X-Ray Crystallography and Free Energy Calculations Reveal the Binding Mechanism of A2A Adenosine Receptor Antagonists

Article abstract of DOI:10.1002/anie.202003788

We present a robust protocol based on iterations of free energy perturbation (FEP) calculations, chemical synthesis, biophysical mapping and X-ray crystallography to reveal the binding mode of an antagonist series to the A_2A adenosine receptor (AR). Eight A_2AAR binding site mutations from biophysical mapping experiments were initially analyzed with sidechain FEP simulations, performed on alternate binding modes. The results distinctively supported one binding mode, which was subsequently used to design new chromone derivatives. Their affinities for the A_2AAR were experimentally determined and investigated through a cycle of ligand-FEP calculations, validating the binding orientation of the different chemical substituents proposed. Subsequent X-ray crystallography of the A_2AAR with a low and a high affinity chromone derivative confirmed the predicted binding orientation. The new molecules and structures here reported were driven by free energy calculations, and provide new insights on antagonist binding to the A_2AAR, an emerging target in immuno-oncology.

Full text of DOI:10.1002/anie.202003788

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
www.angewandte.org
Accepted Article
Title: X-Ray Crystallography and Free Energy Calculations Reveal the
Binding Mechanism of A2A Adenosine Receptor Antagonists.
Authors: Willem Jespers, Gregory Verdon, Jhonny Azuaje, María
Majellaro, Henrik Keränen, Xerardo GArcía-Mera, Miles
Congreve, Francesca deFlorian, Chris de Graaf, Andrei
Zhukov, Andrew S Doré, Jonathan S Mason, Johan Åqvist,
Robert M Cooke, Eddy Sotelo, and Hugo Gutierrez-de-Teran
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202003788
Link to VoR: https://doi.org/10.1002/anie.202003788

10.1002/anie.202003788
Angewandte Chemie International Edition
RESEARCH ARTICLE
X-Ray Crystallography and Free Energy Calculations Reveal the
Binding Mechanism of A2A Adenosine Receptor Antagonists.
Willem Jespers, Grégory Verdon, Jhonny Azuaje, Maria Majellaro, Henrik Keränen,† Xerardo García-
Mera, Miles Congreve, Francesca Deflorian, Chris de Graaf, Andrei Zhukov, Andrew S. Doré, Jonathan
S. Mason, Johan Åqvist, Robert M. Cooke, Eddy Sotelo and Hugo Gutiérrez-de-Terán*
[*]
Dr. H. Gutiérrez-de-Terán
Department of Cell and Molecular Biology
Uppsala University, BMC
Biomedical Center, Box 596, Uppsala (Sweden)
E-mail: hugo.gutierrez@icm.uu.se
W. Jespers, Dr. H. Keränen, Prof. J. Åqvist
Department of Cell and Molecular Biology
Uppsala University, BMC
Biomedical Center, Box 596, Uppsala (Sweden)
Dr. H. Keränen, present address: H. Lundbeck A/S, Ottiliavej 9, 2500 Valby, Denmark
Dr. G. Verdon, Dr. M. Congreve, Dr. F. Deflorian, Dr. C. de Graaf, Dr. A. Zhukov, Dr. A.S. Doré, Dr. J.S. Mason, Dr. R.M. Cooke
Sosei Heptares,
Steinmetz Granta Park, Great Abington, Cambridge CB21 6DG U.K.
Dr. J. Azuaje, Dr. M. Majellaro, Prof. X. García-Mera, Prof. E. Sotelo
Departament of Organic Chemistry, Faculty of Farmacy
and Centro Singular de Investigación en Química Biolóxica y Materiais Moleculares (CIQUS, Dr. J. Azuaje, Dr. M. Majellaro, Prof. E. Sotelo)
Universidade de Santiago de Compostela, Spain
Supporting information for this article is given via a link at the end of the document.
Abstract: We present a robust protocol based on iterations of free
energy perturbation (FEP) calculations, chemical synthesis,
biophysical mapping and X-ray crystallography to reveal the binding
mode of an antagonist series to the adenosine A2A receptor (AR).
Eight A2AAR binding site mutations from biophysical mapping
experiments were initially analysed with sidechain FEP simulations,
performed on alternate binding modes. The results distinctively
supported one binding mode, which was subsequently used to design
new chromone derivatives. Their affinities for the A2AAR were
experimentally determined and investigated through a cycle of ligand-
FEP calculations, validating the binding orientation of the different
chemical substituents proposed. Subsequent X-ray crystallography of
the A2AAR with a low and a high affinity chromone derivative confirmed
the predicted binding orientation. The new molecules and structures
here reported were driven by free energy calculations, and provide
new insights on antagonist binding to the A2AAR, an emerging target
in immuno-oncology.
energy from site-directed mutagenesis (SDM) data. The idea of in
silico mutagenesis, initially introduced almost three decades ago
by Kollman to study the binding and catalysis of subtilisin,[2] was
recently implemented in computational pipelines that pursue a
systematic characterization of the effect on point mutations on e.g.
ligand-binding or protein stability.[3–5] The combination of both
ligand and residue FEP simulations can provide a full energetic
landscape of the molecular interactions governing protein-ligand
binding, which underlies the design of two complementary
protocols in our lab, namely QligFEP[6] and QresFEP[7], integrated
in the molecular dynamics (MD) software package Q.[8,9]
One area where this approach is particularly promising is
the design of ligands for G-protein-coupled receptors (GPCRs), a
superfamily of seven-transmembrane (7TM) cellular receptors[10]
that mediate the therapeutic effects of about 30% of all marketed
drugs.[11] There is a large amount of SAR and SDM data available
for these receptors, which can be combined with the increasing
growth of structural knowledge of many GPCR targets. The first
integrated approach of ligand and residue FEP simulations was
published by Boukharta et al. to characterize antagonist binding
to the Y1 neuropeptide receptor,[12] which we later expanded to
other GPCR families, including the related neuropeptide receptor
Y2,[13] the orphan receptor GPR139[14] and several members of the
family of adenosine receptors.[15–17] Among the later, the
adenosine A2A receptor (A2AAR) was one of the first GPCRs to be
crystallized[18] and today stands out as one of the better
characterized GPCRs from a structural perspective. Several
structures of the inactive and active forms of the receptor have
been solved within the last decade, and the integration of the
available experimental data has strongly aided ligand design
programs for this receptor.[19,20]
Introduction
Computational estimation of shifts in binding free energy,
associated with ligand modifications or point mutations in the
receptor macromolecule, can provide the missing link between
the structure of a protein-ligand complex and a panel of
experimental binding affinities. Rigorous free energy perturbation
(FEP) methods have been used for decades to understand the
structure-affinity relationships (SAR) around a given chemical
scaffold, and recent advances now allow the use of this technique
routinely in ligand design projects.[1] The same methodology can
be used to analyze this problem from
a complementary
Many A2AAR antagonists have been developed targeting a
number of pathologies,[21–23] including recent clinical candidates
perspective, that is to estimate the gain or loss in binding free
1
This article is protected by copyright. All rights reserved.

10.1002/anie.202003788
Angewandte Chemie International Edition
RESEARCH ARTICLE
in immuno-oncology. A number of these antagonists have been
co-crystallized with the A2AAR, providing unique structural
information which, in combination with the extensive SDM data
available,[24] allow envisaging ligand binding mechanisms and
structure-based drug design (SBDD) programs of antagonist
molecules.[19] However, suboptimal properties of traditional
scaffolds, such as poor pharmacokinetics and low selectivity
profiles, motivate the search of novel chemical entities as A2AAR
antagonists,[25] frequently through high throughput screening
(HTS) campaigns. In these cases, it is not common to obtain a
crystal structure of the receptor-ligand complex, which can
hamper further hit to lead optimization. Instead, approximate
binding modes are often inferred from the experimental data
extracted from (SAR) of ligand series and SDM data,[24] which can
be complemented by computational models of the protein-ligand
complex.[26]
optimization (i.e. the most potent compound Chromone 14
contains a metabolically unstable ester group). Interestingly, this
structure-based optimization was guided by the interpretation of
the BPM data and a computational model of the complex
generated by docking. This led to the proposal of two putative
binding modes compatible with the BPM data. Even if the SAR of
the generated series seemed to favor one of them, the question
remained open due to the lack of an X-ray structure with any of
these compounds in the original study.[31,33]
Here, we initially examine the SPR data available for these
A2AAR antagonist families through a recently developed in silico
mutagenesis tool based on free energy perturbation (FEP)
simulations.[12,15,16] The results pointed to a unified binding mode
of the chromone series, which is here used as a basis to design,
synthesis and pharmacological evaluation of an extended series
of compounds aimed to further explain the underlying SAR of
chromones as A2AAR antagonists. Finally, experimental
structures of two chromone-A2AAR complexes were solved which
confirmed the binding mode hypothesis from the computational
studies.
Results and Discussion
Free Energy Perturbation calculations on existing BPM data.
The BPM data obtained for the three chemotypes of A2AAR
antagonists were collected from reference [28], and relative binding
free energy changes between mutant and WT receptor were
calculated from Ki values (see Table 1, ∆∆ꢀꢁꢅꢂꢆꢃꢇꢄ ). Thereafter,
relative binding free energies for each ligand were calculated
Figure 1. Binding mode and chemical structures of antagonists ZM241385 (A,
crystal structure 4EIY[27]) and triazine 4b (B). The experimental pose of the
triazine (cyan) was superimposed on the same crystal structure of the receptor
shown in panel A (ribbons). Both compounds had been characterized by BPM
(residues labelled and depicted in gray sticks). Receptor-ligand hydrogen bonds
are depicted as magenta lines.
ꢈꢉꢊꢈ
based on 3D models of the receptor-ligand complexes (∆∆ꢀ
,
ꢁꢂꢃꢄ
Table 1). In the case of ZM241385, the model was directly
extracted from the high-resolution structure with the A2AAR (see
Figure 1), and the curated receptor model (see SI Methods) was
used throughout this work. The results show excellent agreement
with the experimental data, with a mean absolute error (MAE) of
0.41 kcal/mol and a correlation coefficient of R2 = 0.94 (Table 1).
Next, the same set of BPM mutations was analyzed for triazine
4g. In this case, the starting configuration of the complex was
obtained by aligning the triazine 4g-A2AAR complex (3UZA) to the
Biophysical Mapping (BPM), is an integrated approach that
has been used with success in antagonist design programs on the
A2AAR and other GPCRs.[28–31] Here, the binding affinity of a
ligand series is evaluated via surface plasmon resonance (SPR)
on a panel of mutant receptors, each bearing a single-point
mutation within the putative binding site.[28] The resulting matrix of
binding affinity shifts from wild type (WT) affinities combined with
SAR data and mapped to a receptor-ligand model provide further
insights in the determinants of binding of the scaffold. The first
application of this technique was based around the co-crystallized
A2AAR antagonist ZM241385 in combination with 8 receptor
mutants (see Figure 1A).23 These mutations involve residues in
curated model described before, retaining only the coordinates of
ꢈꢉꢊꢈ
the ligand (Figure 1B).[34] The calculated free energies (∆∆ꢀ
,
ꢁꢂꢃꢄ
Table 1) were in general good agreement with the experimental
data (MAE = 0.94 kcal/mol; R2 = 0.66). In contrast to the previous
case, two mutations (Met1775.38 and Asn1815.42) show qualitative
discrepancy with the experimental data for this ligand.
Subsequent analysis of the MD trajectories suggests that this is
likely due to a suboptimal representation of the (water mediated)
H-bond network upon mutation, which has previously been
recognized as a challenging factor for this particular scaffold.[35,36]
Once the QresFEP protocol was validated to reproduce the
BPM data on experimentally known structures, we moved on to
the chromone scaffold, for which there is analogous BPM data,
but no crystal structure available. Here, two reasonable binding
modes were generated by molecular docking of chromone 14.
The two poses, denoted as A and B (see Figure 2), form at least
one hydrogen bond with Asn2536.55 (a highly conserved
interaction in AR ligand recognition)[24] and are related by a
symmetry axis along the bicyclic core of the chromone scaffold.
According to the docking score function used, both poses were
direct contact with the ligand, such as N2536.55, L853.33, M1775.38
,
N1815.42 and I662.64 (Ballesteros Weinstein numbering[32] in
superscript), as well as residues not directly in contact with the
ligand, namely S2777.42, Y2717.36 and L167EL2 (see Figure 1). This
approach was extended for 1,2,4 triazines as A2AAR
antagonists,[33] and the binding mode was later confirmed by X-
ray crystallography (see Figure 1B).[30] In the same HTS
campaign, a series of chromones were identified as a novel family
of A2AAR antagonists,[33] and consecutively optimized to yield the
potent and selective Chromone 14 (see Figure 2).[31] At that point,
the lead-optimization program was successful in improving the
affinity of the initial HTS hit, while not focusing on pharmacokinetic
2
This article is protected by copyright. All rights reserved.

10.1002/anie.202003788
Angewandte Chemie International Edition
RESEARCH ARTICLE
energetically equivalent (-9.30 vs -9.17 for pose A and B
respectively) making it difficult at this point to discern the correct
one, in line with the previous binding hypothesis by Andrews et
al.[31,37]
Table 1. Comparison between experimental and calculated relative binding free
energies (∆∆ꢀꢁꢂꢃꢄ, in kcal/mol) for A2AAR mutants
Mutant a
ZM241385
Triazine 4g
ꢅꢆꢇ
ꢁꢂꢃꢄ
ꢅꢆꢇ
b
ꢁꢂꢃꢄ
ꢈꢉꢊꢈ
ꢈꢉꢊꢈ
b
∆∆ꢀ
∆∆ꢀ
∆∆ꢀ
∆∆ꢀ
ꢁꢂꢃꢄ
ꢁꢂꢃꢄ
I66A2.64
L85A3.33
0.14
0.83 ± 0.34
3.30 ± 0.41
0.60 ± 0.31
-0.09 ± 0.44
1.47 ± 0.57
5.81 ± 0.57
0.84 ± 0.74
0.41
1.94 ± 0.34
1.65 ± 0.37
-0.39 ± 0.36
1.66 ± 0.49
-0.63 ± 0.55
5.64 ± 0.56
-0.1 ± 0.68
2.45
0.00
1.09
-0.14
-0.27
0.82
L167A5.28
M177A5.38
N181A5.42
N253A6.55
Y271A7.36
0.14
1.23
≥ 5.86c
≥ 4.36c
1.09
0.41
[a] Data for the mutant receptor constructs reported in reference [28]. [b]
Experimental relative binding free energies were calculated from KD values as
ꢐꢑꢒꢓꢔ
ꢅꢆꢇ
ꢁꢂꢃꢄ
∆∆ꢀ
= ꢋꢌꢍꢎ ꢏ
ꢗ
ꢐꢑꢕꢖ
ꢘwith experimental errors in all cases reported as
approximately 0.1 pKD unit, i.e. less than 0.1 kcal/mol [28]. [c] Binding affinity of
the ligand to the (mutant) receptor was lower than the experimental threshold
(pKD < 5 in all cases). Errors are standard error of the mean (s.e.m.) over a total
of 10 replicates
In pose A (Figure 2A), Asn2536.55 forms an H-bond with the
nitrogen in the 4-methylthiazole group, leaving the carbonyl group
potentially exposed to the internal water network stabilized by
residues in TM7, as observed in the A2AAR crystal structure with
ZM241385[27] (see Supporting Information Figure S1). Conversely,
in pose B (Figure 2B) this carbonyl forms an additional hydrogen
bond with N2536.55. We hypothesized that in principle, the
symmetry axis would allow exchanging substituents at R6 and R7
between the binding poses, something that we would explore later
in this work (see below).
Figure 2. Putative binding modes A (green) and B (magenta) of Chromone 14
to the A2AAR (H-bonds in magenta). (C) Experimental and calculated changes
in binding free energies for each mutation in the BP. The error bars correspond
to the s.e.m. of the replica calculations for the calculated values, or are adjusted
to the reported value of 0.1 pKD unit in the case of experimental data.[28]
Design, synthesis and pharmacological evaluation of
chromone derivatives.
Our strategy to select the most reliable binding mode was to
compute the effect on ligand binding of the 8 mutations from the
BPM panel[28] for each pose, and compare the results with the
experimental values. The results (Figure 2C, Table 1 and
Supporting Information Table S1) highlight pose A as the binding
mode with the best correlation to experimental data. A MAE of
0.50 kcal/mol was observed for this pose, which is comparable to
the results obtained for the co-crystallized antagonist ZM241385.
In addition, the correlation coefficient of R2 = 0.74 falls between
those observed for ZM241385 and triazine 4g. Conversely, the
corresponding values calculated on pose B are much higher
(MAE = 1.53 kcal/mol) and the correlation is completely lost (R2 =
0.03). Additionally, the computed loss of binding affinity upon the
N2536.55A mutation is closer to experiment in pose A than in pose
B, indicating that the additional H-bond between Asn2536.55 and
the carbonyl in pose B would not contribute to more favorable
binding free energies. From the MD simulations, we further
observed that the binding modes are not related by a symmetry
axis as initially hypothesized, and the substituents at positions R6
and R7 are exploring different positions in the binding site, making
them no longer readily interchangeable.
On the basis of the binding mode hypothesis assessed by
the FEP calculations, we designed a small collection of chromone
derivatives (Scheme 1), aiming to systematically explore two
different variables on A2AAR affinity: methylation at position 2
(series 5) and the aforementioned effect of switching the
oxygenated function (-OH or -OCOMe) from the original position
7 (series 4) to position 6 (series 8), swapping the alkyl substitution
(Pr/H) accordingly. A collection of 11 molecules was prepared as
depicted in synthetic Scheme 1. Briefly, the key 2-hydroxyphenyl
ketones 3 were obtained by either the Hoesch method[31] or
Claisen condensation,[38] employing phenols 1 or esters 6 and
thiazole derivatives 2a and 2b as reactive precursors respectively.
Treatment of ketones 3 with orthoesters (formate or acetate)
enabled the efficient chromone core formation,[39] thus defining
the substituent pattern at position 2 (H or Me). Finally, the required
acetates were prepared by reaction of the corresponding phenols
with acetyl chloride. The full synthetic methodology is provided in
the SI.
The affinity of these compounds for the WT A2AAR was then
evaluated with the same SPR assay used to generate the BPM
data, using the A2AAR-STAR2 construct (see SI methods).[40] The
data confirmed that methylation at position 2 (compounds 5a-d)
is generally unfavorable for binding, compared to the parent
3
This article is protected by copyright. All rights reserved.

10.1002/anie.202003788
Angewandte Chemie International Edition
RESEARCH ARTICLE
series (compounds 4a-d). This is in agreement with previous work
by Andrews et al.,[31] where methylation of the low affinity
compound 4a (corresponding to chromone 8 as reported by
Andrews et al.[31], with pKi = 5.7) resulted in a relative decrease in
affinity of 0.6 log units (Chromone 12, pKi = 5.1). We observe a
surprising was the observation of a similar effect when the
substituents between R6 and R7 are swapped (compounds 8a-d),
which again was most pronounced for the high affinity compound
4d (see Table 2), with a drop of affinity of 2.7 log units for the
corresponding methylated derivative 8d.
similar difference for this pair of compounds (Table 2, DpKD
=
0.85), this effect is amplified in the case of the high affinity
precursor (see 4d as compared to 5d, DpKD = 2.7). More
R5
R4
HO
N
NC
+
Me
S
OH
1a) R4 = H, R5 = H
1b) R4 = Pr, R5 = H
1c) R4 = H, R5 = Me
2a
1. BF3.OEt2 (8eq)
2. HCl(g), rt, 4h
3. H2O, reflux
4. NH4OH
Me
Me
N
Me
R5
O
O
N
R6
O
R5
O
O
N
CH(OCH3)3
Piperidine, Py
MeC(OCH3)3
Piperidine, Py
R6
HO
R5
HO
R6
HO
S
S
S
80ºC, 2h, MW
80ºC, 2h, MW
OH
Me
3a) R5 = H, R6 = H
3b) R5 = Pr, R6 = H
3c) R5 = H, R6 = Me
4a) R5 = H, R6 = H
4b) R5 = H, R6 = Pr
4c) R5 = Me, R6 = H
5a) R5 = H, R6 = H
5b) R5 = H, R6 = Pr
5c) R5 = Me, R6 = H
CH3COCl
Py, 80ºC, 4h
CH3COCl
Py, 80ºC, 4h
Me
Me
O
N
O
N
R6
O
R6
O
S
O
S
O
Me
O
Me
Me
O
5d) R6 = Pr
4d) R6 = Pr
Me
2b
Me
N
Me
O
N
Pd(P(o-Tol)3)2Cl2
O
O
N
S
Me
P(o-Tol)3
Et2NH
MeO
Pr
MeO
R4
MeO
R4
LiNH(Me3Si)2
S
S
OMe
OMe
(CH3CH2CH2)4Sn
THF
THF
OMe
7c
OMe
0ºC to RT
100ºC
7a) R4 = H
7b) R4 = Br
6a) R4 = H
6b) R4 = Br
Me
Me
Me
O
N
O
N
O
N
CH(OCH3)3
Piperidine, Py
BBr3
CH2Cl2
MeO
R4
HO
R4
HO
R7
S
S
S
120ºC, 2h, MW
-78ºC to RT
12h
OMe
OH
O
3d) R4 = H
3e) R4 = Pr
8a) R7 = H
8b) R7 = Pr
7a) R4 = H
7c) R4 = Pr
CH3COCl
Py, 80ºC, 4h
Me
O
N
Me
O
S
O
R7
O
8c) R7 = H
8d) R7 = Pr
Scheme 1. Structure and synthetic pathways employed to assembly chromones 4, 5 and 8.
4
This article is protected by copyright. All rights reserved.

10.1002/anie.202003788
Angewandte Chemie International Edition
RESEARCH ARTICLE
Computational evaluation of the proposed binding modes.
this is the case: the negligible calculated free energy difference
between the two poses for caffeine is in line with the equally
populated dual binding mode in the crystal structure with the
A2AAR (PDB code 5MZP, Figure 3A). In contrast, the single
binding mode observed in the crystal structure of theophylline
(blue color in Figure 3A) is energetically favored by 1.6 kcal/mol.
Subsequent application of the same strategy in the generated
chromone series showed a similar energy gap between the two
binding poses considered for the simplest chromone in our series
(4a, Figure 3B), with pose A being 1.7 kcal/mol more favorable
than pose B. Notably, this energy gap increases significantly for
the highest affinity compound 4d, suggestive of an optimal
anchoring of the R6 acetate and R7 n-propyl substituents in pose
A (Table 3).
From the pharmacological data on the expanded series of
chromones and the initial MD simulations, it appears that the two
binding poses differ more than just purely on the rotation axis of
the chromone scaffold.
Table 2. SPR affinity data for the series of Chromone derivatives synthesized
in this work.
Me
Me
R5
O
O
N
O
O
N
R6
R6O
R7
S
S
R7O
R2
R2
4,5
8
Table 3. Calculated free energy difference between two alternative poses for
A2AAR antagonists
Substituents
[a]
Compound
pKD
R2
R5
H
R6
H
R7
Pose
comparison
A → B
Ligand perturbation
4a[b]
4b[b]
4c
H
H
H
H
H
H
5.95
6.20
5.80
Ligand 1 (H) → Ligand 2 (CH3)
H
C3H7
DDGexp
(kcal/mol)
DDGcalc
DDGcalc
(kcal/mol)
Ligand 2
Ligand 1
(kcal/mol)[a]
CH3
H
H
Caffeine
0.47 ± 0.49
1.59 ± 0.87
1.65 ± 0.93
8.76 ± 0.81
-
Caffeine
5a
-
-
4d[b]
(Chromone 14)
H
C3H7
COCH3
8.60
Theophylline
0.6 [b]
0.31 ± 0.32
2.06 ± 0.72
1.66 ± 1.09
5a[b]
5b
5c
CH3
CH3
CH3
CH3
H
H
H
H
C3H7
H
H
H
5.10
5.42
5.36
5.90
5.50
5.70
5.90
4a
4d
1.16 ± 0.1 [c]
3.68 ± 0.1 [d]
5d
CH3
H
H
[a] Calculations performed in the selected pose A (see text). [b] DGbind (caffeine
– theophylline), extracted from ChEMBL[44]. [c] DGbind (5a – 4a) and [d] DGbind
(5d – 4d), data from Table 2.
5d
8a
8b
8d
C3H7
H
COCH3
H
H
H
H
H
C3H7
C3H7
H
H
COCH3
[a] Experimental errors in all cases reported as approximately 0.1 pKD unit, i.e.
less than 0.1 kcal/mol [b] Compounds previously reported in ref [31]
The following step was then to investigate whether all
compounds in the series would adopt the preferred binding mode
A, or if binding mode B is accessible by some of the compounds,
depending on the pattern of substitutions. Such a hypothesis was
experimentally observed for caffeine, which in contrast to other
xanthine derivatives presents a dual binding mode to the A2AAR
(see Figure 3A). Indeed, an isoenergetic dual binding mode for
caffeine was previously hypothesized on the basis of free energy
calculations,[41] before experimental observation of dual-
occupancy crystal structures in complex with the A2AAR.[42,43]
Conversely, the X-ray structure of the N7-demethylated analogue
theophylline shows that this molecule adopts only one of these
binding modes, where the acidic hydrogen at position N7 makes
an additional H-bond contact with N2536.55 (Figure 3). We thus
evaluated whether our recently developed dual-topology protocol
QligFEP was suitable to capture this different behavior, by a direct
estimation of relative binding free energies between the two poses,
in analogy to the efficiency of this protocol to compare
topologically unrelated ligands (e.g. scaffold hopping).[6] The
results for the A2A-xanthine system shown in Table 3 show that
Figure 3. (A) Dual binding mode of caffeine, as extracted from the A2AAR crystal
structure with the A2AAR (PDB code 5MZP). Colour code is green (binding mode
A) and magenta (binding mode B). (B) modelled binding modes of chromone 4a,
following the same colouring scheme as in panel A.
We consequently retained the selected binding pose for
each scaffold (i.e. xanthine and chromone) and computed the
affinity shifts observed upon methylation. The calculations
reproduced the experimental decrease in affinity observed for
caffeine, as compared to the N7 demethylated analogue
theophylline (Table 3). Similarly, the corresponding FEP
calculations capture the experimentally observed negative effect
on A2A affinity due to methylation on R2 in the original chromones
series 4 (see series 5, see Table 2).[31] The two pair comparisons
5
This article is protected by copyright. All rights reserved.

10.1002/anie.202003788
Angewandte Chemie International Edition
RESEARCH ARTICLE
performed (4a → 5a and 4d → 5d, see Table 3) qualitatively
capture this effect, which is experimentally more pronounced in
hindrance of the new methyl group with Asn2536.55, the core
scaffold of 5d moved approximately 1.5 Å away from the original
pose A of 4d while maintaining the interaction between the
nitrogen in the 4-methylthiazole group and the key asparagine
residue.
the last case. Notably,
a
comparison of the endpoint
configurations of the 4d → 5d FEP transformation provide the
structural reason of this effect: to avoid the induced steric
Figure 4. (A) Crystal structures of the A2AAR and compound 4d (PDB code: 6ZDR), ligand shown in sticks and sodium ion shown as a sphere. Electron densities
of chromones 4d (B) and 5d (C; PDB code: 6ZDV). Omit maps are 2Fo-Fc at 1 sigma (light blue mesh) and Fo-Fc at 3 sigma (green mesh). Binding mode of
compound 4d (D) and 5d (E); ligands and the conserved residue N2536.55 shown as sticks, water molecules in red spheres.
X-ray Crystallography.
The only exception is Y2717.36, which slightly rotates outward
served to accommodate the alkyl tail of the chromone at R6 (see
SI Figure S2A), an induced-fit effect that was previously observed
for other A2AAR ligands.[34] The structure with the high affinity
ligand 4d shows outstanding agreement with the computational
model (see Figure 5A) with an RMSD of 0.67 Å between the
docked and experimental poses, and is indeed highly similar to
previously reported binding modes of the compound.[31,37] The
core of the scaffold is anchored by an H-bond between the
nitrogen in the 4-methylthiazole ring (coplanar with the chromone
scaffold) and N2536.55, and complemented by a p-p stacking with
F168EL2, in line with the binding pattern observed for many other
A2AAR antagonists.21 The ester at position R7 is stabilizing the
Glu1695.30 – His2647.29 ionic pair, which closes the EL2-EL3
interface, while the propyl at R6 makes contacts with Tyr2717.53
and Met2707.52. Given the good resolution of the structure, we
could observe a well-defined water network of the interface
between the carbonyl moiety of the chromone core and the
receptor (Figure 4D). While most water positions are conserved
In an effort to unequivocally reveal the binding mode of the
chromone scaffold, the A2AAR-STAR2 construct[28] was
crystallized with compound 4d (PDB code: 6ZDR) and its 2-
methyl analogue 5d (PDB code: 6ZDV). The structures were
obtained following the in meso soaking approach (see methods),
and could be refined down to a resolution of 1.92 and 2.13 Å
respectively. Statistics for data collection and refinement are
given in Supporting Information Table S2. The overall structure of
the A2AAR receptor is highly similar to previously solved structures
with other antagonists (see Figure 4A), with an RMSD of 0.46 Å
for the Ca trace as compared to the ZM241385 structure (PDB
4EIY, calculated using PyMOL[45]). The two structures show clear
positive omit density at 1s for the presence of the chromone
compounds in the orthosteric binding site (Figure 4B and C), in
both cases adopting binding mode A (Figure 4D and 4E). All
residues in the binding site show a conserved rotameric state
between the two structures and the A2AAR-ZM241385 complex.
6
This article is protected by copyright. All rights reserved.

10.1002/anie.202003788
Angewandte Chemie International Edition
RESEARCH ARTICLE
as compared to the A2AAR-ZM241385 complex, ligand 4d
displaces a number of water molecules observed in the first
hydration shell around that ligand (see Supporting Information
Figure 2B). Some of these waters were previously associated to
a high-energy or “unhappy” state,[31] which could partially explain
the high affinity of this particular compound.
initial binding mode hypothesis was generated based on the
exploration of BPM and SAR data of the original chromone series
using FEP. This constituted the basis for the design, synthesis
and evaluation of an expanded series of chromone derivatives.
The experimental results were conveniently interpreted with the
aid of a second iteration of FEP calculations, which reinforced the
binding hypothesis. Finally, X-ray crystallography experimentally
confirmed this binding mode, supporting the rational design of
these compounds. These structures, combined with the FEP
calculations, provide structural and energetic insights in the
determinants of high affinity binding of the chromone scaffold
series. We expect the presented workflow to be of general
applicability in structure-based drug design, particularly in the
case of GPCRs where structures of receptor-ligand complexes
are increasingly available.
The A2AAR-5b complex (Figure 4E) further shows that
methylation at position R2 displaces the chromone core by 1.79 Å,
in accordance with observations from the FEP simulations (Table
3 and Figure 5B, RMSD = 1.24 Å between docked and
experimental ligand configuration). The 4-methylthiazole ring of
compound 5d remains almost co-planar with the chromone
scaffold (torsional angle of 16 degrees), and the H-bond
interaction between the nitrogen in the and Asn2536.55 is
maintained. Indeed the methylation does not disrupt the position
of Asn2536.55, which remains linked to Glu1695.30 via a conserved
water molecule. Similarly, the water molecule deeper in the
binding crevice remains, mediating a common anchoring point
Acknowledgements
between the carbonyl group of the two ligands and His2767.43
.
However, the relative displacement of the chromone core on 5d
situates the substituents at R6 and R7 in sub-optimal pockets
(Figure 4D), displacing two of the water molecules described for
compound 4d, while a new water molecule in the 5d complex
replaces the role of the acetate in 4d (see Figure 5D) in the
stabilization of the Glu1695.30 – His2647.29 ionic pair. This could
explain why the inclusion of an acetyl moiety in compound 5d
could not recover the affinity of the parent 5b (DpKD = 0.48, Table
2) as much as it was observed for the 4b/4d pair (DpKD = 2.6).
Finally, the crystal structures offered the opportunity to revise the
This work was financially supported by the Swedish Research
Council; Consellerıa de Cultura, Educación
e
Ordenación
́
Universitaria
of
the
Galician
Government (Grant
ED431B2017/70); Centro Singular de Investigación de Galicia
accreditation 2016–2019 (Grant ED431G/09), and the European
Regional Development Fund (ERDF). Additional support from the
Swedish strategic research program eSSENCE is acknowledged.
The computations were performed on resources provided by the
Swedish National Infrastructure for Computing (SNIC). This
research program has been developed in the frame of the
European COST action ERNEST (Grant CA 18133) and
GLISTEN (Grant CA 1207).
4d
→
5d FEP transformation, which was somehow
underestimated (see Table 3). However, the calculated value
using the starting pose from the experimental coordinates did not
change significantly, (DDGcalc = 1.54 ± 1.22 kcal/mol) indicating
that the docking pose was an accurate enough starting point for
the FEP calculations.
Keywords: Adenosine receptors • Biophysical mapping (BPM) •
Free energy perturbation (FEP) • G protein-coupled receptor
(GPCR).
[1]
[2]
[3]
Z. Cournia, B. Allen, W. Sherman, J. Chem. Inf. Model. 2017, 57,
2911–2937.
S. N. Rao, U. C. Singh, P. A. Bash, P. A. Kollman, Nature 1987,
328, 551–4.
W. Jespers, G. V. Isaksen, T. A. H. Andberg, S. Vasile, A. Van
Veen, J. Åqvist, B. O. Brandsdal, H. Gutiérrez-De-Terán, J. Chem.
Theory Comput. 2019, 15, 5461–5473.
[4]
T. B. Steinbrecher, M. Dahlgren, D. Cappel, T. Lin, L. Wang, G.
Krilov, R. Abel, R. Friesner, W. Sherman, J. Chem. Inf. Model. 2015,
55, 2411–20.
[5]
[6]
[7]
V. Gapsys, S. Michielssens, D. Seeliger, B. L. de Groot, J. Comput.
Chem. 2015, 36, 348–54.
Figure 5. Crystal structure (orange) and modelled coordinates (cyan) of (A) the
highest affinity compound 4d (PDB code: 6ZDR) and (B) the methylated
derivative 5d (PDB code: 6ZDV) with the A2AAR. H-bond interactions are
indicated in magenta.
W. Jespers, M. Esguerra, J. Åqvist, H. Gutiérrez-de-Terán, J.
Cheminform. 2019, 11, 1–16.
W. Jespers, G. V. Isaksen, T. A. H. Andberg, S. Vasile, A. Van
Veen, J. Åqvist, B. O. Brandsdal, H. Gutiérrez-De-Terán, J. Chem.
Theory Comput. 2019, 15, 5461–5473.
Conclusion
[8]
[9]
J. Marelius, K. Kolmodin, I. Feierberg, J. Åqvist, J. Aqvist, J. Mol.
Graph. Model. 1998, 16, 213–225.
We describe a robust workflow to iteratively improve
receptor-ligand binding models, based on mapping of available
experimental data onto structural information via free energy
calculations. We applied this protocol to provide new insights in
the binding mode of a recent series of A2AAR antagonists. An
P. Bauer, A. Barrozo, M. Purg, B. A. Amrein, M. Esguerra, P. B.
Wilson, D. T. Major, J. Aqvist, S. C. L. Kamerlin, SoftwareX 2018, 7,
388–395.
[10]
A. L. Hopkins, C. R. Groom, Nat. Rev. Drug Discov. 2002, 1, 727–
7
This article is protected by copyright. All rights reserved.

10.1002/anie.202003788
Angewandte Chemie International Edition
RESEARCH ARTICLE
30.
Robertson, A. Zhukov, et al., J. Med. Chem. 2012, 55, 1904–1909.
P. Rucktooa, R. K. Y. Cheng, E. Segala, T. Geng, J. C. Errey, G. A.
Brown, R. M. Cooke, F. H. Marshall, A. S. Doré, Sci. Rep. 2018, 8,
41.
[11]
[12]
[13]
A. S. Hauser, M. M. Attwood, M. Rask-Andersen, H. B. Schiöth, D.
E. Gloriam, Nat. Rev. Drug Discov. 2017, 16, 829–842.
L. Boukharta, H. Gutiérrez-de-Terán, J. Åqvist, PLoS Comput. Biol.
2014, 10, e1003585.
[34]
[35]
[36]
[37]
A. Bortolato, B. G. Tehan, M. S. Bodnarchuk, J. W. Essex, J. S.
Mason, J. Chem. Inf. Model. 2013, 53, 1700–1713.
A. Bortolato, B. G. Tehan, R. T. Smith, J. S. Mason, in Methods Mol.
Biol., Humana Press Inc., 2018, pp. 207–232.
B. Xu, S. Vasile, S. Østergaard, J. F. Paulsson, J. Pruner, J. Åqvist,
B. S. Wulff, H. Gutiérrez-De-Terán, D. Larhammar, in Mol.
Pharmacol., American Society For Pharmacology And Experimental
Therapy, 2018, pp. 323–334.
C. J. Langmead, S. P. Andrews, M. Congreve, J. C. Errey, E.
Hurrell, F. H. Marshall, J. S. Mason, C. M. Richardson, N.
Robertson, A. Zhukov, et al., J. Med. Chem. 2012, 55, 1904–9.
K. S. Gudmundsson, B. A. Johns, S. H. Allen, Bioorganic Med.
Chem. Lett. 2008, 18, 1157–1161.
[14]
A. C. Nøhr, W. Jespers, M. A. Shehata, L. Floryan, V. Isberg, K. B.
Andersen, J. Åqvist, H. Gutiérrez-de-Terán, H. Bräuner-Osborne, D.
E. Gloriam, Sci. Rep. 2017, 7, 1–9.
[38]
[39]
[40]
[15]
[16]
[17]
H. Keränen, H. Gutiérrez-de-Terán, J. Åqvist, PLoS One 2014, 9,
e108492.
M. Deodhar, K. Wood, D. S. Black, N. Kumar, Tetrahedron Lett.
2012, 53, 6697–6700.
H. Keränen, J. Åqvist, H. Gutiérrez-de-Terán, Chem. Commun.
2015, 51, 3522–3525.
A. Zhukov, S. P. Andrews, J. C. Errey, N. Robertson, B. Tehan, J.
S. Mason, F. H. Marshall, M. Weir, M. Congreve, J. Med. Chem.
2011, 54, 4312–4323.
W. Jespers, A. Oliveira, R. Prieto-Díaz, M. Majellaro, J. Åqvist, E.
Sotelo, H. Gutiérrez-De-Terán, Molecules 2017, 22, DOI
10.3390/molecules22111945.
[41]
[42]
Y. Liu, S. K. Burger, P. W. Ayers, E. Vöhringer-Martinez, J. Phys.
Chem. B 2011, 115, 13880–13890.
[18]
V.-P. Jaakola, M. T. Griffith, M. A. Hanson, V. Cherezov, E. Y. T.
Chien, J. R. Lane, A. P. Ijzerman, R. C. Stevens, Science 2008,
322, 1211–7.
R. K. Y. Cheng, E. Segala, N. Robertson, F. Deflorian, A. S. Doré, J.
C. Errey, C. Fiez-Vandal, F. H. Marshall, R. M. Cooke, Structure
2017, 25, 1275-1285.e4.
[19]
[20]
[21]
[22]
[23]
[24]
A. Jazayeri, S. P. Andrews, F. H. Marshall, Chem. Rev. 2017, 117,
21–37.
[43]
[44]
[45]
A. S. Doré, N. Robertson, J. C. Errey, I. Ng, K. Hollenstein, B.
Tehan, E. Hurrell, K. Bennett, M. Congreve, F. Magnani, et al.,
Structure 2011, 19, 1283–93.
H. Gutiérrez-de-Terán, J. Sallander, E. Sotelo, Curr. Top. Med.
Chem. 2017, 17, 40–58.
B. B. Fredholm, A. P. IJzerman, K. A. Jacobson, J. Linden, C. E.
Müller, Pharmacol. Rev. 2011, 63, 1–34.
A. Gaulton, L. J. Bellis, a P. Bento, J. Chambers, M. Davies, A.
Hersey, Y. Light, S. McGlinchey, D. Michalovich, B. Al-Lazikani, et
al., Nucleic Acids Res. 2012, 40, D1100-7.
C. E. Müller, K. A. Jacobson, Biochim. Biophys. Acta 2011, 1808,
1290–308.
The PyMOL Molecular Graphics System, Version 1.4 Schrödinger,
LLC
J.-F. Chen, H. K. Eltzschig, B. B. Fredholm, Nat. Rev. Drug Discov.
2013, 12, 265–86.
W. Jespers, A. C. Schiedel, L. H. Heitman, R. M. Cooke, L. Kleene,
G. J. P. van Westen, D. E. Gloriam, C. E. Müller, E. Sotelo, H.
Gutiérrez-de-Terán, Trends Pharmacol. Sci. 2018, 39, 75–89.
C. M. Richardson, R. J. Gillespie, D. S. Williamson, A. M. Jordan, A.
Fink, A. R. Knight, D. M. Sellwood, A. Misra, Bioorg. Med. Chem.
Lett. 2006, 16, 5993–7.
[25]
[26]
[27]
V. Lounnas, T. Ritschel, J. Kelder, R. McGuire, R. P. Bywater, N.
Foloppe, Comput. Struct. Biotechnol. J. 2013, 5, e201302011.
W. Liu, E. Chun, A. A. Thompson, P. Chubukov, F. Xu, V. Katritch,
G. W. Han, C. B. Roth, L. H. Heitman, A. P. IJzerman, et al.,
Science 2012, 337, 232–6.
[28]
A. Zhukov, S. P. Andrews, J. C. Errey, N. Robertson, B. Tehan, J.
S. Mason, F. H. Marshall, M. Weir, M. Congreve, J. Med. Chem.
2011, 54, 4312–4323.
[29]
[30]
M. Congreve, C. J. Langmead, J. S. Mason, F. H. Marshall, J. Med.
Chem. 2011, 54, 4283–311.
M. Congreve, S. P. Andrews, A. S. Dore, K. Hollenstein, E. Hurrell,
C. J. Langmead, J. S. Mason, I. W. Ng, B. Tehan, A. Zhukov, et al.,
J. Med. Chem. 2012, 55, 1898–1903.
[31]
[32]
[33]
S. P. Andrews, J. S. Mason, E. Hurrell, M. Congreve,
Medchemcomm 2014, 5, 571.
J. A. Ballesteros, H. Weinstein, Methods Neurosci. 1995, 25, 366–
428.
C. J. Langmead, S. P. Andrews, M. Congreve, J. C. Errey, E.
Hurrell, F. H. Marshall, J. S. Mason, C. M. Richardson, N.
8
This article is protected by copyright. All rights reserved.

10.1002/anie.202003788
Angewandte Chemie International Edition
RESEARCH ARTICLE
Entry for the Table of Contents
Insert graphic for Table of Contents here.
In molecular design, structural, pharmacological and chemical information can be interconnected by computational estimations of
binding free energies. Using a combined FEP approach to examine both mutagenesis and ligand SAR, we designed new analogues
of the A2AAR antagonist series of chromones. Subsequent crystal structures supported the rational design of these compounds,
linking the structural and energetic understanding on ligand binding.
Institute and/or researcher Twitter usernames: @hugogdtc
9
This article is protected by copyright. All rights reserved.