Received: 3 January 2022 Revised: 11 April 2022 Accepted: 8 May 2022

DOI: 10.1002/jcc.26938

RESEARCH ARTICLE

Evaluating the conformational space of the active site of D2

dopamine receptor. Scope and limitations of the standard
docking methods

Rodrigo D. Tosso1 | M. Natalia C. Zarycz1 | Ayelén Schiel1 |

Luisa Goicoechea Moro1 | Héctor A. Baldoni2 | Emilio Angelina3 | Ricardo D. Enriz1

1
Facultad de Química, Bioquímica y Farmacia,
Universidad Nacional de San Luis, Instituto Abstract
Multidisciplinario de Investigaciones We report here for the first time the potential energy surfaces (PES) of

gicas, San Luis, Argentina
Biolo
2 phenyletilamine (PEA) and meta-tyramine (m-OH-PEA) at the D2 dopamine receptor
Facultad de Química, Bioquímica y Farmacia,
Universidad Nacional de San Luis; Instituto de (D2DR) binding site. PESs not only allow us to observe all the critical points of the
Matemáticas, San Luis, Argentina
surface (minimums, maximums, and transition states), but also to note the ease or dif-
3
Laboratorio de Estructura Molecular y
Propiedades, Facultad de Ciencias Exactas y ficulty that each local minima have for their conformational inter-conversions and
Naturales y Agrimensura, Universidad Nacional therefore know the conformational flexibility that these ligands have in their active
del Nordeste, Instituto de Química Básica y
Aplicada, Corrientes, Argentina sites. Taking advantage of possessing this valuable information, we analyze how
accurate a standard docking study is in these cases. Our results indicate that although
Correspondence
Rodrigo D. Tosso and Ricardo D. Enriz, we have to be careful in how to carry out this type of study and to consider per-
Facultad de Química, Bioquímica y Farmacia, forming some extra-simulations, docking calculations can be satisfactory. In order to
Universidad Nacional de San Luis, Instituto

gicas
Multidisciplinario de Investigaciones Biolo analyze in detail the different molecular interactions that are stabilizing the different
(IMIBIO-SL), CONICET. Ejército de los Andes ligand-receptor (L-R) complexes, we carried out quantum theory of atoms in mole-
950, 5700 San Luis, Argentina.
Email: rodri.tosso@gmail.com and cules (QTAIM) computations and NMR shielding calculations. Although some of
danielenriz@gmail.com. these techniques are a bit tedious and require more computational time, our results

Funding information
Consejo Nacional de Investigaciones
Científicas y Técnicas; Universidad Nacional de
San Luis, Grant/Award Number: PROICO:
02-1418
demonstrate the importance of performing computational simulations using different
types of combined techniques (docking/MD/hybrid QM-MM/QTAIM and NMR
shielding calculations) in order to obtain more accurate results. Our results allow us
to understand in details the molecular interactions stabilizing and destabilizing the
different L-R complexes reported here. Thus, the different activities observed for
dopamine (DA), m-OH-PEA, and PEA can be clearly explained at molecular level.

KEYWORDS
D2 dopamine receptor, phenyletilamine, potential energy surfaces, QTAIM analysis, resonance
assisted hydrogen bond

1 | I N T RO DU CT I O N development of the macromolecular perturbation theory of Bel-

The key-lock concept of Emile Fischer from 1894 to understand the
formation of the ligand-receptor complex (L-R) is a fundamental pillar
of medicinal chemistry. This initial concept was modified and correctly
adapted by Daniel Koshland's theory of induced fit,[1–5] which allowed
us to understand the dynamic nature of this process. The
leau[6,7] and the activation-aggregation theory of Changeux and co-
workers[8] and Karlin[9] has allowed us to understand even better the
complexity of this essential process for medicinal chemistry at molec-
ular level.
In molecular modeling studies, docking methods are widely used
to evaluate the formation of L-R complexes. Currently there are many

J Comput Chem. 2022;1–15. wileyonlinelibrary.com/journal/jcc © 2022 Wiley Periodicals LLC. 1

2 TOSSO ET AL.
options and different programs designed to carry out this type of
study. Among several we can mention Autodock,[10] Pathdock,[11]
[12]
Rosetadock; reference 13 gives an extensive review of current
methods.[13] Although most of these programs give satisfactory
results for this type of study, at least in exploratory and preliminary
stages, some limitations have been reported for this type of
[13,14]
approach. The docking programs present two main limitations:
the conformational space is reduced by imposing limitations to the
system (a rigid receptor and fixed bond angles and lengths in the
ligand) and the simplified scoring function (often based on empirical
free energies of binding) is used to scores poses quickly at each step
of the conformation search. Both of these are limitations, and users
must employ tools such as molecular dynamics or free energy pertur-
bation if a more realistic conformational search or energy prediction is
necessary. These tools are complementary with computational docking
methods, since docking methods generally search a larger conforma-
tional space, but more advanced methods can predict conformation
and energy more accurately within a local area of the conformational
[14]
landscape. However, even taking all possible care, it is clear that
docking is an approximate approach and therefore caution is necessary
and must be taken as exploratory and not a definitive technique.
In general, docking studies of L-R complexes face three different
scenarios: (i) rigid ligands (without rotatable bonds) bound to a narrow
binding site with scarce flexibility, (ii) flexible ligand (possessing sev-
eral rotatable bonds) bound to a narrow binding site and with scarce
flexibility and (iii) flexible ligand bound to a broad and flexible binding
site. Docking calculations have serious limitations to correctly evalu-
ate type (iv) complexes. A study carried out by our research group on
flexible ligands acting as sphingosine kinase 1 (SphK1) inhibitors
showed that not only docking calculations have limitations, but also
molecular dynamics simulations have limitations for evaluating this
type of complexes.[15] Our results show that it is necessary to increase
the number of simulations and perform clustering processes in order
to be able to evaluate this type of L-R complex satisfactorily. In con-
trast, in the case of type (i) L-R complexes, docking calculations com-
bined with relatively simple MD simulations are highly satisfactory
and allow us to obtain good correlations between theoretical results
and activity data obtained from experimental measurements. In fact, if
these simulations are complemented with quantum theory of atoms in
molecules (QTAIM) calculations[16–18] so that to evaluate molecular
interactions more accurately, the correlations are excellent.[19] Exam-
ples for this complex type (i) are alkaloids structurally related to
galantamine acting as acetylcholinesterase (AchE) inhibitors. It should
be noted that very good correlations between theoretical calculations
and experimental data were obtained for these complexes.[19,20] On
the other hand complexes type (ii), which probably are the most com-
mon in medicinal chemistry, present an intermediate situation.
Although docking calculations sometimes provide very relevant infor-
mation, there are some questions that arise: Is there a loss of relevant
information when applying standard docking calculations? How this
missing information affect the correct interpretation of the conforma-
tional behavior of the ligand in its active site? Is it possible to correct
this problem employing more precise methods? Even if it is possible
to correct it, how expensive in terms of calculation time it might be? Is
it possible to carry out this type of more detailed study in a simple
and routinely way? In order to answer all these questions, we have
carried out a comparative study performing standard docking calcula-
tions versus a comprehensive systematic conformational analyses
using conformational potential energy surfaces (PESs) of the ligands.
For this study, it is very important to carefully select the type of
ligands to be studied, as well as the molecular target, particularly tak-
ing into account their structural characteristics. Thus, for this study
we have selected the following compounds: dopamine (DA),
phenyletilamine (PEA), and meta-tyramine (m-OH-PEA) (Figure 1).
There are numerous advantages to select such compounds: (a) they
are compounds of great interest in medicinal chemistry, (b) reliable struc-
tural data are available for the molecular target (D2DR),[21] (c) data on
biological activities are available for these ligands acting on D2DR,[22]
(d) we have previously studied DA interacting in the active site of
D2DR;[23] in addition, we have reported new ligands for this receptor,
some of them very powerful and selective with respect to D1DR.[24–26]
In other words, we have experience in this molecular target (e) the struc-
tural characteristic of these ligands allows to perform a systematic con-
formational study since their conformations are mainly determined by
two dihedral angles (ϕ and ψ in Figure 1), (f) from a structural point of
view they represent adequately ligands of complexes type (ii) since they
are flexible but at the same time they have only three dihedral angles,
being two of them the determinants of the spatial ordering, (g) structural
differences between the ligands are convenient, in fact they are phenyl-
ethylamines with different hydroxylation patterns; note that PEA does
not have hydroxyl groups in its structure, dopamine has two of them
from its catechol ring, while m-OH-PEA has only one phenolic hydroxyl
in meta position. The advantage of using structurally closely related com-
pounds is that the effects of small structural changes on the properties
of the compounds may then be determined. The importance of hydroxyl
groups for agonist binding and receptor activation has been previously
investigated.[27–35] These studies showed that the number and place-
ment of hydroxyl groups on the agonist can be an important determinant
of agonist action. Small changes in agonist structure may affect the pre-
cise interactions with the receptor and hence agonist binding and recep-
tor activation. The question that arises is: are the standard docking
F I G U R E 1 Schematic structure of molecular systems analyzed in
this study: PEA (R1 and R2 = H), m-OH-PEA (R1 = OH, R2 = H), and
DA (R1 and R2 = OH). The main torsional angles (ϕ and ψ) are shown
in this figure.
TOSSO ET AL.
calculations capable of showing these differences? As mentioned above
one of the objectives of this work is to carry out a comparative study
between performing standard docking calculations versus a study using
a systematic conformational analysis of the ligands analyzing their
respective conformational PES. However, this is not the only objective
of this work, another important goal of this study is to analyze in detail
the different molecular interactions that stabilize and destabilize the
complexes of these ligands. It is clear that this information allows us to
have a better understanding of the conformational behavior of these
ligands in the active site of the receptor and from this information be in
a position to explain the different agonist activity reported for these
compounds. In order to analyze molecular interactions as accurately as
possible we have used two specific techniques to evaluate the strengths
of such interactions: calculations using the QTAIM[16–18] and Nuclear
[36]
Magnetic Shielding Constants. We have used both techniques in
studies of different biological systems[37–39] and such studies have dem-
onstrated that these methods are sufficiently accurate to understand the
different stereo-electronic intricacies involved in the formation of the
different molecular complexes.
2 | COMPUTATIONAL DETAILS
2.1 | Docking calculations
The receptor structure corresponds to the following PDB code:
[21]
6CM4. Risperidone was removed and missing residues Met140,
Leu141, Tyr142, Asn143, Lys362, Leu363, and Cys443 were incorpo-
rated in the structure with Modeller 9.20.[40] The docking studies
were carried out using AutoDock 4.2.6 and all graphic manipulations
[10]
and visualizations were performed with AutoDock Tools 1.5.6.1.
The grid dimensions were 40, 40, and 40 for the X, Y, and Z-axes,
respectively, in the active site region with a resolution of 0.375 Å, and
some residues (Asp114, Val115, Cys118, Ser193, Ser194, Ser197,
Phe389, and Phe390) were considered flexible. Nonpolar hydrogen
atoms were merged, and Gasteiger charges were assigned for the
compounds. All torsions of the ligands and flexible residues were
allowed to rotate during docking. The number of poses collected was
200, whereas the maximum number of generations and energy evalu-
ation was set to 27,000 and 5
107, respectively. Finally, docked
conformations were clustered using a tolerance of 2 Å root-mean-
square deviations (RMSD).
2.2 | Molecular dynamics simulations
The three lowest energy conformations of the complex PEA/D2DR of
the most populated cluster obtained from docking calculations were
chosen to carry out the MD simulations. In the case of the complex
m-OH-PEA/D2DR, the MD simulations were performed with the low-
est energy conformation of each one of the three most populated
clusters. The calculations were carried out using periodic boundary
conditions and octahedral simulation cells with explicit water
3
employing the TIP3P model.[41] About 25,000 water molecules were
added to the box and, in order to neutralize the system, 19 chloride
ions were included. The all-atom force field ff99SB[42] and the general
Amber force field[43] were used to describe the receptor and the
ligands, respectively. The force field parameters of PEA and m-OH-
PEA were produced by the antechamber program. The particle mesh
Ewald method[44] was applied using a grid spacing of 1.2 Å, a spline
interpolation order of four, and a real space direct sum cutoff of 10 Å.
The SHAKE algorithm[45] was applied to allow for an integration time
step of 2 fs. During simulations all alpha carbons from the protein
backbone were kept almost fixed with a harmonic force constant of
100 kcal/mol Å2, whereas the temperature was 310 K. Three MD
simulations of 30 ns were performed for each starting conformation
obtained from docking calculations of each L-R complex, under differ-
ent starting velocity distribution functions. The NPT ensemble was
employed using Berendsen coupling to a baro/thermostat (target
pressure 1 atm, relaxation time 0.1 ps). The total energy of the system,
the RMSD of the alpha carbons of D2DR, and the RMSD of ligands
were calculated in order to evaluate the stability of the complexes.
The MD simulations were performed using the AMBER16 software
package,[46] while post MD analysis was carried out with program
CPPTRAJ.[47] In order to evaluate the stability of the complex during
the 30 ns of simulation, the total energy of the system (Figure S1), the
RMSD of the alpha carbons of D2DR (Figure S2a), and the RMSD of
each ligand (Figure S2b) were calculated.
Considering the main goal of our study, it could be advisable to
include entropy in QM calculations. In particular, in those cases of
structurally complex systems where active sites are large and have
some flexibility. These calculations are also important for ligands
which by themselves are quite flexible and can be moved sufficiently
before to fit into the binding pocket. A great structural variability of
the ligands (different structural scaffolds) is another situation in which
inclusion of entropy in QM calculations might be important. D2DR
could be considered as a simple system from a structural point of
view. The active site of D2DR is relatively small and tight and there-
fore it has a limited flexibility. Regarding the selected ligands, their
structural variability is scarce and their molecular flexibility is limited.
Thus, taking into account these considerations and the expensive
computational cost of including entropy to obtain the PESs, we think
that is not essential to incorporate entropy in this case. Anyway, the
entropy calculation was done in MD simulations. The entropic contri-
bution to the binding free energy (TΔS) was included by estimating
the interaction entropy.[48–50] This approach was used as in our previ-
ous paper.[51]
2.3 | QM and ONIOM QM/MM calculations
QM and QM/MM calculations were performed using the Gaussian16
software,[52] and the surfaces were created using the Matplotlib
python library.[53] The relaxed PES scans (with geometry optimization
at each point) were obtained rotating the dihedral angles ϕ and ψ of
each ligand 360 degrees with a step size of 15.
4 TOSSO ET AL.
2.4 | Potential energy surfaces
An average conformation from the last 10 ns of the MD simulations
were considered as the starting structures to get the surface. To carry
out these calculations, a reduced model was built considering all the res-
idues located to a distance of 10 Å from the ligand. Calculations were
[54]
performed using a two layered ONIOM method, where the ligand
was considered at a high-level of calculation (B3LYP/6-31G*)[55–59]
while the residues of the receptor were treated at a lower level of calcu-
lation by molecular mechanics using the Amber force field.[43] The back-
bone atoms of D2DR were kept fixed.
2.5 | Full optimizations
The most representative points of the PES of each complex were con-
sidered to be fully optimized (including the dihedral angles ϕ and ψ)
with the exception of the backbone atoms of D2DR, which were kept
fixed. An ONIOM calculation was performed considering three
regions in the system. The ligand was included in the high-level
(HL) layer and it was calculated with a B3LYP/6-31G* level of
theory.[55–59] The side-chains of the residues of the binding site
(Asp114, Val115, Cys118, Ser193, Ser194, Ser197, Phe389, and
Phe390) were considered in the mid-level (ML) layer, which was calcu-
lated at a semiempirical level (PM6).[60] Finally, the rest of the residues
were incorporated in the low-level (LL) layer and considered with
[61]
molecular mechanics using the Amber force field. H atoms were
used as link atoms to complete the valence of the bonds crossing
between the layers of the ONIOM QM/MM scheme.
2.6 | QTAIM: Topological analysis of the electron
density distribution
We only present here the essential information needed to understand
the molecular graphs (networks of the bond path in the density topol-
ogy) of L-R complexes, because the use of topological concepts in the
description of intra/intermolecular interactions is well documented in
the standard literature.[62]
The electron charge density, ρ(r), is a physi-
cal quantity that has a definite value at each point in space. QTAIM
analysis is based on the critical points (CPs) of this electronic density
distribution. The topological properties of a scalar field, such as
ρ(r) might be summarized in terms of their CPs, that is, the points rc
where r ρ(r) = 0. CPs are classified according to their type (ω, σ) by
stating their rank (ω) and signature (σ). The rank is equal to the number
of nonzero eigenvalues of the Hessian matrix of ρ(r) at rc, while the
signature is the algebraic sum of the signs of the eigenvalues of this
matrix.
CPs of the (3, 1) and (3, +1) types describe saddle points, while
the (3, 3) is a maximum, and (3, +3) is a minimum in the field. Among
these CPs, the (3, 1) or bond critical points (BCP) are the most rele-
vant ones since they are found between any two atoms linked by a
chemical bond. Therefore, the determination of BCP allows us to find
bond paths connecting these points with bonded nuclei. The opti-
mized geometries obtained from the PES of PEA/D2DR and m-OH-
PEA/D2DR were used to set up reduced 3D model systems of ligands
in complex with D2DR. These models were constructed from the
selected structures by keeping only the receptor residues that interact
directly with the ligands. In each case, the following 24 residues were
considered: Val87, Val91, Phe110, Val111, Asp114, Val115, Cys118,
Thr119, Ile184, Phe189, Ser193, Ser194, Ser197, Phe198, Trp386,
Phe389, Phe390, His393, Tyr408, Phe411, Thr412, Trp413, Gly415,
and Tyr416.
The charge density of reduced model systems was then com-
puted by DFT methodology with the PBE hybrid functional and
6-31G* as a basis set, as implemented in the Gaussian16 package.[52]
The topological analysis of charge density was performed in the con-
text of the QTAIM[63] with the help of Multiwfn software.[64] This
type of calculations has been used in recent works since it ensures a
reasonable compromise between the wave function quality required
to obtain reliable values of ρ(r) and the computer power available,
considering the extension of the system in study.[15,65–68]
2.7 | NMR nuclear magnetic shielding constants
Both theoretical calculations and experimental analysis of NMR spec-
troscopic parameters have demonstrated to be one of the main tech-
niques for the determination of molecular structure, conformation
and to describe intermolecular interactions[69–83] Nuclear magnetic
shielding constant is one of the main NMR parameters, since it
depends on the chemical environment of individual nuclei and there-
fore determines the position of the signal in the spectrum.
Theoretical aspects of NMR nuclear magnetic shielding and of dif-
ferent computational methods for calculating it have been described
extensively in the literature.[36] However, one should mention that
according to nonrelativistic Ramsey's theory the nuclear magnetic
shielding is given as the sum of a diamagnetic and a paramagnetic
term which do not depend on the spin of electrons. The diamagnetic
contribution is associated to spherical charge distributions, while the
paramagnetic term results from non-spherical charge distributions of
p or electrons of higher angular momentum.
Molecular interactions of the ligand with residues belonging to
the active site of it receptor were studied by analyzing the variation
of the NMR parameters of the ligand nuclei by using reduced models
from the optimized L-R geometries, as was done in reference 23.
Thus, for a nucleus of interest of the ligand, the Δσ was obtained as
the difference between the σ of the nucleus in the reduced model and
the σ of the atom in the isolated ligand. In order to describe HB-type
interactions between the ligand and the D2DR, Δσs of the nuclei of
the residues involved in the HBs were additionally computed as the
difference between the σ of the nucleus in the reduced system and σ
of the atom at the isolated residue.
In order to perform the NMR analysis, the following residues
were included in the reduced system: Asp114, Val115, Cys118,
Ser193, Ser194, Ser197, Phe198, Trp386, Phe389, Phe390, Hie393,
TOSSO ET AL.
Thr412, Tyr416. Selected residues fulfill the condition that some of
their atoms are located at a distance less than 5 Å from the ligand,
since the residues placed at a distance larger than that mentioned do
not yield significant variations in the nuclear magnetic shielding of the
ligand nuclei.
NMR nuclear magnetic shielding constants were calculated at the
DFT level using the B3LYP exchange-correlation functional,[55,56]
employing the gauge independent atomic orbitals (GIAOs) method[84–86]
and the cc-pVTZ. basis set.[87,88] All NMR calculations were performed
using the Gaussian16 program package.[52]
3 | RESULTS AND DISCUSSION
3.1 | Conformational study using PES
One of the best ways to perform a systematic conformational study is
analyzing the PES. Previously, we have used this type of study not
[89–91]
only in amino acids or small peptides, but recently we used it to
study the conformational behavior of DA in the active site of
D2DR.[23] Information obtained in this recent article will be very use-
ful to compare with the data reported in the present work. Thus, the
first step in this work was to perform the conformational study of m-
OH-PEA and PEA by using PES of these ligands at the D2DR binding
pocket.
It is important to keep in mind that D2DR has not been
crystalized with m-OH-PEA or PEA; therefore, with the purpose to
carry out QM/MM calculations in the first stage, we performed dock-
ing calculations, we chose three structures and then we performed
MD simulations. We took the last 10 ns from each simulation and
chose an “average structure”; next we compared the torsional angles
ϕ and ψ obtained for the ligands and due that their values are similar
we chose one of them as starting structure for the L-R complex. Once
F I G U R E 2 3D PES (A) and 2D PES (B) obtained for the complex m-OH-PEA/D2DR. Letters (A-D) denotes the minima in the PES, whereas
the numbers (1–3) indicate the minima obtained from docking calculations. The conformations 1, 2, and 3 correspond to the clusters I, II, and III,
respectively. Coloring represents energy, with red color indicating high values and deep blue indicating the lowest energy; green and yellow
colors indicate intermediate values. Color-code is shown at the right.
5
obtained these initial structures, QM/MM calculations were made to
get the PES (see calculation methods). This study was carried out in
two stages. First, the PES was obtained keeping fixed only two tor-
sional angles (ϕ and ψ angles in Figure 1). In fact, both angles were
rotated every 15 in the scan. In a second step, the main conforma-
tions obtained in the PES were fully optimized (including ϕ and ψ
angles) (see more details at calculation methods). Figure 2 shows the
3D and 2D PES obtained for m-OH-PEA located at the active site of
D2DR. In this way, the relative energy considers not only the confor-
mational energy of m-OH-PEA but also the interactions between the
ligand and the receptor.
If we compare the surfaces obtained for m-OH-PEA with that
surface previously reported for DA,[23] it is evident that this surface is
less intricate than that of DA. In this case, more valley areas with flat
areas are observed in which the different conformations can be inter-
converted more easily and the peaks of energy maximum areas are
clearly lower than in DA. This result is reasonable taking into account
that DA possesses two OHs in the catechol ring, which allows a
greater number of interactions, especially in the area of the serine
cluster, and this makes DA mobility within the binding pocket signifi-
cantly more restricted than that observed for m-OH-PEA. Another
important difference observed is the energies of the transition states
required for the conformational interconversions; while for m-OH-
PEA they are in the order of 4.5 kcal/mol (Table 1), in the case of DA
the energy requirement in some cases were more than 10 kcal/mol
(more than double). This clearly shows that the conformational flexi-
bility that m-OH-PEA has in the active site of D2DR is markedly
greater than that of DA.
In Figure 2A, the four most significant local minima observed in
this 3D surface have been marked (A-D). The conformational energies
obtained for the four minima are shown in Table 1, and the location of
these minima within the surface can be better appreciated in the 2D
image (Figure 2B). In our previous paper, we demonstrated the
6 TOSSO ET AL.

importance of performing the complete optimization of the complexes
in order to obtain more accurate energies; therefore, our next step
was to optimize the four complexes including the torsional angles ϕ
and ψ (Table 1, right side).
When we perform the complete optimization of these four min-
ima (Table 1, right side), it is clear that A and B conformations con-
verge to the same minimum (the global one). This is not a quirky
result; in fact, conformations A and B shown in the PES as slightly dif-
ferent spatial orderings can be attributed to an artifice because angles
ϕ and ψ were kept fixed, but when they were relaxed both con-
formers converge to the same point of the surface. Another interest-
ing data to remark is that the energy gaps among the different
conformations are extremely low. This added to the also very low
energy values required for the conformational interconversions clearly
indicate the high conformational flexibility that m-OH-PEA possesses
in the binding pocket of D2DR. It is interesting to note that all the
energetically preferred conformations obtained for m-OH-PEA have a
spatial arrangement very similar to that of the I conformation reported
[23]
for DA as the possible biologically relevant conformation.
them present a partially extended spatial arrangement (one angle in
trans position and the other in gauche). The significant difference
between m-OH-PEA and DA is that in the case of the endogenous
ligand, the global minimum is located in a well of relatively deep
potential and to be able to pass from this conformation to another,
more than 10 kcal/mol are required. In contrast, in the case of m-OH-
PEA the global minimum is located in a fairly flat area and only needs
4 kcal/mol to change to the other two conformers which are practi-
cally isoenergetic. It is clear that m-OH-PEA has a marked greater
conformational flexibility than DA in the binding pocket. This can be
attributed to the fact that DA establishes much stronger H-bond
interactions with the serine cluster, making the mobility of the ligand
in its active site much more difficult. These interactions are discussed
in detail in the next section.
Figure 3 shows the 3D and 2D PES obtained for PEA located at
the active site of D2DR.
As we expected the surface obtained for PEA is also less intricate
than that reported for DA, showing more valley areas with flat areas
in which the different conformations might be interconverted more
easily and the areas of maximum energy are also clearly lower than in
DA. In fact, we expected that the PES of PEA might be even more flat
than that obtained for m-OH-PEA. However, our results show that
All of this is true in a great part of the surface, but not in the entire surface.
The surface obtained for PEA shows clearly fewer areas with peaks
and maximums in general have less energy than that observed in the
PES of m-OH-PEA (compare Figures 2 and 3). However, the particular
area where the global minimum and the local minima are located for

TABLE 1 Different conformations obtained for m-OH-PEA/D2DR complexes.

Data obtained from points of PESa (angles φ and ψ were restricted) Data obtained after full optimization of points of PESa

Conformation ϕ ( ) ψ ( ) Relative energy (kcal/mol) ϕ( ) ψ ( ) Relative energy (kcal/mol)

A 172.71 85.99 0.00 179.88 97.17 0.00
B 172.71 100.99 0.40 179.9 97.16 0.00
C 172.29 124.01 1.81 179.94 128.79 0.00
D 172.29 49.01 3.07 179.89 55.00 0.01
a
The PES was calculated considering the reduced model.

F I G U R E 3 3D PES (A) and 2D PES (B) obtained for the complex PEA/D2DR. Letters (A-F) denote the minima in the PES, whereas the
numbers (1–3) indicate the minima obtained from docking calculations. Coloring represents energy, with red color indicating high values and deep
blue indicating the lowest energy; green and yellow colors indicate intermediate values. Color-code is shown at the right.
TOSSO ET AL. 7

PEA is a slightly more intricate than the m-OH-PEA equivalent zone.
This can be observed by comparing the energy gaps obtained
between the different minimums, as well as the energy requirement
of the transition states for the conformational interconversions
(Table 2).
The six most significant local minima observed in this 3D sur-
face have been marked as A-F in Figure 3A. The conformational
energies obtained for these six minima are shown in Table 1, and
once again the location of these minima within the surface can be
better appreciated in the 2D image (Figure 3B). In the same way as
that observed in m-OH-PEA, conformers A and B converge in the
same potential well (same critical point) when the two torsional
angles ϕ and ψ are optimized. In this way, five conformers are the
spatial arrangements that PEA might adopt in the D2DR binding
pocket. A significant difference between the results obtained for
PEA with respect to DA and m-OH-PEA is that its global minimum
is a conformation with a fairly folded spatial arrangement (both
dihedrals in gauche position). In addition to the A conformation, the
D conformant also adopts this type of spatial ordering. In any case,
PEA can also adopt conformations similar to those obtained for DA
and m-OH-PEA (partially extended forms as C, E, and F con-
formers). On the other hand, our simulations suggest that the con-
formational flexibility that PEA possesses in the binding pocket is
markedly greater than that of DA, but slightly less than that of m-
OH-PEA. Once again, the fact that PEA can easily establish both
folded and extended conformations can be attributed to the fact
that it does not have OH groups in its ring and therefore cannot
establish hydrogen bonds with the cluster of serines; this allows
that the ligand adopts these different types of spatial ordering
without significant energetic requirements. A detailed description of
the interactions stabilizing the different complexes is given in the
following section.
Possessing the complete surface allows us to carry out a full
exploratory study for all possible conformations of the different
ligands. Thus, taking advantage of such information our next step
was to analyze how accurate a standard docking study is in these
cases. The docking study was carried out using a standard tech-
nique; however, some extra cares were taken into account. For
example, the number of conformations used was extensive (200)
and some amino acids at the binding site were considered flexible
in such a way as to facilitate this study trying to obtain the best
possible results (see method section). The results obtained for m-
OH-PEA show that practically all the points can be grouped in three
families (families I-III in Figure S3). If we consider only one structure
(the energetically preferred one), this minimum is located in a zone
relatively near to the minima of the PES (see structure 1 in
Figure 2B). However, the torsional angles of this structure
suggested by docking has not exactly the same values that those
obtained in the PES (compare torsional angles of structure 1 in
Table 3 with those of conformers A and B in Table 1). If we con-
sider the energetically preferred structure for each family (structures
1, 2, and 3 in Figure 2B), the location of these minima does seem
to cover the area of the minima obtained on the surface quite well.
Next, we refined these three structures obtained from docking cal-
culation by using MD calculations. Structures 1 and 2 simulated by
MD calculations give conformations possessing torsional angles very
similar to that of the global minimum obtained in the PES (con-
formers A and B) (Tables 1 and 3).

TABLE 2 Different conformations obtained for the PEA/D2DR complexes.

Data obtained from points of PESa (angles φ and ψ were restricted) Data obtained after full optimization of points of PESa

Conformation ϕ( ) ψ ( ) Relative energy (kcal/mol) ϕ ( ) ψ ( ) Relative energy (kcal/mol)

A 71.03 94.35 0.00 72.48 91.54 1.07
B 71.03 55.65 1.91 72.95 85.79 0.00
C 146.03 79.35 2.50 152.63 69.98 3.25
D 71.03 79.35 3.67 66.87 85.24 3.74
E 161.03 55.65 5.12 161.36 63.99 5.61
F 71.03 4.35 5.15 68.16 2.17 5.95
a
The PES was calculated considering the reduced model.

TABLE 3 Torsional angles of the preferred forms of m-OH-PEA and PEA obtained from docking analysis and after MD simulations.

m-OH-PEA PEA

Ligand angles after docking Ligand angles after MD Ligand angles after docking Ligand angles after MD
calculations simulations calculations simulations

Conformation ϕ ( ) ψ ( ) ϕ( ) ψ ( ) ϕ ( ) ψ ( ) ϕ( ) ψ ( )

1 80.34 104.44 164.39 88.36 114.51 48.01 141.7 86.88
2 98.16 43.92 178.28 93.51 100.12 68.8 173.37 93.71
3 106.16 107.81 69.12 93.31 132.16 40.22 157.89 89.51
8 TOSSO ET AL.

In contrast when we perform MD simulations starting from struc-

ture 3, we obtain a more folded spatial ordering that does not corre-
spond to any minima at the PES. It should be noted that structure
3 corresponds to family III, which is not the most populated nor ener-
getically preferred family. On the other hand, if we are interested only
in the global minimum, it is not necessary to optimize three candidate
structures since only optimizing any structure from families I or II, we
can obtain a structure very similar to that of the global minimum. Our
results indicate that for a relatively simple surface such as m-OH-PEA,
the docking calculations are actually an efficient approach. The only
danger would be to consider as the biologically relevant conformation
only the minimum energy structure obtained in the docking study
(without further refinement using MD calculations), since it has a spa-
tial arrangement somewhat different from that of the true global F I G U R E 4 Charge density molecular graph of m-OH-PEA
minimum. conformer A at the D2DR binding pocket. Small red spheres are the
bond critical points (BCPs) and yellow lines connecting BCPs to the
Results from docking are even better in the case of PEA; for this
neighboring interacting atoms are the bond paths (BPs).
molecule the docking calculations grouped practically all the most
populated forms in just one family (Figure S4). For PEA, we also simu-
lated the three energetically preferred structures obtained from dock-
ing by using MD calculations. These structures are located in the PES
with numbers 1–3 (Figure 3B). In this case, if a simple optimization is
carried out by means of MD calculations; all the conformations
obtained are very similar to those obtained as global minimum in the
PES (Tables 2 and 3). These results indicate that for structurally rela-
tively simple systems like studied here, docking analysis is an ade-
quate tool, but to perform MD simulations could help to obtain even
more accurate results.

3.2 | Molecular interactions stabilizing the

different complexes with D2DR

F I G U R E 5 Sum of charge density values at the bond critical

3.2.1 | Charge density analysis on relevant
conformations of the ligands
Figure 4 shows the charge density molecular graph of the most stable
conformation of m-OH-PEA (conformer A) at the D2DR binding
pocket. Other relevant conformers are shown in Figures S5 and S6.
m-OH-PEA adopts a similar binding mode and establishes nearly the
same intermolecular interactions in all conformations. It is anchored
to receptor binding pocket mainly by two strong N–H•••O hydrogen
bonds with TM3 residue Asp114, as evidenced by the bond paths
connecting ammonium protons of the ligand to carboxylate oxygen
atoms of Asp114. At the other end of the ligand molecule, m-OH
group acts simultaneously as H-bond donor to the backbone carbonyl
oxygen atom of TM5 residue Ser197 and as H-bond acceptor of the
hydroxyl H atom of the same residue. m-O atom is also engaged in
other interactions with the backbone of Val115 and with residue
Thr119. Ligand ring atoms, on the other hand, establish hydrophobic
interactions with residues Cys118, Phe198, Phe390, and Trp386.
Due to the high flexibility of phenylethylamine moiety, the m-OH
group, which is oriented towards the extracellular side of the receptor
(m-OH up) in the analyzed conformations of m-OH-PEA, could be
points (BCPs) of L-R interactions. Contributions of each part of the
ligand molecule are depicted in different colors. For m-OH-PEA and
PEA, the averaged charge density contributions among all analyzed
conformers are depicted.
equally pointing towards the intracellular side of the transmembrane
bundle (m-OH down). If we compare with D2DR agonist apomor-
phine, which has a rigid structure, with less degrees of freedom for
positioning the OH group within the receptor binding pocket
(Figure S7), we can see that m-OH in apomorphine is also in the up
conformation, thus supporting our calculations that indicate that this
is the most stable orientation.
Bar plot in Figure 5 shows the contribution of the different parts
of the ligands to the anchoring into the D2DR binding pocket. The
contributions were calculated by summing up the charge density
values at the BCPs of all intermolecular interactions, except those
involving two hydrogen atoms H•••H since they are suggestive of
steric clashes and therefore, they do not contribute to ligand anchor-
ing. Since PEA and m-OH-PEA do not experience significant changes
among the minima in their respective PES, the averaged contributions
among all conformations are depicted for those two ligands.
TOSSO ET AL.
F I G U R E 6 Surface representation of the ligand orthosteric site
(cyan) and deep hydrophobic subpocket (red) in the D2DR receptor.
m-OH-PEA phenolic ring is overlapping the deep hydrophobic
subpocket surface.
Undoubtedly, protonated amine group makes the greatest contri-
bution to ligand anchoring in all complexes. Moreover, as evidenced
by the total height of the stacked bars, the ligand binding strength
decreases as the number of OH groups attached to the phenylethyl-
amine moiety decreases, from DA to m-OH-PEA to PEA.
Unlike m-OH-PEA and PEA, DA experiences significant confor-
mational changes among the stable minima in the PES.[23] Bar plot in
Figure 5 shows the charge density contributions for two relevant con-
formers of DA, F, and I. In conformer I, DA binds at the receptor
orthosteric site and interacts with the serine residues from TM5 seg-
ment. In conformer F, the catecholic ring is anchored in a deep hydro-
phobic subpocket that is located below the orthosteric site, towards
the intracellular side of TM segments. Both sites are depicted in
Figure 6.
As judged by the total charge density sum for those conformers,
the anchoring of DA into the deep hydrophobic subpocket seems to
be comparable in strength to the anchoring of DA into the orthosteric
site. While the catecholic OH atoms are better positioned for inter-
acting with the cluster of serine residues in conformer I, as indicated
by the greater contribution of the m-OH and p-OH interactions, they
are compensated by the additional hydrophobic interactions that the
ligand phenyl ring can establish within the deep hydrophobic sub-
pocket (Phe contribution, Figure 6).
On the other hand, considering the two binding modes described
for DA, m-OH-PEA adopts a conformation more similar to conformer
F of DA in which ligand aromatic ring is directed towards the deep
hydrophobic subpocket at the bottom of the receptor extracellular
cavity (Figure 6). This finding makes sense considering that m-OH-
PEA has only one OH group to interact with the serine residues at the
orthosteric site, hence the tendency of m-OH-PEA for anchoring into
the deep hydrophobic subpocket would be even greater than for DA.
At first glance, these results might seem somewhat counterintui-
tive because DA and m-OH-PEA are agonists of D2DR and yet they
seem to prefer a conformation that is more typical for antagonists,
especially in the case of m-OH-PEA. Furthermore, it is well known
9
F I G U R E 7 Charge density molecular graph of PEA conformer A
at the D2DR binding pocket. Small red spheres are the bond critical
points (BCPs) and yellow lines connecting BCPs to the neighboring
interacting atoms are the bond paths (BPs)
that ligand anchoring at the deep hydrophobic subpocket stabilizes
the D2DR receptor in an inactive state.[21] However, if we consider an
induced fit mechanism for receptor activation, agonists might first
bind to the deep hydrophobic subpocket and from there they could
trigger the molecular switches that promote receptor activation, and
the ligand translocation to the orthosteric site.[92]
Regarding PEA ligand, Figure 7 shows the charge density molecu-
lar graph of the most stable conformation of PEA at the D2DR binding
site. Figures S8 and S9 show the molecular graphs for other stable
minima on the PES of PEA.
Similar to other aminergic ligands, PEA protonated amine also
forms the characteristic double H-bond with Asp114, which is consid-
ered as the driving force for ligand anchoring into D2DR binding
pocket. Also, a strong intramolecular N–H•••π H-bond between the
protonated amine group and C2 of the phenyl ring stabilizes a quite
folded conformation of PEA. This folded conformation, mainly due to
the gauche torsion angle φ, determines a different binding mode of
PEA with respect to their hydroxylated counterparts m-OH-PEA and
DA, both having more extended conformations. In all the analyzed
conformers, PEA is folded towards the upper part of the binding
pocket, with the phenyl ring atoms interacting with the cluster of ser-
ine residues at TM5, mainly Ser193, as well as other residues from the
outermost part of the TM bundle like Phe189, His393, and Tyr408.
As PEA only fills the uppermost part of the orthosteric site, the
residue side chains get somewhat reconfigured so as to fill the space
left unoccupied by the ligand. In particular, the side chain of TM5 resi-
due Phe198 is protruding into the orthosteric site, as can be seen
more clearly in Figure S9. This is an important conformational change
when compared with complexes of the hydroxylated ligands in which
Phe198 is in the interface region between TM5 and TM6 transmem-
brane segments.
Finally, another distinctive characteristic of some PEA conformers
are the face-to-face stacking interactions of the ligand with Phe389,
as evidenced by the bond paths directly connecting the carbon atoms
10 TOSSO ET AL.

of both rings (Figure 7 and Figure S8). This type of ring interactions is
more rarely observed among the hydroxylated counterparts of PEA,
possibly due to the higher electron density of the catecholic/phenolic
ring that would repel with the electron density from another facing
ring.[93]
In summary, the three ligands analyzed so far form the character-
istic salt bridge with residue Asp114 that drives the ligand binding into
the receptor binding pocket. However, apart from the driving interac-
tion they show quite different behaviors and binding modes. DA can
adopt two distinct binding modes, either it binds at the orthosteric
site where it can strongly interact with the serine cluster or at the
deep subpocket where it mainly interacts with hydrophobic residues.
Both binding modes have comparable stabilities, as judged by the
charge density analysis. However, when the p-OH is removed from
DA, the equilibrium between those two opposing interaction forces
gets broken and the ligand is driven towards the deep hydrophobic
subpocket, the only binding site observed for m-OH-PEA. On the con-
trary, the non-hydroxylated analog PEA seems to be unable to fully
penetrate into the receptor binding pocket since in all the conformers
it is anchored at the top of the orthosteric site.

3.2.2 | NMR nuclear magnetic shielding calculations

NMR nuclear magnetic shieldings strongly depend on the electronic

cloud closest to the nucleus though it will also be affected by electron
clouds surrounding the molecule of the shielded nucleus. Variations of
this property will provide useful information on subtle changes in the
electron density closest to the nucleus of interest. This makes nuclear
magnetic shielding constants an excellent tool for investigating inter-
molecular interactions. We have recently used this approach success-
fully to determine the biologically relevant conformation of DA.[23]
Discussions performed in the previous sections suggest that the
hydroxylation pattern of phenylethylamines affects the interactions of
the ligands with the receptor active site. To better understand the
effect of hydroxyl substituents in the aromatic ring on such molecular
interactions, we conducted a comparative study of the modification
of the nuclear magnetic shielding of DA, m-OHM-PEA and PEA nuclei
when they are complexing with D2DR. This task was carried out by
using reduced models of the optimized L-R geometries, which for sim-
F I G U R E 8 Variations of nuclear magnetic shieldings (Δσ) for
plicity are called L-Asp114-ResS, where L represents the ligand and
non-hydrogen nuclei of conformer I of DA, conformer A of
Asp114-ResS the set of residues selected from the active site, namely, m-OH-PEA and conformer A of PEA in reduced systems. (A) L-asp
Asp114- Val115- Cys118- Ser193- Ser194- Ser197- Phe198- Trp386- (L represents the ligand), (B) L-ResS (L-Val115-Cys118-Ser193-
Phe389- Phe390- Hie393-Thr412-Tyr416. Receptor residues interac- Ser194-Ser197-Phe198-Trp386-Phe389-Phe390-Hie393-Thr412-
tions with the ligand cause nuclear magnetic shieldings variations of its Tyr416) and (C) L-asp-ResS, calculated at the GIAO/B3LYP/cc-pVTZ
level of approximation.
atoms, Δσ. The larger the interaction of the residues with a given ligand
nucleus, the larger the Δσ of the nucleus.
Our main NMR results obtained for the global minimum of DA of this residue is not exactly the same on nuclei of the three ligands
(conformer I), m-OH-PEA (conformer A), and PEA (conformer A) studied. This can be seen in Figures 8A and 9A, where the results
are shown in Figures 8 and 9. Further details are provided in corresponding to the reduced systems formed by each ligand and
Tables S1–S3. Our calculations for the three selected ligand structures Asp114, L-Asp114, are shown. Thus, results obtained for m-OH-PEA
show that the main variations in the shielding of most of their nuclei show that its interaction with Asp114 decreases the shielding of two
are produced by their interaction with Asp114. Although the impact hydrogen atoms belonging to the amino group as well as the σ(N),
TOSSO ET AL.
F I G U R E 9 Variations of nuclear magnetic shieldings (Δσ) for
hydrogen nuclei of conformer I of DA, conformer A of m-OH-PEA and
conformer A of PEA in reduced systems. (A) L-asp (L represents the
ligand), (B) L-ResS (L-Val115-Cys118-Ser193-Ser194-Ser197-
Phe198-Trp386-Phe389-Phe390-Hie393-Thr412-Tyr416) and
(C) L-asp-ResS, calculated at the GIAO/B3LYP/cc-pVTZ level of
approximation.
indicating formation of a double hydrogen bridge. In the case of DA,
the deshielding suffered by the N atom is twice that observed for the
same atom included in m-OH-PEA. Furthermore, unlike m-OH-PEA,
where the negative Δσ of hydrogen atoms are 7.9 and – 6.1 ppm, in
this case, the largest variation is more than three times than the minor
11
(3.5 times). This implies that the two HBs resulting from DA-Asp114
interaction are more asymmetric than those observed in the m-OH-
PEA-Asp system.
Regarding PEA/D2DR complex, the deshielding suffered by the N
atom is less than that of DA and larger than that of m-OH-PEA, while
the deshielding of two of the hydrogen atoms of the NH3 + group
show a similar pattern to DA. Moreover, in the case of PEA, a
decrease in σ (H2) and σ (C2) is also observed, which means that one
of the carboxylic oxygens of Asp114 in addition to being involved in a
hydrogen bond with the NH3 group also forms a hydrogen bond type
interaction with a CH group belonging to the aromatic ring. This is
possible due to the fairly folded arrangement adopting by PEA, unlike
DA and m-OH-PEA.
Besides to shielding variations of atoms directly involved in
hydrogen bonds formed by Asp114 and each ligand, these interac-
tions also produce appreciable variations of shielding of several nuclei
of the ring and of the ethylene group, as well as of the catecholic oxy-
gens. This indicates that such hydrogen bonds cause long range
effects that give rise to changes in the electronic environment of the
most distant nuclei, even to the hydroxyl substituents.
NMR results collected in Figures 8A,B and 9A,B show that
shielding variations of ligands nuclei in the reduced system formed by
the ligand and the set of residues selected from the active site exclud-
ing Asp114, L-ResS (Val115- Cys118- Ser193- Ser194- Ser197-
Phe198- Trp386- Phe389-Phe390-Hie393-Thr412-Tyr416), are
smaller in comparison to those of the reduced systems formed by the
ligand and Asp114.
Inspection of results obtained for L-ResS reduced system consid-
ering m-OH-PEA as ligand reveals that interactions presented in this
case cause an appreciable deshielding of m-O10 and N atom. The
shielding changes caused by the interaction of each residue of the
reduced system with m-OH-PEA, shown in Table S1, indicate that the
deshielding of the catecholic oxygen occurs due to the combined
effect of Val115, Cys118, Ser193, and Ser197, while the observed
decrease of σ(N) is mainly produced by Tyr416. Taking into account
the geometric disposition of those residues with respect to m-OH-
PEA, the mentioned variations of σ for the L-ResS system suggest the
existence of several weak HB-type and electrostatic interactions
between the ligand and residues of the active site.
Thus, there is a repulsive interaction between the carbonyl oxy-
gen of Val115 and the catecholic oxygen of the ligand and two very
weak HB-type interactions Cys118-S    H8a-C8 and Cys118-S   
H8b-C8 where the S atom acts as a hydrogen acceptor, confirmed by
the slight decreases of σ(H8a) and σ(H8b) by 0.24 and – 0.27 ppm,
respectively. The carbonyl oxygen of Ser193 is also involved in an
electrostatic repulsion interaction with the oxygen of the ligand,
although this interaction is weaker than that of Val115. The most
important interactions of the ligand in this reduced system are HBs
formed between m-O10 and Ser197, namely, m-O10 – H10a   
O = C-Ser1197 and p-O10    HO-Ser197. The existence of
such interactions is corroborated by the decreases of σ(H10a) by
0.75 ppm and the deshielding of 1.2 ppm undergoing by the
hydrogen belonging to the OH of Ser197.
12
On the other hand, our theoretical NMR results corresponding to
PEA conformer in the reduced system L-ResS show small or negligible
shielding variations of atoms belonging to phenyl ring, except σ(C6), which
undergoes a decrease of 4.0 ppm. As suggested by results displayed in
Table S2, this shielding variation is mainly caused by a weak interaction
between the aromatic ring of Phe389 and the region of the ligand ring in
which C6 is located. It is also important to mention the deshielding suf-
fered by the N atom and one of the hydrogen bonded to it (H9c), which
are caused by a HB type interaction (NH3 +) H9a    O=C-Tyr416.
We now turn to the analysis of DA interactions with residues of
the active site when considering the system DA-ResS. In this case, the
most important shielding variation is that suffered by m-O11. The
detailed analysis of NMR results and of spatial arrangement of DA in
relation to the active site residues show that shielding variations of
ligand's nuclei are caused by different kind of interactions of DA with,
Ser193, Ser194, Ser197, Phe198, Phe389, and Phe490 (Figures 8, 9 and
Table S3). Although in some cases the effects of different residues on
the same ligand atom yield shielding variations of opposite sign, occa-
sionally giving a final variation close to zero. The latter does not imply
the absence of interactions, but rather a combined effect of these, as
observed in the case of Δσ (m-O10). As reported in our previous
work,[23] there are two very weak HBs interaction where Ser194 is
involved, that is, m-O10  HO–Ser194 and m-O10  Hα–Ser194.
Whereas Ser197 is part of two relatively stronger hydrogen bonds
involving p-O11, namely p-O11  HO-Ser197 and p-O11–H11a  OH-
Ser197. The decrease of shielding obtained for the H of the Ser197
hydroxyl group and for H11a by 1.7 and 1.1 ppm, respectively, is in
agreement with the existence of these interactions.
It should also be mentioned that the NMR results show the exis-
tence of a weak HB type interaction C5-H5a    O = C-Val115
between H5a and the carbonyl oxygen of Val115. The presence of
such interaction is confirmed by the decreases of σ(H5a) and σ(C5) by
0.4 ppm and 2.66 ppm, respectively.
In brief, for the three analyzed ligands, our NMR results suggest
that the salt bridge formed between Asp114 and the amino group is
very strong. Such strength is similar in the case of DA and PEA and
slightly lower for m- OH-PEA. This fact is reflected not only by
changes of σ of hydrogen atoms involved in the salt bridges, but also
by shielding variations of nitrogen atoms.
At the same time, our NMR data show that the hydroxylation pat-
tern of phenylethylamines affects the degree of interaction of the
ligand ring with the active site. Thus, shielding variations suggest that
the larger the number of hydroxyl groups present in the ring, the
larger the degree of interaction of the ring with the active site and the
lower the flexibility of the ligand. Consequently, the flexibility of the
ligand decreases in the following order DA > m-OH-PEA>PEA.
3.3 | Influence of the catecholic hydroxyls in the
affinity of the ligands
There is much experimental evidence and theoretical support indicat-
ing the central role of the interaction between the tertiary nitrogen
TOSSO ET AL.
group and Asp114 for D2DR agonists. Our detailed study on this
interaction in the case of DA, m-OH-PEA, and PEA clearly indicates
that it is the driving interaction for these ligands, giving quantitative
data in this regard.
Looking at the different dopaminergic activities reported for
these ligands, it is clear that hydroxyl substitution pattern of the
ligands influences their affinities for the receptor. Payne et al[22]
reported that the non-hydroxylated compound, PEA, binds with a
moderate affinity to D2DR (350 mM), addition of a 3-hydroxyl
group (m-OH-PEA) increases affinity 13-fold; whereas combination
of the 3- and 4-hydroxyl groups (DA) increases affinity 40-fold com-
pared with the unhydroxylated molecule.[22] It is important to
remark that our simulations are in a complete agreement with those
experimental results, not just from a qualitative but also a quantita-
tive point of view. With only three available points it has no statisti-
cal value to try to establish a correlation, but it is clear that both
QTAIM and NMR calculations give us an excellent trend for these
three ligands. Our simulations also indicate that the interaction of
the catecholic hydroxyls with a network of serine residues establish
a kind of synergism, so that the ligand is positioned more favorably
in the binding site, optimizing interactions with aromatic residues.
These results are an additional support to those reported previously
by Coley et al.[34]
At this stage of our study and based on the information obtained
(PESs as well as QTAIM and NMR calculations), it is possible to pro-
pose what could be the biologically relevant conformations of m-OH-
PEA and PEA. It is clear that the greater molecular flexibility that these
ligands possess in the active site with respect to DA, makes it more
difficult to suggest only one conformation as the active one. However,
considering our results, it is reasonable to think that a conformation
of type A or B could be the active conformation of m-OH-PEA. It is
interesting to note that this spatial ordering of m-OH-PEA is not very
similar to the active conformation proposed for DA,[23] but this spatial
ordering is very similar to that reported for conformer F of DA. As the
QTAIM calculations showed, these results are a consequence of the
fact that m-OH-PEA has only one OH, and therefore less capacity to
interact with the serine cluster that stabilizes the conformation I of
DA. In this way, the ligand is located at the bottom and is anchored in
the hydrophobic pocket, similar to the conformation F of DA.
Figure S10 shows the spatial overlap of these two ligands in which it
is possible to appreciate the different spatial ordering of conformation
A of m-OH-PEA with conformation I of DA. In turn, Figure S11 dis-
plays the similar spatial ordering of conformation A of m-OH-PEA
with conformation F of DA.
In the case of PEA, the situation is somewhat different because
the molecular flexibility of this ligand at the active site is very large,
since it practically has only one marked interaction (the main interac-
tion with Asp114), while the rest of the molecule can adopt different
conformations at the active site space. Although based on our results
we can propose conformations A or B as the most relevant, in this
case we must be cautious because it is clear that PEA can spatially
rearrange itself in the active site with relative ease. Figure S12 shows
the spatial overlap of the A conformation of PEA with the form I of
TOSSO ET AL.
DA displaying the different spatial ordering of this ligand in the active
site of the receptor. These results are in line with the biological activi-
ties reported for these compounds; the different spatial ordering of
m-OH-PEA with respect to DA could explain at least in part its lower
activity; while the significantly lower activity of PEA with respect to
DA could be attributed to its marked flexibility and greater difficulty
in anchoring to the serines cluster.
4 | C O N CL U S I O N S
It is clear that the extensive study of L-R complexes is not a simple
or fast task, particularly if one wants to obtain reliable results that
can be compared with experimental data. In the particular case of
ligands with a reduced number of rotatable bonds and especially
when two of them are mainly responsible for the spatial ordering of
the molecule, it is clear that obtaining the PES of the ligand in the
active site of the receptor is the best way to get a complete picture
of the spatial behavior of those compounds at their active sites. In
fact, to obtain the PES not only allows us to observe all the critical
points of the surface (minimums, maximums and transition states),
but also permits us to know the ease or difficulty that these local
minima have for their conformational interconversions; in other
words, to know the conformational flexibility that a ligand has in its
active site. It should be noted that it is not possible to obtain this
information from standard studies such as docking calculations nei-
ther using a combination of docking calculations and MD simula-
tions, unless they were carried out in an extremely exhaustive way.
Although a study by obtaining PES cannot yet be considered a rou-
tine study and requires some extra effort, in the case of this type of
complex, such effort is justified considering the information they
provide. Anyway, it is interesting to note that our results show that
performing a standard docking study with certain care allows to
obtain satisfactory results in the case of L-R complexes with these
structural characteristics. However, it must be pointed out that this
type of study should be taken as an exploratory and preliminary
analysis.
On the other hand, the results presented here show the impor-
tance of performing computational simulations using different types
of combined techniques employed in molecular modeling (docking/
MD/hybrid MM- QM/QTAIM and NMR shielding calculations).
Certainly, some of these techniques are a bit tedious and require
not only computation time, but is also time consuming to perform a
correct and detailed analysis of the molecular interactions involved in
the stabilization and destabilization of the L-R complexes. Neverthe-
less, such efforts are well justified when we are interested in
obtaining a very detailed, specific, and reliable information on the dif-
ferent molecular interactions involved in the stabilization of the differ-
ent complexes. Thus, the use of combined techniques presented here
has allowed us to explain in a qualitative and quantitative way the dif-
ferent affinity that DA, m-OH-PEA, and PEA have for D2DR and thus
understand the different biological activities of these compounds at
the molecular level.
13
ACKNOWLEDG MENTS
Grants from Universidad Nacional de San Luis (UNSL-Argentina)
(PROICO: 02-1418) partially supported this work. AS thanks a post-
doctoral fellowship of CONICET-Argentina. RDT, MNCZ, EA, HA, and
RDE are members of CIC-CONICET (Consejo Nacional de Investigaciones
Científicas y Técnicas).
DATA AVAILABILITY STAT EMEN T
The data that support the findings of this study are available from the
corresponding author upon reasonable request.
OR CID
Rodrigo D. Tosso https://orcid.org/0000-0003-0798-2003
M. Natalia C. Zarycz https://orcid.org/0000-0001-9838-5778
Ricardo D. Enriz https://orcid.org/0000-0002-4846-9452
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