonde MI, Taura J, Paoletta S, Gakh AA, Chakraborty S, Hernando J, Fernandez-Duenas-
V, Jacobson KA, Gorostiza P, Ciruela F (2014) Photomodulation of G protein-coupled aden-
osine receptors by a novel light-switchable ligand. Bioconjug Chem 25:1847–1854
140. Broichhagen J, Schonberger M, Cork SC, Frank JA, Marchetti P, Bugliani M, Shapiro AM,
Trapp S, Rutter GA, Hodson DJ, Trauner D (2014) Optical control of insulin release using a
photoswitchable sulfonylurea. Nat Commun 5:5116
141. Kokel D, Cheung CY, Mills R, Coutinho-Budd J, Huang L, Setola V, Sprague J, Jin S, Jin YN,
Huang XP, Bruni G, Woolf CJ, Roth BL, Hamblin MR, Zylka MJ, Milan DJ, Peterson RT
(2013) Photochemical activation of TRPA1 channels in neurons and animals. Nat Chem Biol
9:257–263
142. Schonberger M, Trauner D (2014) A photochromic agonist for mu-opioid receptors. Angew
Chem Int Ed Engl 53:3264–3267
143. Stein M, Middendorp SJ, Carta V, Pejo E, Raines DE, Forman SA, Sigel E, Trauner D (2012)
Azo-propofols: photochromic potentiators of GABA(A) receptors. Angew Chem Int Ed Engl
51:10500–10504
144. Engers DW, Field JR, Le U, Zhou Y, Bolinger JD, Zamorano R, Blobaum AL, Jones CK,
Jadhav S, Weaver CD, Conn PJ, Lindsley CW, Niswender CM, Hopkins CR (2011) Discov-
ery, synthesis, and structure-activity relationship development of a series of N-(4-acetamido)
phenylpicolinamides as positive allosteric modulators of metabotropic glutamate receptor
4 (mGlu(4)) with CNS exposure in rats. J Med Chem 54:1106–1110
145. Rovira X, Trapero A, Pittolo S, Zussy C, Faucherre A, Jopling C, Giraldo J, Pin JP,
Gorostiza P, Goudet C, Llebaria A (2016) OptoGluNAM4.1, a photoswitchable allosteric
antagonist for real-time control of mGlu4 receptor activity. Cell Chem Biol 23:929–934
Index
A Apex 3D, 105

A70108, 138 Apocytochrome b562, 18
Activation, 53 Apomorphine (APO), 127–131, 134
Adenosine receptors (ARs), 75 iso-APO, 128
A1, 4, 33, 240 Aquaporin-2 (AQP2) channel, 169
A2A, 3, 4, 13, 17, 33, 80, 85, 230 Arginine vasopressin (AVP), 168
A3, 240 Aripiprazole, 166
Adenylyl cyclase, 54 Aroylaminoethyloctahydro-
Adhesion (class B) family, 67, 103 pyrazinopyridoindoles, 152
Adrenergic receptors (ADRs), 17, 74, 165, 242 Arrestins, 1, 20, 35, 38, 53, 67, 90, 104, 165,
alpha-1, 110, 119 181, 196, 217, 234
beta-1, 5, 14, 33, 240 Autism, 258, 259
beta-2, 3, 6, 14, 17, 18, 33, 55, 57, 69, 104, p-Azido-L-phenylalanine (AzF), 200
109, 167, 240, 242 Azobenzene, 269, 273
Agonists, 13, 65, 258
biased, 164, 173, 174, 219
inverse, 13, 20, 39, 54, 75, 107, 126, 165, B
171, 174 Bacteriorhodopsin, 108
AH-11110A, 113 Benign prostate hyperplasia (BPH), 110
Allostery, 53, 67, 168 Benzhydrylamines, 141
Alloswitch-1, 272, 273 BI-167107, 18
Alpha-factor, 201 Biased agonists, 164, 173, 174, 219
Ambroxol, 167 Bioluminescence RET (BRET), 219–244
Amiloride, 170 Biovia, 105
6-Amino-5,6,7,8-tetrahydronaphthalene-2,3- BMY-7378, 115
diol (ADTN), 128 Bromocriptine, 127
Amino acids, unnatural (UAAs), 195–212 Bromotopsentin, 113
Analog based approaches (AAA), 105
Angiotensin receptors (ATR), 5, 165
Antagonists, 35, 54, 60, 65, 67, 73, 95, C
110–130, 141 Calcium-sensing receptor, 167
neutral, 35, 107 Cannabinoid receptors (CB), 7
Antidiuretic hormone, 163 type 2 (CB2), 30
Antihistamines, 80, 140–144, 147–149, 152 CAPRI, 85
285
286 Index
Carazolol, 74, 126 3,5-Difluorotyrosine (F2Y), 208

Cardiac dysfunction/failure, 54, 74, 82, 104, 6,7-Dihydroxy-2-aminotetralin, 128
166 5,6-Dihydroxy-2-(di-n-propylamino)tetralin,
Carvedilol, 166 128
CC chemokine receptors (CCRs), 7 2,3-Dihydroxypropyl-(7Z)-tetradec-7-enoate
CCR2, 7, 60 (MAG7.7), 14
CCR5, 7, 18, 202, 204, 207, 210, 211 Diphenhydramine, 140
CCR9, 7, 19, 60 Diphenylhydramines, 141
CXCR1, 30 DISCO, 105
Centbutindole, 131 Discretamine, 113
Cetirizine, 140 Docking, 1, 65–95, 117–137, 148–152, 260
Chaperones, pharmacological, 13, 17, 18, screens, 65, 83
163–175 Dodecyl β-D-maltoside (DDM), 13, 35
Chemokines, 200 Dopamine, 79, 101, 127
4, 20 receptors (DR), 8, 17, 20, 78, 127, 165, 190,
receptors, 30 209, 230, 236
Chlorpheniramine, 140 agonists, 127
Cholesteryl hemisuccinate (CHS), 13 Double electron–electron resonance (DEER),
Chronic obstructory pulmonary disease, 81 36
Comparative modeling, 65 Doxepin, 80
Comparative molecular field analysis 2DQSAR, 105
(CoMFA), 105 Drug design, structure-based, 65
Comparative molecular similarity index Drug discovery, 65, 90, 95, 163
analysis (CoMSIA), 105 Dynamics, 53
Congenital nephrogenic diabetes insipidus
(cNDI), 163
Corticotropin-releasing factor receptors, 11, E
200, 203 E90K receptor, 167
Corynanthine, 111, 117 Endosomal signaling, 181
Crystallizablity, enhancing, 15 Endothelin receptor B, 8
CSAR, 85 Energy landscape, 27
CVX15, 88 Epinastine, 140
CXC chemokine receptors (CXCRs), 80 Epinephrine (adrenaline), 74
CXCR1, 30 Ergoline, 90
CXCR4, 80, 88, 200, 201, 236 Ergotamine, 89
CXCR7, 236 Escherichia coli, 3, 9, 27, 30, 41, 198, 204, 236
Cyclazosin, 120 50 -N-Ethylcarboxamidoadenosine (NECA), 4,
Cyclooxygenase inhibitors, 170 5, 19, 77
Cyproheptadine, 140 Extracellular domains (ECD), 15, 187, 253,
256, 259
D
DBSCAN, 243 F
D3DR, 88 Fabry disease, 167
Deglycosylation, 16 Fabs, 2
Detergents, 13, 18, 35, 46, 104, 230 Fexofenadine, 140
solubilization, 1 Fluorescence correlation spectroscopy (FCS),
Diabetes, 81, 104, 203 219, 240
insipidus, congenital nephrogenic, 163, 168 Fluorescence cross-correlation spectroscopy
type 2, 81 (FCCS), 240
Dibenzocyclooctyne (DIBO), 208 Fluorescence recovery after photobleaching
Dicentrine, 111 (FRAP), 239
Diffraction, 1, 18, 20, 242 Fluorescent assay, 195
Diffraction limited fluorescence, 221, 222, N-Formyl peptide receptor (FPR), 241
241, 242 Fragment-based lead discovery (FBLD), 65, 76
Index 287
Free electron laser (FEL), 20 I

Förster (or fluorescence) RET (FRET), 182, IBF28145, 140
188, 206, 219–244 ICI118551, 126
time-resolved, 225 Indomethacin, 170
Frizzled/Smoothened (class F) family, 67, 103 Inflammation, 104, 140, 200
Interaction fingerprints (IFPs), 80
Ion channels, 54, 167, 197, 209, 224,
G 253–255, 273
GABA receptors, 243 Ion modulation, 266
GALAHAD, 105
GASP, 105
Gaucher disease, 167 J
Genetic code expansion (GCE), 195 JF-NP-26, 273
GG818, 112 JNJ7777120, 166
Ghrelin receptor (GhrR), 206
Glucagon receptor, 11, 17, 60, 184, 187
Glutamate, 84, 253 L
caged, 272 LA-PTH, 189
(class C) family, 67, 103, 230 Lauryl maltose neopentyl glycol (MNG3), 13
metabotropic (mGluRs), 3, 18, 20, 84, 230, Leukotriene B4 receptor (BLT2), 36
242, 253, 255 Ligand-based drug design (LBDD), 104–110,
Glycosylations, 13 151
GPR109a receptor, 165 Ligand-gated ion channels, 197, 209, 253, 255
G-protein binding, 53 Ligands, allosteric, 45, 59, 67, 79, 83, 257,
G protein-coupled receptor (GPCR), crystal 262, 274
structures, 195 binding, 53
interacting proteins (GIPs), 104 fluorescent, 226–230, 237, 240
interactome, 195 light-operated, 269
kinases (GRKs), 107 orthosteric, 229, 259, 262
molecular modeling, 101 photo-caged, 271, 272
non-rhodopsin, 1 photoswitchable, 269–273
structure prediction, 69 Lipidic cubic phase method (LCP), 14
G protein-independent signal transducers, 107 Lipinski’s rule, 72
Growth hormone secretagogue receptors Llama nanobodies, 267
(GHSR), 30, 206 Loratadine, 140
Low urinary tract symptoms (LUTS), 110
Lumi4-terbium (Lumi4-Tb), 234
H Luminescence-based strategy developments,
HEK293 N-actetylglucosaminetransferase I, 16 217
Herkinorin, 166 LY-2940680, 90
Hiphop, 105 Lysophosphatidic acid receptor (LPA1), 8
Histamine, 101 Lysosomal storage disorders (LSDs), 167
receptors (HRs), 8, 80
antagonists, 140
HIV, 80, 201 M
Homology modeling, 65 M1 muscarinic acetylcholine receptor
Hydrochlorothiazide, 170 (CHRM1), 8
7-Hydroxy-2-(di-n-propylamino)-tetralin, 128 Maltose neopentyl glycol (MNG-3), 35
3-(3-Hydroxyphenyl)-N-n-propylpiperidine MAP kinases, 54
(3-PPP), 128 Maraviroc, 202
Hypocalcemia, 181 MCF14/MCF18/MCF57, 171
Hypoparathyroidism, 189 Mepyramine, 140
Hypothetical active site lattice (HASL), 105 Mequitazine, 140
288 Index
Metabotropic glutamate receptors (mGluRs), 3, analogs, long-acting (LA-PTH), 189

18, 20, 84, 230, 242, 253, 255 receptor (PTHR), 181
Methyluropidil, 110, 112 related peptide (PTHrP), 182
Mianserine, 140 Parkinson’s disease (PD), 75, 78
Microcrystals, 1 Perdeuteration, 27
Microswitches, 29 Pergolide, 127
Migalastat, 167 Pharmacological chaperones, 13, 17, 18,
Misacylated suppressor tRNA, 197 163–175
MK 0354, 166 Pharmacology, optogenetic, 253
Molecular docking, 65, 71 Pharmacophore, 101
Molecular dynamics (MD), 30 PHASE, 105
Monoacylglycerol (MAG), 14 PHCCC, 268
Monoclonal antibodies (mAbs), 203, 267 Phenbenzamines, 141
Monoolein (MAG9.9), 14 Phenindamine, 140
Muscarinic receptors (MRs), 81 Phentolamine, 112
Phospholipases, 54
Phosphorylations, 13, 169, 199, 208, 209, 228,
N 231
NAN-190, 120 Photo-activation light microscopy (PALM),
Nanobodies, 2, 17, 36–39, 55, 58, 227, 267 220, 222, 242–244
Negative allosteric modulators (NAMs), 80, Photobridges, 204
229, 257, 264, 272 Photo-caged ligands, 271, 272
Neurokinin, 200 Photocrosslinking, 184, 195, 199–204, 210,
Neurotensin, 14, 20 211
receptor, 9, 14 Photopharmacology, 253, 271
Neuroinflammation, 259 Photoswitchable orthogonal remotely tethered
Neurotransmitters, 67, 82, 253, 272 ligands (PORTL), 270
New chemical entities (NCEs), 105 Photoswitching, 242, 269–274
Niguldipine, 110 Piperoxan, 140
NMR, solution-state, 27 Positive allosteric modulators (PAMs), 44, 92,
Nociceptin/orphanin FQ receptor 229, 262, 267
(NOP/ORL-1), 9 Pramipexole, 127
Noradrenaline, 74 Prazosin, 112
Protease-activated receptor, 10
Protein–protein interactions, 217
O Purinoceptors (P2Y1/P2Y12), 10
Octahydropyrazinopyridoindoles, 140, 150 Pyrococcus abysii glycogen synthase (PGS), 18
OHDPAT, 127, 128, 135, 139
OHNPA, 137, 138
OPC31260 (mozavaptan), 171 Q
OPC41061 (tolvaptan), 171 Quinpirole, 139
Opioids, receptors, 10, 29, 33, 38, 45, 57, 81,
165
OptoGluNAM, 274 R
Optogluram, 273 Renilla luciferase ( Rluc), 223
Optopharmacology, 253 Resonance energy transfer, 217
Orexin receptors, 9, 10 Retromer, 181, 183, 188
type 1 (Ox1), 9 Rhodopsin, 2, 55, 69, 85, 108, 119, 121, 147,
type 2 (Ox2), 10 188, 205
Orthosteric ligands, 229, 259, 262 (class A) family, 57, 67, 85, 103, 108, 201,
254
Risperidone, 117
P Ropinirole, 127
Pan-assay interference compounds (PAINS), 72 RS17053, 120
Parathyroid hormone (PTH), 181 RS100975, 112
Index 289
S Tolvaptan, 163
Salmeterol, 166 Total internal reflection microscopy (TIRF),
SAMPL, 85 241
SANT1, 90 Trace amine-associated receptor 1 (TAAR1),
Schizophrenia, 78, 259 92
Secretin (class B) family, 67, 103 Transducin, 106, 107
Serial femtosecond crystallography (SFX), 20 Transmembrane (TM) domains, 13, 19, 106,
Serotonin (5-hydroxytryptamine), 20, 78, 89 203, 229, 253, 274
receptors, 11, 60, 82–92 Transmembrane (TM) helices, 16, 29, 55, 67,
Silodosin, 110, 112 201, 205
Single-particle tracking (SPT), 241 Triprolidine, 140
super-resolution microscopy, 217 TRV027, 166
SKF38393, 137 TRV130, 166
SKF104856, 113
Smoothened receptors (SMO), 12, 85, 89
SNAP8719, 113 U
Solubilization, 13 UK-432097, 16, 77
Spatio-temporal network, 217 Unnatural amino acids (UAAs), 195–212
Spectroscopic analysis, 195 Urocortin-1, 203
Sphingosine-1-phosphate receptor, 11
Spiperone, 112
SR49059, 171 V
SR121463, 170 VA999089, 173
SSR149415, 172 Vaptans, 163
Stability/stabilization, 1, 3 Vasopressin receptors, 163, 167
Ste2p, 200 Virtual screening (VS), 65, 106
Strain-promoted [3+2] alkyne-azide VPA985, 171
cycloaddition (SpAAC), 206 VU0155041, 268
Structure–activity relationship, 195 VU0357121, 263
Structure based (direct) drug design/discovery VU0409551, 265
(SBDD), vi, 104 VU0415374, 268, 273
Substance P (SP), 201 VU0418506, 268
Substituent spanned space (SSS), 105 VUF10214, 166
Sumanirole, 137
W
T WB4101, 112
T4 lysozyme (T4L), 18
T140, 201
Tamsulosin, 116 X
Target immobilized NMR screening (TINS), X-linked genetic pathology, 163, 168
76 X-ray crystallography, 1–20, 71, 104, 131, 184,
Terfenadine, 140 195
Tetramethylrhodamine, 182
Therapeutic rescue, 163
Thermal stability, receptors, 19 Z
Thioredoxin, 37 ZINC lead-like library, 78
Timolol, 126 ZM241385, 76, 85

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Topics in Medicinal Chemistry 30

Topics in Medicinal Chemistry (TMC) covers all relevant aspects of medicinal

More information about this series at http://www.springer.com/series/7355

Structure and Function

ISSN 1862-2461 ISSN 1862-247X (electronic)

© Springer Nature Switzerland AG 2019

G protein-coupled receptors (GPCRs) are localised at the plasma membrane of

agonists is presented by Bernard Mouillac and Christiane Mendre. In addition, they

IGF, CNRS UMR, INSERM, University Guillaume Lebon

1. Venkatakrishnan AJ et al (2013) Molecular signatures of G-protein-coupled

3. Tate CG, Schertler GFX (2009) Engineering G protein-coupled receptors to

23. Congreve M et al (2012) Discovery of 1,2,4-triazine derivatives as adenosine A

39. Goudet C, Rovira X, Llebaria A (2018) Shedding light on metabotropic gluta-

Structures of Non-rhodopsin GPCRs Elucidated Through X-Ray

Modulation of Metabotropic Glutamate Receptors by Orthosteric,

Structures of Non-rhodopsin GPCRs

Chady Nasrallah and Guillaume Lebon

Keywords Detergents solubilization, Diffraction, GPCRs, Micro-crystals,

C. Nasrallah and G. Lebon (*)

5 GPCR Sub-Micrometer and Micrometer-Size Crystals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

G Protein-Coupled Receptors (GPCRs) are the largest class of human extracellular

2 Large-Scale Production of GPCRs

Fig. 1 Successful stabilization strategies enhancing GPCRs crystallization. (a) Structures of

Table 1 Structures of non-rhodopsin GPCRSs solved by X-ray crystallography

FQ receptor phase 1-yl]propyl)pyrrolidine-2-carboxamide (ANT) A) [63]

SB-674042 (ANT) R 4ZJC (2.83

mutations phase benzonitrile (ANT) A) [27]

3 Solubilization of GPCRs Prior to Crystallization

Before proceeding to purification that is required to set up crystallization trials,

detergent synthetized based on DDM scaffold with further constraints on detergent

4 Enhancing GPCRs Crystallizablity

4.1 Sample Homogeneity

4.1.1 Ligands Screening

4.1.3 N and C Termini Domains

GPCRs exhibit remarkably conserved protein architecture with seven transmembrane

4.2 Binding Partners Stabilizing Receptor Conformation

receptor following immunization process and to guarantee optimal receptor confor-

4.3 Increasing Receptor Hydrophilic Area

Highly crystallizable soluble proteins replacing disordered segments within

were obtained, has proven to be a powerful alternative to in surfo crystallization

4.4 Increasing Receptor Thermal Stability

Another successful approach to stabilize GPCRs for structural studies consists of

thermostabilized ß1-AR receptor was solved bound to a wide variety of ligands

5 GPCR Sub-Micrometer and Micrometer-Size Crystals

The main difficulty to solve GPCR structures is to obtain well-diffracting crystals of

All the implementation and developments in the area of GPCR crystallography

Acknowledgments The authors acknowledge program ATIP (2013–2016), CNRS, FRM,

1. Lagerstrom MC, Schioth HB (2008) Structural diversity of G protein-coupled receptors and

4. Ghosh E, Kumari P, Jaiman D, Shukla AK (2015) Methodological advances: the unsung

28. Miller-Gallacher JL et al (2014) The 2.1 A resolution structure of cyanopindolol-bound

79. Hanson MA et al (2012) Crystal structure of a lipid G protein-coupled receptor. Science

104. Olofsson L et al (2014) Fine tuning of sub-millisecond conformational dynamics controls

NMR Spectroscopy for the

Marina Casiraghi, Jean-Louis Banères, and Laurent J. Catoire

Abstract G protein-coupled receptor (GPCR)-mediated signal transduction has a

Keywords Energy landscape, Escherichia coli, GPCR, Perdeuteration, Solution-

M. Casiraghi and L.J. Catoire (*)

3 Solution-State NMR Investigations of GPCR Conformational Landscapes: State

the thermodynamics and kinetics of the conformational transitions [5]. Following

1.1 What Crystals Tell Us?

1.2 The Picture Needs to Be Completed

Crystal structures obtained so far have largely contributed to our understanding of

extensively the energy landscape, by determining the different conformations that