Sensor Issues

pubs.acs.org/acssensors

Harnessing the Versatility of Optical Biosensors for Target-Based

Small-Molecule Drug Discovery
Tim Kaminski, Anders Gunnarsson, and Stefan Geschwindner*
Discovery Sciences, Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Pepparedsleden 1, S-43183 Mölndal,
Sweden

ABSTRACT: Optical biosensors entered target-based small-molecule drug discovery

more than two decades ago and have since transformed into a value-adding
See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

component in the decision-making process. Here, we briefly highlight the major

application areas of optical biosensors and focus on desirable profiles of such platforms
in order to ensure their effective use in small molecule drug discovery. Furthermore,
we will emphasize current technology-based constraints and discuss experimental
strategies to address these limitations as well as provide a view of necessary technology
Downloaded via 141.0.9.202 on February 2, 2020 at 12:24:30 (UTC).

improvements for next generation platforms.

KEYWORDS: drug discovery, small molecule, kinetics, single molecule, surface-plasmon resonance, waveguide-grating,
biolayer interferometry

■ CURRENT USE AND IMPACT OF OPTICAL

BIOSENSORS IN TARGET-BASED
SMALL-MOLECULE DRUG DISCOVERY
Optical Biosensors have seen widespread application in target-
based drug discovery following the appearance of the first
commercial platforms in the early 1990s.1,2 Instruments based
on surface plasmon resonance (SPR),3 optical waveguide
grating (OWG),4 or biolayer interferometry (BLI)5 are
predominantly applied in early drug discovery focusing on
small-molecule screening and profiling, antibody character-
ization and selection in addition to biomarker detection, of
which the latter two will not be discussed here. The major
screening of much smaller compound libraries (<2000) and
typically monitors compound binding directly by employing,
e.g., fluidics-based SPR7 or BLI.5 This has recently gained in
importance, as FBDD has become a valuable alternative to
traditional hit finding approaches like high-throughput screen-
ing (HTS) and typically involves screening campaigns using
smaller compound fragments (∼200 Da) that are binding
weakly (Kd ∼ mM) to the target of interest.8 Thanks to their
improved sensitivity, optical biosensors are effectively used
alongside more traditional biophysical methods like nuclear
magnetic resonance (NMR) or X-ray crystallography to fuel
FBDD campaigns with novel chemistry.

application areas in the small molecule space aim to identify
and characterize the interaction of compounds with their target
protein, which encompass (i) primary screening, (ii) validation
of target engagement, (iii) identification of the molecular mode
of action (MoA), along with (iv) detailed characterization of
compound binding such as binding kinetics and thermody-
namics. A summary of established and currently emerging
biosensor platforms that are used in the context of small
molecule drug discovery is provided in Table 1, including their
primary application areas as well as the associated information
content.
■
VALIDATION OF TARGET ENGAGEMENT
Validation of primary hits or the assessment of target
engagement is predominantly used to confirm HTS actives.
This often starts alongside HTS assay development and thus
prior to any screening campaign, in order to assess the quality
of the assay (typically biochemical) and to understand
limitations as well as potential sources for HTS assay artifacts.9
Even more important is the assessment of constructive target

■
PRIMARY SCREENING
Primary screening using optical biosensors can utilize plate-
based OWG systems to enable screening of relatively large
compound libraries (>10 000). The experimental flexibility
allows equally for a biochemical4 or a cell-based readout,6 with
the notion that the later rather reflects the consequences of
compound binding through changes in cell morphology. In
contrast, Fragment-based drug discovery (FBDD) requires
engagement of compounds identified by HTS, as those typically
serve as starting points for extended and resource-intensive
medicinal chemistry campaigns.10 In this context, it is
important to note that target engagement is required but
often not sufficient to elicit a pharmacological response.
Received: November 15, 2016
Accepted: December 15, 2016
Published: December 15, 2016

© 2016 American Chemical Society 10 DOI: 10.1021/acssensors.6b00735

ACS Sens. 2017, 2, 10−15
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ref
■ UNDERSTANDING THE MOLECULAR MoA

11

12
3

4
To confirm compound-target engagement is only one of many
critical factors to drive compound development and ultimately

higher reagent consumption, de-

lower sensitivity, higher reagent

lower sensitivity, higher reagent

pendence on conformational
ensure clinical success. Equally important is to understand and
Table 1. Selection of Already Established and Currently Emerging Optical Biosensing Technologies Applied during Target-Based Small-Molecule Drug Discovery

confirm the compound-mechanism hypothesis, which means

limitations

building a detailed understanding of the molecular MoA.13 This

information can often help to bridge from simplistic in vitro
lower throughput

lower throughput
consumption

consumption observations to pharmacodynamics activity in a biologically

relevant disease setting. In the case of the α7 nicotinic

change
acetylcholine receptor, it is known that positive allosteric
modulators can provide a pharmacological advantage compared
affinity, kinetics, stoichi- to direct agonists.14 By tweaking the conditions in a biosensing
affinity, conformational
modynamics, stoichi-
information content

experiment, one is able to clearly distinguish compounds that

affinity, kinetics, ther-

directly act via the agonist binding site and those that modulate
affinity, kinetics

receptor activation via allosteric sites and thus might offer a

higher therapeutic potential.15 Such mechanistic studies
changes
ometry

ometry

frequently complement target engagement studies, as they

affinity

typically build on similar assay formats and platforms.

■
screening including whole cells, mode of
action studies including pathway anal-

screening (for conformational changes),

BINDING KINETICS AND THERMODYNAMICS

screening, target engagement, mode of

screening, target engagement, mode of

screening, target engagement, mode of

Finally, optical biosensors can provide a detailed character-

main application areas

ization of the dynamics of binding. A good example relates to

the generation of binding kinetic data, which can be used to
mode of action studies

drive structure−kinetic relationships during the compound

optimization process.16 Kinetic information is frequently
action studies

action studies

action studies

deduced through microfluidics-based biosensor platforms like

SPR, as the optimized fluidics and the high sampling rates allow
for an exact description of association and dissociation
ysis

processes. Information about extended dissociation processes

can eventually highlight possibilities to enhance compound
like SPR, detects refractive index changes within an evanescent field near the sensor surface, due

efficacy and safety and thus may help to ensure therapeutic

incident light couples into the waveguide resulting in a narrow band of reflected wavelengths,

induced conformational changes lead to a different relative orientation of the dye, which is
second-harmonic generation sensitive dyes are conjugated to surface-tethered targets; ligand-
analyzes the change in interference pattern of white light reflected from reference and active
detects changes in refractive index close to the sensor surface induced by analyte binding to

success if the pharmacokinetic features are compatible.17 In

analogy, thermodynamic data is generated by studying the
temperature-dependency of the binding affinity and can
eventually highlight compounds with a favorable enthalpy
allowing to detect changes in the sensing layer containing ligands or cells

profile, albeit the value of such data for more successful

compound design is disputable.18

■ DESIRED SPECIFICATIONS FOR OPTICAL

BIOSENSORS PLATFORMS
detection principles

Previously described application areas have some common

denominators for the optimal application of biosensor plat-
to mass changes induced by complex formation

forms in drug discovery. It can be essentially reduced to three

surface, containing surface-tethered ligand

main factors that make a successful assay: (i) the right

throughput, (ii) the right sensitivity, and (iii) the right reagent
(Figure 1). In order to effectively conduct primary screening
activities, higher throughput becomes a key factor, as the results
from such activities need to feed timely into the decision-
surface-tethered ligands

making process. This becomes even more important when

engaging in FBDD, as there is the frequent desire to conduct
this in parallel to HTS in order to get optimal synergies
between these activities.8 While biochemical assays used for
detected

HTS typically offer the desired throughput, they are frequently

not able to detect weak interactions, which displays a severe
shortcoming particular to FBDD. Consequently, biosensor
platforms need to provide both high detection sensitivity, i.e.,
second-harmonic gener-

ting-coupled interfer-
biolayer interferometry

optical waveguide gra-

surface plasmon reso-

waveguide-based gra-

acceptable signal-to-noise ratios together with an adequate

throughput of >1000 data points per day.
name

Once moving forward in this process, an ideal scenario is the

use of the same or a similar biosensor platform and/or assay
ometry
nance

ation
ting

configuration to continuously support the maturation of weak

binding fragments into more potent chemical leads. Simulta-
11 DOI: 10.1021/acssensors.6b00735
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Figure 1. The three R’s of biosensing representing key factors for
successful generation of biosensor-derived data to support target-based
small-molecule drug discovery: right throughput, right sensitivity, and
right reagent.
neously, one often needs to enable hit validation originating
from alternative hit finding approaches such as HTS.
Consequently, such platforms need to be able to cover a
broad affinity range from millimolar- down to picomolar-
binding affinities. This will ensure such platforms are applicable
throughout the early drug discovery process from hit finding
and validation via lead identification into lead optimization and
beyond.
A key but often neglected aspect is the stringent require-
ments on biosensor-compatible protein reagents since they
make intimate use of the transducer surface. Hence, one of the
most critical aspects of data quality is the ability to tether a
functional target with high stability to the surface. In addition,
the density of tethered functional target must be relatively high,
since the signal amplitude scales with the surface density of the
tethered ligand. This is particular critical in the case of small
molecule interactions due to the relatively low signal generated
upon binding. In summary, optimization of protein reagent is
key for successful deployment of any biosensor platform in
early drug discovery.
There are a number of intrinsic and extrinsic factors that will
influence this optimization workflow. Target stability can be
affected by the buffer and its supplements, making screening for
the right assay conditions often a prerequisite. Frequently, the
manipulation of the target protein itself is critical for success.
This may include addition of short peptide tags (e.g., Avi- or
hexa-His) to allow for directed and thus more controlled target
tethering in contrast to nondirectional covalent- (e.g., amine or
maleimide chemistry) or noncovalent (charge, hydrophobic)
tethering approaches. For more challenging targets such as
membrane-bound receptors, iterative rounds of point mutations
have sometimes been successful.19 While many native receptors
are unstable and rapidly denature at the sensor surface,
thermostabilized mutants of the same receptors can retain
their functionality and stability in a surface-tethered config-
uration for a number of days. Another commonly employed
strategy is the use of truncated proteins containing only the
domain of interest that is targeted with small molecules. In such
cases, it is very important to closely monitor that those
variations and alterations do show similar ligand binding
patterns as to the native protein. Observed discrepancies could
indicate a change in pharmacology when going into an in vivo
model to test the target hypothesis and thus need to be
12
Sensor Issues
addressed. Nevertheless, such modifications are typically
tedious and tend to be very resource-intensive without
guarantee for success.
In summary, the throughput and the sensitivity of any
biosensor platform typically provides a framework for defining
optimal protein reagents or tools in order to meet the
individual project requirements. These can vary quite
substantially and thereby expose both strengths as well as
weaknesses of the individual biosensor platforms used in drug
discovery.
■ STRENGTHS AND WEAKNESSES OF MAIN
BIOSENSOR PLATFORMS IN DRUG DISCOVERY
The choice of optical biosensor platform is heavily dependent
on application and assay requirements. As plate-based
biosensor platforms like OWG offer a similar throughput as
biochemical assays used in HTS, they provide an attractive
alternative for biophysical screening. Nevertheless, those
platforms typically experience relatively low sensitivity when
monitoring direct and specific small molecule binding, which is
in part a consequence of the limited tethering capacity of the
functionalized biosensor surface. Thus, such platforms are more
useful when trying to identify so-called “promiscuous” or
aggregation-based inhibitors, as those signals tend to be much
larger and are easily detectable.20
Changing the assay configuration can in some cases bypass
the sensitivity limitations of OWG platforms and enables the
screening of weakly binding fragments to support FBDD. This
is achieved through directed tethering of a functionalized tool
compound, which still retains good binding competence to the
respective target protein. Challenging the modified biosensor
with the target protein typically leads to a detectable signal, as
the amplitude scales with the change in molecular mass on the
biosensor. This signal can be subsequently modulated by
compounds that compete for the same binding site in solution,
thus this assay configuration is also referred to as inhibition in
solution assay (ISA).21
A different picture emerges for SPR-based instruments.
While OWG platforms provide much higher throughput than
sequentially operating systems (interrogating one compound or
concentration at a time), SPR systems typically offer much
higher sensitivity at the cost of lower throughput. This generally
makes it the platform of choice for driving FBDD campaigns in
a direct binding assay (DBA) configuration, as this allows
reliable detection of weak interactions with small molecules to
surface-tethered proteins.7 As a consequence of the fluidics-
based nature of these platforms, there is the additional
possibility to record binding kinetics data. While plate-based
systems typically operate at equilibrium, which only allows for
affinity (Kd) determination, fluidics-based systems such as SPR
also provide the opportunity for kinetic readout (kon, koff) and
thus offers higher information content. However, even for
platforms with the same optical transducer, e.g. SPR, the
balance between sensitivity and throughput is crucial. Although
“regular” SPR is unrivaled in terms of sensitivity, imaging SPR22
offers accelerated throughput but at the expense of sensitivity,
thus making the latter technology less attractive to small
molecule research. In order to address the sensitivity issue,
some SPR imaging platforms are making use of chemical
microarrays, which have shown to be particular useful for
fragment screening.23 While following similar principles for
sensitivity enhancement as the ISA, kinetic information is more
challenging to assess in this assay configuration.
DOI: 10.1021/acssensors.6b00735
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Whereas throughput and sensitivity in addition to robustness
has continuously improved over time, all biosensor platforms
suffer from different limitations that are intrinsic to their build
up. Besides the sensitivity limit that is dictated by the molecular
mass of the interactants, also throughput can be surprisingly
affected by system-related factors like binding kinetics. As
optimized compounds gain dramatically in potency by several
orders of magnitude once progressed from an initial hit toward
a candidate drug, they frequently display residence times of
hours or even days. For a reliable determination of binding
kinetics, the measurement time thus needs to be extended24
and a complete dissociation is required to present a compound-
free biosensor for subsequent experiments. Even with
throughput-enhancing strategies like single-cycle kinetics,25 it
can easily take 12−24h to obtain affinity and binding kinetics
data for a single compound.
BLI can address some of those limitations, as it elegantly
combines some advantageous features of SPR and OWG by
offering a plate-based readout with the option for kinetic
measurements.26 A range of biosensor tips (8−16) are dipping
into an analyte solution that is presented in a plate that can be
agitated to mimic a fluidics-based scenario for kinetic
measurements. Those sensors can be discarded afterwards to
enable a new round of measurements, thereby eliminating the
need to wait for complete compound dissociation. This strive
for parallelization has also seen some recent advances in the
development of throughput-enhanced SPR platforms, enabling
eight analyte evaluations in parallel.
Despite extensive optimization of protein reagents and assay
conditions, it can sometimes be challenging to make a clear-cut
assessment of the binding specificity. While it is straightforward
to identify compounds that deviate from the expected binding
behavior in terms of saturable, reversible binding following a
1:1 binding model,27 this is often much more difficult to
achieve for compounds that specifically engage with the target.
In this case, one is forced to conduct additional experiments to
look for competition with known, well-characterized tool
compounds or to perform a subsequent ISA experiment as
previously described. This obviously requires readily access to
such tool compounds, which preferably display high potency
and/or can be modified to accommodate a suitable linker for
directed tethering. Both approaches come at the cost of
additional reagent consumption and extended timelines for
decision-making.
■
NEXT-GENERATION BIOSENSOR PLATFORMS
ADDRESSING DRUG DISCOVERY NEEDS
One common denominator in the experimental workflow of
the discussed biosensor platforms is the need for two separate
experiments. First, the sensor surface is brought into contact
with the analyte. Thereby, the convoluted association and
dissociation reaction is measured. Typically, this reaction is
observed until equilibrium coverage of the sensor surface is
reached. Subsequently, the sensor surface is exposed to an
analyte-free solution to monitor the dissociation reaction alone.
The possibility of simultaneous quantification of association
and dissociation rates would obviously represent a great
technology enhancement, which can be realized with single
molecule biosensor platforms.
Such novel biosensor platforms and their potential value for
drug discovery have recently been described by making use of
the ISA configuration.28 While the tool compound is tethered
to the biosensor surface, the target protein is attached to or
13
Sensor Issues
incorporated into the lipid layer of fluorescently labeled
liposomes. Using a standard TIRF microscope, the binding of
the target−liposome complex to the surface can be monitored
with single molecule resolution.With each liposome containing
at maximum a single copy of the target protein, each observed
binding event reflects the interaction of a single target protein
with a single tool compound at the surface. The ability to
monitor each binding and release event at the surface under
equilibrium conditions allows for an estimation of association
and dissociation rates from a single experiment without
microfluidics or alternative liquid handling.29
This has many practical implications such as unrivaled
sensitivity and thus provides a good rationale to eventually
succeed existing biosensor platforms. The requirements on the
tethered tool compounds are less stringent, as low affinity is not
only tolerated but often even desirable, as it increases the
number of observable binding events in a given time frame due
to faster dissociation. Additionally, the use of tool compounds
in conjunction with single molecule sensitivity furnishes the
opportunity to distinguish specific from nonspecific binding
events, as they will show different kinetic profiles that can be
deconvoluted. This is essentially impossible with technologies
that are based on ensemble measurements like SPR, OWG, or
BLI. Furthermore, the use of liposomes offers the possibility to
study ligand binding even to labile membrane proteins in an
almost nativelike environment, without the need of extensive
protein purification or stability-enhancing mutations.29 This
strategy does not exclude soluble proteins, as they can be
tethered to liposomes following similar strategies as for
traditional biosensor experiments.
Finally and most importantly, performing experiments at the
single molecule level has surprising consequences for the limit
of detection and thus the sensitivity. By increasing the
observation time one is able to detect more binding and
release events to the biosensor. Therefore, the lower limit of
detection is constantly decreasing with increasing measurement
time and is not a fixed and platform-dependent parameter
anymore.
■
CONCLUSION AND OUTLOOK
Over recent years, optical biosensors have been positioned as a
vital part in small molecule drug discovery. The continuous
improvements of existing technologies have expanded their
application areas significantly. For example, state-of-the-art SPR
systems have now reached a level of sensitivity that readily
allows direct monitoring of small molecule interactions even at
suboptimal assay conditions (e.g., only a fraction of the tethered
target is functional). However, new platforms are also starting
to emerge in the drug discovery arena. One example is single
molecule microscopy, where evanescent-field illumination
combined with surface functionalization provides a readout at
the level of individual molecules. This provides unique
opportunities such as the possibility to monitor binding
kinetics at equilibrium in a plate-based format, thereby
combining the benefits from plate-based (throughput) and
flow-based systems (kinetics) in one platform. Another
advantage lies in the extremely high sensitivity, which reduces
protein consumption and allows measurements across a wide
affinity range. This together with the significantly reduced
reagent consumption furnishes the opportunity to generate
unique value for small molecule drug discovery.
DOI: 10.1021/acssensors.6b00735
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■ AUTHOR INFORMATION
Corresponding Author
*E-mail: stefan.geschwindner@astrazeneca.com.
ORCID
Stefan Geschwindner: 0000-0002-2154-8345
Author Contributions
The manuscript is written through contributions of all authors.
All authors have given approval to the final version of the
manuscript.
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
We would like to acknowledge all colleagues within the global
AstraZeneca Biophysics community for inspiration and their
continuous strive to unleash the full potential and maximise the
value of optical biosensors in drug discovery. Furthermore, we
would like to thank the internal AstraZeneca postdoc program
for the great support it has given to Tim Kaminski and Anders
Gunnarsson. In addition, we want to thank Helen Boyd for
input on the manuscript.
■
ABBREVIATIONS
BLI, biolayer inteferometry; DBA, direct binding assay; FBDD,
fragment-based drug discovery; HTS, high-throughput screen-
ing; ISA, inhibition in solution assay; MoA, mode of action;
OWG, optical waveguide grating; SPR, surface plasmon
resonance; TIRF, total internal reflection fluorescence
■
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15 DOI: 10.1021/acssensors.6b00735
ACS Sens. 2017, 2, 10−15