Evaluation of molecular modeling of agonist binding in light of the crystallographic structure of an agonist-bound A2A adenosine receptor

Article

pubs.acs.org/jmc

Evaluation of Molecular Modeling of Agonist Binding in Light of the

Crystallographic Structure of an Agonist-Bound A_2AAdenosine

Receptor

Francesca Deflorian,^†T. Santhosh Kumar,^†Khai Phan,^†Zhan-Guo Gao,^†Fei Xu,^‡Huixian Wu,^‡

Vsevolod Katritch,^‡Raymond C. Stevens,^‡and Kenneth A. Jacobson*^,†

^†Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney

Diseases, National Institutes of Health, Building 8A, Room B1A-19, Bethesda, Maryland 20892-0810, United States

^‡Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037,

United States

S

* Supporting Information

ABSTRACT: Molecular modeling of agonist binding to the

human A_2Aadenosine receptor (AR) was assessed and

extended in light of crystallographic structures. Heterocyclic

adenine nitrogens of cocrystallized agonist overlaid corre-

sponding positions of the heterocyclic base of a bound

triazolotriazine antagonist, and ribose moiety was coordinated

in a hydrophilic region, as previously predicted based on

modeling using the inactive receptor. Automatic agonist

docking of 20 known potent nucleoside agonists to agonist-

bound A_2AAR crystallographic structures predicted new

stabilizing protein interactions to provide a structural basis

for previous empirical structure activity relationships consistent with previous mutagenesis results. We predicted binding of novel

C2 terminal amino acid conjugates of A_2AAR agonist CGS21680 and used these models to interpret effects on binding affinity of

newly synthesized agonists. ^D-Amino acid conjugates were generally more potent than L-stereoisomers and free terminal

carboxylates more potent than corresponding methyl esters. Amino acid moieties were coordinated close to extracellular loops 2

and 3. Thus, molecular modeling is useful in probing ligand recognition and rational design of GPCR-targeting compounds with

specific pharmacological profiles.

(UK-432,097, Chart 1) and crystallization in lipidic cubic phase

(LCP).^16,18In the second case, A_2AAR was thermostabilized by

four point mutations in the receptor, allowing its crystallization

in complex with the much smaller agonists adenosine 4 and its

derivative 5′-N-ethylcarboxamidoadenosine 14 (NECA).¹⁷

Despite the differences in complex composition and crystal

packing, all these A_2AAR structures display very similar

activation-related changes on the intracellular side, while

additional differences specific for the binding of the bulky

agonist 1 to the A_2AAR are located at the extracellular surface,

mainly within the extracellular loops (ELs) 2 and 3.

There is still significant interest in predicting binding modes

of agonists based on the more prevalent inactive state

structures. This approach has resulted in several studies over

the past few years targeting agonists of the β₂adrenergic

receptor⁸and A_2AAR,^19,20as reviewed by Katritch et al.²¹Now,

with experimental structures of agonist complexes of the human

A_2AAR available, it is possible to evaluate the quality of these

previous models of agonist binding and to discern which

INTRODUCTION

■

Despite the great importance in human biology and clinical

applications of G protein-coupled receptors (GPCRs),¹⁻³

crystallography has only recently started to overcome

technological barriers to yield the first high resolution

structures for this membrane protein family.⁴⁻⁸While initially

a number of GPCR structures were resolved in an inactive state

stabilized by antagonist or inverse agonist, four GPCRs have

also been resolved recently in the active state: bovine opsin,⁹⁻¹²

β₂- and β₁-adrenergic receptors,¹³⁻¹⁵and human A_2Aadenosine

receptor (AR).^16,17Among the conformational changes induced

by agonists are common movements and rotations of

transmembrane domains (TMs) 3, 5, 6, and 7. These helical

rearrangements enlarge a crevice in the intracellular interface of

the receptor, facilitating G protein binding and activation.

The crystal structures of the human A_2AAR have been solved

in complexes with several different agonists using two very

different approaches to stabilize the active state of the receptor.

In the first case, A_2AAR was stabilized by extensive interactions

with a bulky (∼780 Da), conformationally selective agonist 2-

N-(3-(1-(pyridin-2-yl)piperidin-4-yl)ureido)ethyl-N⁶-(2,2-di-

phenylethyl)-5′-N-ethylcarboxamidoadenosine-2-carboxamide 1

Received: October 28, 2011

Published: November 21, 2011

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structure has many of the features of activated GPCRs,

including characteristic helical movements and microswitches

on the intracellular side.²⁴The active state of the receptor was

also supported by observed pharmacological behavior in

modulation of nucleoside binding by sodium ions.¹⁶

Chart 1. Structures of Agonist (1) and Antagonist (2) AR

Ligands That Were Co-crystallized with the Human A_2AAR

Previously, various residues located mostly in TMs 3, 5, 6,

and 7 of the A_2AAR were predicted by modeling to be involved

in agonist recognition (Supporting Information Table S1).¹⁹In

this model, Thr88 (3.36), Phe168 (EL2), Asn181 (5.42),

His250 (6.52), Asn253 (6.55), Ser277 (7.42), and His278

(7.43) are located in proximity to the potent, nonselective

agonist 14 and are involved in ligand interactions (the numbers

in parentheses correspond to the Ballesteros−Weinstein

indexing system).²⁵The importance for agonist binding of

residues located in the TM regions and in EL2 was supported

by site-directed mutagenesis data obtained for the A_2AAR²⁶and

also for closely related A₁and A₃AR subtypes.^27,28In agreement

with the indications from molecular modeling and pharmaco-

logical studies, all of these critical residues are located in

proximity to the adenosine core of the agonist ligand 1 in the

recent crystallographic structure of the A_2AAR with 1 bound

(designated here 1−A_2AAR).¹⁶The previous docking orienta-

tion¹⁹of both agonist ligands, adenosine 4 and 5′-uronamide

14, in the inactive state of the human A_2AAR⁴was nearly

identical to the binding mode of the adenosine moiety of the

agonist 1 in the TM region as determined by X-ray

crystallography (Figure 1).

A specific mode of overlay of both small agonists 4 and 14

with the position of antagonist 4-[2-[7-amino-2-(2-furyl)-1,2,4-

triazolo[1,5-a][1,3,5]triazin-5-yl-amino]ethylphenol 2

(ZM241,385) inside the inactive A_2AAR (designated here 2−

A_2AAR) was predicted,^4,19and this correspondence was

compared with the position of 1 in the cocrystallized

structure.¹⁶The modes of overlay and interactions with specific

amino acid residues in the X-ray structure 1−A_2AAR and in the

predicted model are shown in Supporting Information. The

adenine moiety of the agonists and the triazolotriazine fragment

of 2 have very similar positions inside the binding site and are

involved in similar interactions with the receptor, i.e. a π−π

stacking interaction with Phe168 (EL2) and H-bonding of the

exocyclic amine with Asn253 (6.55). The position of the

carboxylate of Glu169 (EL2) has shifted in the 1−A_2AAR X-ray

structure from the exocyclic amine of adenine in docked

agonists 4 and 14 to the urea group of the extended C2 side

chain of 1. This side chain shift is considered to occur only after

the binding of agonists with bulky substituents at the N⁶, such

as 1, and was not expected in the earlier modeling

predictions,¹⁹possibly because no N⁶and C2 substitutions

were examined. In the recent structure of 14 bound to a

thermostabilized A_2AAR, Glu169 (EL2) does not shift away

from the (unsubstituted) exocyclic amine.¹⁷The adenine C2-

position of the modeled agonists 4 and 14 was oriented toward

the extracellular part of the receptor, as found in the 1−A_2AAR

structure and later confirmed in the crystal structures of the

A_2AAR in complex with 4 (designated here 4−A_2AAR) and 14

(designated here 14−A_2AAR). The ribose moiety of 4 and 14

docked to 2−A_2AAR was predicted to be in proximity to

hydrophilic residues in TM3 and TM7, i.e. Thr88, Ser277, and

His278. The docking poses of 4 and 14 were able to predict the

interactions of the ribose ring with residues in TM3, TM6, and

TM7 but could not predict the backbone changes of the TMs

upon binding with agonists, as shown in the agonist-bound

crystal structures.

approaches are more likely to accurately predict the binding

modes of other known agonists (3−22, Table 1). The binding

modes of other agonists not yet crystallized in complex with the

A_2AAR are of current interest, such as 1-[6-amino-9-

[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-

purin-2-yl]-N-methylpyrazole-4-carboxamide 10 (CVT-3146,

regadenoson, Lexiscan), a short-acting adenosine A_2AAR

agonist already approved as a diagnostic coronary vasodilator.²²

The objectives of this study were 3-fold: (1) Because much

of the docking of biologically relevant GPCRs will still require

use of the inactive basal conformational state of a given

receptor, we have evaluated the accuracy of previously

predicted interactions of A_2AAR agonists^19,20in light of the

complexes recently crystallized; (2) docking predictions were

made for a wide range of known A_2AAR agonists by extending

the structures of agonist complexes crystallized. This helps to

interpret already elucidated structure−activity relationships

(SARs) in this chemical series in terms of predicted interactions

with the A_2AAR receptor; (3) we predicted docking of novel C2

terminal amino acid conjugates of A_2AAR agonist 2-[p-(2-

carboxyethyl)phenyl-ethylamino]-5′-N-ethylcarboxamidoade-

nosine 16 (CGS21680) and used these models to interpret

effects on the measured binding affinity of the newly

synthesized agonists.

RESULTS

■

Evaluation of Nucleoside Binding to the A_2AAR:

Predicted Model Based on Inactive State vs Crystallo-

graphic Structure. Because of the activation-related con-

formational changes, receptor modeling of an agonist binding

to a template representing the inactive or basal state of a GPCR

is especially challenging and may require additional validation.²³

To assess the quality of such docking, we have compared the

docking of adenosine derivatives in our previous study¹⁹using

the inactive, basal conformation of the A_2AAR⁴(PDB code:

3EML) with the newly reported X-ray structure of an agonist-

bound receptor (PDB code: 3QAK).¹⁶The agonist-bound

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Table 1. Structures and Affinities of Agonists of the A_2AAR Used in Docking to the Crystallographic Structure of the Agonist-

Bound Receptor (Unless Noted, X = O; Y = N; R′, R″ = H)

a

K_ior IC₅₀values are from refs 22, 29, 35−45, 50.

Evaluation of Binding of Known Active Nucleosides at

the A_2AAR: Predictions Based on Agonist-Bound

Receptor Complexes. Redocking of Cocrystallized Ago-

nist 1. To investigate the ability of molecular docking to

reproduce an experimentally observed ligand binding mode, the

potent A_2AAR agonist 1, formerly in clinical trials by Pfizer for

chronic obstructive pulmonary disease,²⁹was redocked to the

crystal structure of the 1−A_2AAR. Different automated docking

approaches with various protocols and distance constraints

were tested in order to choose the docking methods that were

capable of retrieving a docking pose for 1 with a root-mean-

square deviation (rmsd) less than 2 Å from the published

crystal structure.¹⁶The peculiar molecular properties of 1 and

the shape and electrostatic characteristics of the binding cavity

of the active receptor, with a big portion of the ligand exposed

to the solvent, make automated docking challenging.

Compound 1 breaks three of Lipinski’s rule-of-five³⁰for the

druglikeness with 7 H-bond donors, 11 H-bond acceptors, and

a molecular weight of 778 Da. The numerous rotatable bonds

in 1 define a high conformational freedom of the ligand, and

usually a compound’s high flexibility negatively affects the

accuracy of automated docking.³¹Moreover, the shape of the

receptor’s binding pocket in the 1−A_2AAR structure shows a

large portion of the cocrystallized ligand in the extracellular

media. Some automated docking protocols can perform poorly

in retrieving accurate poses for partially buried ligands.³¹Those

issues might account for some of the difficulties in reproducing

the cocrystallized orientation of 1 in the binding site of the 1−

A_2AAR structure with standard automated docking procedures

such as Glide³²or MOE.³³

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Figure 1. (a) The newly determined structure of the A_2AAR is shown surrounding its synthetic agonist 1.¹⁶The helices (color sequence from red to

blue) and the slim connecting loops represent the receptor protein, winding back and forth through the cell membrane. The central ribose moiety

(red) of the agonist binds in a hydrophilic region and is critical for activation of the receptor, while the adenine heterocycle (blue) binds in a

hydrophobic region. The top (tan-colored) C2 and N⁶substituents of the agonist, facing the outside of the cell, effectively fill the remaining spaces in

the binding site and stabilize the receptor in order to obtain a crystallized structure. (b) Similar view of the agonist docking model of Ivanov et al.

using the inactive A_2AAR structure.^4,19The potent nonselective agonist 14 is present in the binding site. (c) Superposition of the agonist 1 (carbons

colored in green) in 1−A_2AAR (represented as ribbon colored in green), the antagonist 2 (carbons colored in orange) in 2−A_2AAR (represented as

ribbon colored in orange), and the predicted docked pose of 14 as in Ivanov et al. (carbons colored in cyan).

Unexpectedly, the Glide docking was unable to find a

docking pose for 1 with an rmsd lower than 2 Å compared with

the corresponding crystal structure, even after the use of

distance restraints to force the formation of the key H-bond

interactions in the binding site. One docking protocol among

the tested ones able to retrieve an accurate pose of 1 closer than

an rmsd of 2 Å from the experimental conformation was the

MOE docking with the pharmacophore placement method,

where a pharmacophore model was used to guide the docking

process. Pharmacophore-based docking is a useful way to

include the known ligand−receptor interactions in the docking

procedure. In the pharmacophore query used in this docking

study (refer to the methodology paragraph for more details),

four pharmacophore features were used: the projections of the

N⁶H-bond donor and N⁷H-bond acceptor of the adenine ring

on Asn253 (6.55), and the projections of the OH groups at 2′

and 3′ position of the ribose ring on His278 (7.43) and Ser277

(7.42), respectively (Supporting Information Figure S2). These

ligand−receptor interactions are considered to be critical for

the binding of 1 in the binding pocket of the A_2AAR and are

expected to be conserved for all the adenosine-like agonists.

The most favorably MOE-docked pose of compound 1 in the

binding pocket of 1−A_2AAR had an rmsd of 0.42 Å from the X-

ray conformation. The second and the third best poses had a

rmsd of 1.29 Å and 1.63 Å, respectively, and showed the

adenosine core and the N⁶-diphenylethyl moiety correctly

positioned in the binding site, but with the terminal end of the

C2-substituent in a slightly different orientation (Supporting

Information Figure S2). It should be noted that the majority of

the docking solutions had an rmsd greater than 2 Å due to the

misplacement of the long and highly flexible chain at the C2-

position of 1 even if the adenosine core of the compound was

correctly positioned in the binding pocket.

Molecular Docking of Diverse Known Agonists to Agonist-

Bound A_2AAR Crystal Structures. A diverse set of nucleosides

that are potent and/or selective agonists to the A_2AAR was

assembled (Table 1).^{22,29,35−45}These 20 adenosine derivatives

contain modifications of the ribose moiety, a nucleobase

substitution, or combinations thereof. They include clinical

candidates, such as 2-[2-(4-chlorophenyl)ethoxy]adenosine 6

(MRE-0094, sonedenoson),⁵⁵2-((cyclohexylmethylene)-

hydrazino)adenosine 9 (WRC-0470, binodenoson),⁵⁶13

(UK-371,104),³⁶[trans-4-{3-[6-amino-9-(N-ethyl-β-D-ribofura-

n o s y l u r o n a m i d e ) - 9 H - p u r i n - 2 - y l ] p r o p - 2 - y n y l } -

cyclohexanecarboxylic acid methyl ester 19 (ATL-146e,

apadenoson),³⁹[1S-[1a,2b,3b,4a(S*)]]-4-[7-[[2-(3-chloro-2-

thienyl)-1-methylpropyl]amino]-3H-imidazo[4,5-b]pyridyl-3-

yl]cyclopentane carboxamide 20 (AMP-579),⁴⁴4-{3-[6-amino-

9-(5-cyclopropylcarbamoyl-3,4-dihydroxytetrahydrofuran-2-yl)-

9H-purin-2-yl]prop-2-ynyl}piperidine-1-carboxylic acid methyl

ester 21 (ATL-313),³⁹(2R,3R,4S,5R)-2-(6-amino-2-{[(1S)-2-

hydroxy-1-(phenylmethyl)ethyl]amino}-9H-purin-9-yl)-5-(2-

ethyl-2H-tetrazol-5-yl)tetrahydro-3,4-furandiol 22 (GW

328267X, which also binds to the A₃AR),^45,57and the approved

clinical diagnostic agent 10. Various agonists used as research

tools and molecular probes of the A_2AAR (such as the amine

congener 17 and its irreversibly binding isothiocyanate

derivative 18)⁴³are also included. Various 2-hexynyl derivatives

of adenosine, e.g. 5 and 15, were found to maintain high affinity

at the A_2AAR.⁴⁰The 2-hexynyl 5′-truncated agonist 3, a 4′-

thioadenosine, maintains affinity and selectivity at the A_2AAR.³⁸

Compounds 11 and 12, containing the same N⁶-(2,2-

diphenylethyl) group as 1, were among the first A_2AAR

agonists identified.⁴²Several other 2-ethers, i.e. the bulky

naphthyl derivative 7,^41,50or 2-thioether derivatives, i.e.

cyclohexylethyl derivative 8 that was converted to a prodrug

form,³⁷were included. Each of these agonists was sequentially

docked to the 1−A_2AAR or 14−A_2AAR crystal structures. A

variety of substituents at the 5′, C2, and N⁶positions were

chosen (i) to allow the characterization of the different

subpockets, (ii) to define where those substituents locate in the

After extensive comparison of Glide and MOE, we also found

that redocking of 1 using the ICM-Dock module of Molsoft³⁴

reproduced the position of the agonist in the X-ray structure

with a rmsd value within 1 Å.

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Figure 2. Docking poses of 4 ((a,b) with carbon atoms colored in green and orange) and 14 ((c) with carbons colored in yellow) in the binding site

of the agonist-bound 1−A_2AAR structure. The key H-bond interactions between the compounds and the residues of the binding pocket are

highlighted as dotted lines. Adenosine 4 may assume an orientation of the 5′-OH group that points toward H250, like the docking mode in (a), or

with the 5′-OH group pointing and interacting with T88, docking mode in (b). The uronamide group of 14 interacts with both H250 and T88. The

H-bond interactions between the adenosine moiety of the agonists and the key residues N253, S277, and H278 as well as the hydrophobic

interactions with F268, L249, and I274 are believed to be conserved for all the agonists in our list.

binding cavity of the receptor, and (iii) to highlight the

relevance of receptor residues important for the binding and

activity of those compounds.

and 14, between Ser277 (7.42) and the 3′-OH group and

between His278 (7.43) and the 2′-OH group of the ribose

moiety. Favorable van der Waals (vdW) contacts further

stabilized the poses of 4 and 14, involving residues Val84

(3.32), Leu85 (3.33), Trp246 (6.48), and Leu249 (6.51)

located in close proximity to the ribose moiety, and Ile274

(7.39), Met270 (7.35), Phe168 (EL2), and Met177 (5.38),

which embedded the adenine core of the two agonists.

In the optimized binding pose of 14, the 5′ carboxamide

group, in common with 1, was firmly locked in the cavity by H-

bond interactions with His250 (6.52) on one side and Thr88

(3.36) on the other side, while the attached hydrophobic ethyl

group was embedded by favorable vdW interactions with

residues Leu85 (3.33), Gln89 (3.37), Ile92 (3.40), Met177

(5.38), Asn181 (5.42), Cys185 (5.46), Val186 (5.47), and

Trp246 (6.48) (Figure 2c).

The 5′ substituent of 4 consists of a hydroxymethyl group. A

high mobility of the small OH group in the 5′-substitution

pocket of 1−A_2AAR appeared in the docking results for

hydroxymethyl agonists 4−13, with two different orientations

of the OH group. The more favorably scored docking

orientation of 4 was characterized by an interaction of the 5′-

OH group with Thr88 (3.36), where the threonine was acting

as H-bond acceptor (Figure 2b). The other most common

docking orientation was pointing the 5′-OH group toward

His250 (6.52) (Figure 2a). Both docking placements of the 5′-

OH group were plausible because the hydroxyl group could

closely resemble the role of the uronamide NH group of the

cocrystallized 1, donating the H-bond interaction to Thr88

(3.36), or, at the same time, it could be placed where the

uronamide carbonyl oxygen was found in cocrystallized 1,

accepting an H-bond interaction from His250 (6.52). After

optimization of both representative docking orientations of 4 in

the binding pocket of 1−A_2AAR, the lowest energy con-

formations of the ligand were engaged in a H-bond interaction

with His250 (6.52) and not Thr88 (3.36). This energy trend

was maintained also for the other agonists having a 5′-OH

group. For all the agonists on our list with a hydroxyl group at

the 5′-position, the two different docking orientations of the

OH were considered for the further MCMM optimization. All

of these agonists showed the same trend as 4, i.e., the

An automated docking approach was used to place the

molecules in the rigid binding cavities of 1−A_2AAR or 14−

A_2AAR after removal of the cocrystallized ligand, using, as for

compound 1, different techniques and protocols in order to

find a placement able to reproduce the common interactions

with the adenosine moiety shown by 1 in the crystal structure.

The indispensable chemical features needed by an agonist to

activate the A_2AAR are shared by all the agonists in our list: the

aromatic nature of the nucleobase for the π−π stacking with

Phe168 (EL2) and the stabilizing interactions with Leu249

(6.51) and Ile274 (7.39), the H-bond acceptor nitrogen atom

at N⁷and the exocyclic amino group of the adenine core to

interact with Asn253 (6.55), and the two hydroxyl groups at 2′-

and 3′-positions to interact with Ser277 (7.42) and His278

(7.43). These molecular characteristics of the nucleoside

agonists are crucial for the ligand recognition at the receptor

site and are considered essential for the molecules’ biological

activity.

Docking poses with the correct critical interactions with

Phe168, Asn253, Ser277, and His278 in the binding cavity of

the receptor were retrieved and further optimized by means of a

Monte Carlo multiple minimum (MCMM) conformational

search of the ligands and the residues with a radius of 4 Å from

the docked poses in order to simulate the complementary

adjustments of bound ligand. The final docking solutions

showed structural characteristics of the agonists that might not

be necessary for the activation of the receptor but clearly are

crucial for the potency and the selectivity of those compounds.

The native agonist 4 and potent nonselective 5′-uronamide

14 were docked in the rigid binding site of 1−A_2AAR and then

optimized along with the binding cavity residues located within

4 Å from the docked poses. Because both of these agonists were

unsubstituted at the N⁶and C2 positions, the docking poses of

4 and 14 highlighted the crucial anchoring interactions with the

binding site of A_2AAR that are expected to be common among

all the agonists in our list. Strong H-bond interactions were

observed between the carboxamide group of Asn253 (6.55) and

the primary N⁶amino group and the N⁷of the adenine ring of 4

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energetically favored conformations showed the 5′-OH group

engaged with His250 (6.52). The binding energies between the

individual residues in the binding site of 1−A_2AAR, and the

minimized docking poses of 4 were calculated and listed in

Table 2. When the 5′-OH of 4 was pointing toward Thr88, the

The importance of water has been recently demonstrated by

the 4−A_2AAR structure,¹⁷where the interaction between the 5′-

OH group of the cocrystallized adenosine 4 and the residues

His250 (6.52) and Asn181 (5.42) is mediated by a structured

water molecule (HOH 2017 in the PDB entry 2YDO¹⁷).

From the docking results in 1−A_2AAR and from the 4−

A_2AAR complex, the small and flexible 5′-OH group of agonists

4−13 could assume different orientations in the 5′ subpocket of

A_2AAR, and a water molecule could fit as well in the pocket to

enhance the contacts of the hydroxyl group with polar residues

in the binding cavity. For compounds 21 and 22,^39,45instead,

the 5′-substituent was bulky and rigid, and the residues in this

5′-subpocket needed to adjust during the MCMM optimization

in order to accommodate the cyclopropylcarboxamido group of

21 and the tetrazole moiety of 22. The movement of the

residues in the 5′-subpocket were minimal without any major

reorientation of the residues' side chains, nevertheless the need

of a slightly bigger pocket was evident during the automated

docking of compounds 21 and 22. The cyclopropyl ring of 21

was embedded by favorable hydrophobic interactions with

residues like Leu85 (3.33), Ile92 (3.40), and Trp246 (6.48),

while His250 (6.52) and Thr88 (3.36) coordinated the 5′

amide moiety with H-bond interactions. At the same time,

hydrophilic residue side chains in the 5′-subpocket were able to

stabilize the bulky tetrazole ring of 22. The predicted binding

mode of 22 showed H-bond interactions with Thr88 (3.36)

and Gln89 (3.37), and Asn181 (5.42) was found in proximity

to the 5′ moiety of 22.

Table 2. Energy Contributions to Binding at the A_2AAR

Associated with Individual Amino Acid Residues in

Proximity to the Co-Crystallized Ligand in the Binding Site

a

minimized docked

pose A (toward

H250), 4

minimized docked

pose B (toward

T88), 4

minimized docked

pose, 14

residue Tot En

vdW

Tot En

vdW

Tot En

vdW

V84

−7.26

−6.22

−6.63

−6.74

−6.51

−30.45

−3.40

−9.26

−7.11

−15.38

−1.81

−3.08

−5.27

−14.54

−2.61

−4.54

−8.65

−6.51

−28.40

−30.91

−46.15

−9.31

−8.69

−13.86

−5.16

−42.14

−6.84

−16.86

−28.44

−18.70

−7.19

−6.64

−4.62

−30.93

−3.75

−8.55

−6.28

−15.25

−4.41

−3.58

−5.16

−14.28

−3.21

−4.35

−10.03

−17.21

−29.98

−30.77

−45.86

−10.11

−11.58

−14.55

−36.64

−44.03

−7.23

−9.26

−15.61

−5.95

−30.60

−3.39

−10.83

−9.99

−15.35

−4.60

−3.98

−5.36

−14.64

−1.81

−4.93

L85

T88

−10.18

−30.17

−46.96

−10.99

−9.66

−13.31

−27.87

−43.30

−7.07

F168

E169

M177

W246

L249

H250

N253

M270

I274

−17.35

−30.83

−18.31

−17.77

−33.07

−17.13

S277

H278

a

Bold font refers to residues that form an H-bond with the ribose

Compared to the antagonist-bound A_2AAR structure, Glu169

in the EL2 assumed a different and specific rotameric

orientation of the side chain in the 1−A_2AAR structure.

Glu169 is oriented toward the C2-substituent of 1 in order to

engage in an H-bond interaction with the urea moiety of the

agonist. In 2−A_2AAR, the carboxyl group of Glu169 is oriented

toward the binding site, interacting with the exocyclic amine

group of the antagonists and further stabilized by the H-bond

contact with His264 in EL3. The shifted orientation of the

Glu169 side chain in the 1−A_2AAR structure is believed to be

very specific for agonists sharing with 1 both a bulky substituent

moiety.

contribution to the binding energy by the proximity of His250

(6.52) was small and predominantly a vdW contribution due to

the distance between the residue and the ligand. The energy

contribution of Thr88, instead, was still substantial even when

the 5′-OH group was engaged with His250, making this

orientation the energetically most favored pose after

optimization. Furthermore, the predicted energy contributions

of each contact residue did not take into account the presence

of water molecules which may impact the interaction network.

Figure 3. Docking poses of the C2 derivatives 7 ((a) with carbon atoms colored in orange) and 10 ((b,c) with carbon atoms colored in orange and

green, respectively) in the optimized binding site of the 1−A_2AAR structure. The key H-bond interactions between the compounds and the residues

of the binding pocket are highlighted as dotted lines. The bulky and hydrophobic naphthyl moiety of 7 could fit in the binding site of 1−A_2AAR only

after the rotation of Glu169 side chain toward the adenine moiety of the ligand, engaging in a strong H-bond interaction with the exocyclic amine

group of 7 and relieving the steric clash with the C2 substituent of the agonist. The rotamer flexibility of Glu169 in the 1−A_2AAR structure is shown

with the two possible binding modes of 10. The carboxyl group of Glu169 could interact with the exocyclic amine group of the ligand (b) or with the

amide carbonyl group at the C2 substituent of 10 (c).

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Figure 4. (a) Docking poses of N⁶derivatives 11 (with carbon atoms colored in green), 12 (with carbons in orange), and 13 (with carbons colored

in cyan) in the binding site of the 1−A_2AAR structure. The bulky N⁶aromatic rings of the agonists are located in a hydrophobic pocket at the

extracellular region of TM6, TM7, EL2, and EL3. (b) Predicted binding orientation of 20 in the binding site of 1−A_2AAR. The key H-bond

interactions between the compounds and the residues of the binding pocket are highlighted as dotted lines.

at the N⁶position and H-bond donor groups in the C2-

substituent. In the case of agonists nonsubstituted at the N⁶

position, Glu169 can be expected to assume a 2-like rotameric

orientation of the side chain in order to H-bond with the

exocyclic amine. The prediction of the rotameric adjustment of

Glu169 toward the adenine primary amine has been confirmed

by the 14−A_2AAR and 4−A_2AAR structures, where the

rotameric state of Glu169 and the conformation of EL3 are

very similar to the ones observed in the 2−A_2AAR complex.

Here we also considered this assumption for agonists with

hydrophobic and bulky substituents at the C2 position (α-

naphthylethyl ether derivative 7 among others⁴¹). After the

docking of 7 in the rigid 1−A_2AAR structure, the steric and

electrostatic clashes between the ligand naphthyl substituent

and Glu169 were preventing the retrieval of low energy docking

poses. After MCMM optimization of the docked pose of 7 and

the residues in the proximity, a rotation of the Glu169 carboxyl

chain occurred, relieving the clashes with the ligand and

enhancing the binding interactions with an additional strong H-

bond interaction with the exocyclic amine (Figure 3a). The

intuitive supposition that for N⁶nonsubstituted agonists,

Glu169 should contribute favorably to the binding energies

through an H-bond interaction with the exocyclic amine was

emphasized by the prominent energy contribution of Glu169 in

anchoring the adenine moiety of the small and nonsubstituted

agonists 4 and 14, as shown in Table 2. All the agonists in our

list with a primary amine at the N⁶position were docked in the

optimized binding site of the 1−A_2AAR structure with a 2-like

rotamer of Glu169.

analysis of the docked poses in the 1−A_2AAR structure. The

docking poses of compound 10 are shown in Figures 3b,c. The

4-methylamide pyrazole chain of compound 10 was protruding

toward the extracellular part similarly to the 2-substituent of 1

in the crystal structure. Interestingly, the pyrazole moiety of

compound 10 was about the same length as the ethylamide

linker of compound 1 with respect to the overlap of the NH

group of the methylamide moiety of compound 10 with one of

the NH groups of the urea moiety in compound 1. After

optimization of the docking pose of 10 and the binding site

residues of 1−A_2AAR, the carboxyl group of Glu169 was still

oriented to interact with the C2-amide of agonist 10. In this

predicted pose of compound 10, the carboxyl group of Glu169

acted as H-bond acceptor from the NH group of the same

amide, while the hydroxyl group of Tyr271 (7.36) acted as H-

bond donor in favor of the carbonyl group of the methylamide

chain of the agonist. This double H-bond lock of the C2-chain

stabilized the agonist 10 in the active mode inside the binding

cavity in a manner similar to 1, although less strongly. Beside

the favorable electrostatic interactions, hydrophobic residues in

the extracellular part of the pocket were embedding the

substituent at the C2 position of the purine moiety, such as

Leu267 in EL3 and Leu167 in EL2 that are in proximity of the

methyl group of the proximal amide. On the other hand, the

optimized binding orientation of 10 in the 1−A_2AAR binding

site with a 2-like rotamer of Glu169 led to a similar

conformation of the ligand with the H-bond interaction

between the C2-side chain and the hydroxyl group of Tyr271

(7.36), and no further rotation of Glu169 toward the amino

group at the C2-substituent, while the interaction between

Glu169 and the purine exocyclic amine of 10 was maintained

(Figure 3b). We interpreted this finding as the ambivalent

ability of Glu169 to interact with both H-bond donor groups of

10 in order to stabilize the molecule in the binding cavity of the

receptor. The rotamer flexibility of Glu169 in the 1−A_2AAR

structure might be favored by the distance between Glu169 and

His264 (EL3) and the consequent lack of the H-bonding

The diagnostic vasodilator 10, an adenosine derivative

containing a primary amine at the N⁶position and a hydrophilic

side chain at the C2 position,²²was docked in both the binding

sites of the 1−A_2AAR structure before and after the rotameric

optimization of Glu169. The contributions to the binding

energy of Glu169 to the docked pose of 10 in both binding

sites, shown in Table 2, were prominent in both docked poses.

Moreover, no rotation of the Glu169 side chain was observed

after optimization by means of MCMM conformational search

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interaction between these two residues as shown in the 2−

A_2AAR complex.

was instead oriented toward the extracellular part of TM6,

reaching Thr256 (6.58) (Figure 4b). An unusual feature of

compound 20 is also the substitution of the classical ribose of

the adenosine-like agonists with a cyclopentyl ring. Never-

theless, the conformation of the 5-membered ring was similar

to the one assumed by the ribose ring of the other agonists in

our list, thanks to the strong H-bond interactions between the

substituents at 2′, 3′, and 5′ positions and the residues Ser277

(7.42), His278 (7.43), His250 (6.52), and Thr88 (3.36) in the

binding pocket.

A widely used A_2AAR agonist 16 shows a long and flexible

chain at the C2-position ending with a carboxyl acid group.

Among the docking poses of compound 16 in 1−A_2AAR, some

had an H-bond interaction between the carboxyl group of the

ligand and the hydroxyl group of Tyr271 (7.36), but the lowest

energy conformation had the terminal carboxylate group free in

the extracellular media. The C2 aromatic ring of 16 was located

in the space between Tyr271 (7.36), Leu267 (7.32), and

Leu167 (EL2), embedded by favorable hydrophobic inter-

actions with these residues, but more possible orientations of

the C2 side chain were found due to the flexibility of the chain

and the open solvent exposed space between the upper part of

TM7 and EL2.

On the other hand, favorable interactions of the rotamer of

Glu169 in the 1−A_2AAR X-ray structure contributed to the

stabilization of the predicted binding modes for those agonists

having substitution at the N⁶position. A secondary amine at N⁶

of the adenine core is prevented from interacting with the

Glu169 side chain because the only available hydrogen atom is

already engaged in an H-bonding interaction with Asn253

(6.55). Beside compound 1, in our list there are four other

agonists that are substituted at the N⁶position (Figure 4).

Compound 11,^42,46a monosubstituted adenosine derivative

that was one of the first agonists to display any degree of A_2AAR

selectivity, is similar to 1 at N⁶but is lacking the C2-substituent.

The most favorable binding poses for compound 11 showed

the diphenyl moiety oriented similarly to the orientation of the

group in the 1−A_2AAR X-ray structure. As in the case of 1, the

diphenyl moiety of compound 11 had extensive hydrophobic

interactions with the receptor. Met270 (7.35) stabilized both

phenyl rings from above with hydrophobic interactions. Tyr271

(7.36) and Ala273 (7.38) were in close proximity to one

aromatic ring of agonist 11 offering good hydrophobic

interactions, while Thr256 (6.58), Ile252 (6.54), and Ala256

(EL3) were stabilizing the other phenyl ring. Met174 (5.35)

was found in close proximity to the ethyl linker between N⁶and

the phenyl rings (Figure 4).

The chirality of the unevenly substituted diphenyl derivative

12 was studied,^42,46and both diastereomers were docked in the

binding cavity of the receptor. The best poses for the

diastereomer of agonist 12 having an (R) configuration of the

N⁶group showed an orientation of the phenyl rings similar to

the docking pose of 11, with the methyl-substituted ring close

to Tyr271 (7.36) and below Met270 (7.35), while the

dimethoxy-substituted ring was closer to the EL2 residues.

The methyl group of the first ring could be found pointing

toward TM6 and TM7, close to Ile252 (6.54) and Ala273

(7.38), and groups on the other ring were found pointing

toward the solvent and close to the methyl group of Thr256

(EL3) and Met270 (7.35) (Figure 4a). The (S) diastereomer of

12, instead, showed an orientation of the diphenyl moiety that

led to a shifted adenine ring weakening the interaction between

the adenine ring and Asn253 (6.55) (Supporting Information

Figure S3). From these results, we suggested that between the

two diastereomers of 12 the (R)-diastereomer should be more

active than the (S).

Also, many conformations of the long and positively charged

C2-chain of amine congener 17 were found after the MCMM

optimization of the docked complex in 1−A_2AAR, and the most

energetically favorable orientation was found with the positively

charged terminal amino group of 17 interacting with both the

carboxyl groups of Glu169 and Asp170 in EL2.

Compound 18 acts at the A_2AAR in a peculiar manner

because the terminal aryl isothiocyanate group of the agonist

C2 chain is assumed to bind irreversibly to the receptor.⁴³The

possible residues that can react with the isothiocyanate group

are lysine or, less likely, cysteine, and because the C2-

substituent is protruding toward the extracellular medium, the

targeted lysine residues should be found in the extracellular part

of the receptor; however, there are only two lysines in the

extracellular portion of the A_2AAR, Lys150, and Lys153, located

in the EL2. Unfortunately, neither is present in the 1−A_2AAR

X-ray structure because that portion of EL2 was missing.

We also studied the docking of compounds 16, 17, and 18,

all members of the same chemical series of long chain C2

derivatives,⁴³in the 14−A_2AAR structure. The region where the

C2-substituent was positioned in the extracellular part of the

binding cavity was formed by residues from the upper part of

TM7 (e.g., Met270 and Tyr271), EL2 (e.g., Glu169 and

Leu167), and EL3 (e.g., His264 and Leu267). This region is

very different between the 1−A_2AAR and the 14−A_2AAR X-ray

structures. In the 1−A_2AAR structure, EL3 is folded away from

the binding site, allowing a more open cavity mouth in order to

adjust to the bulky and aromatic nature of the N⁶substituent of

1. Instead, the smaller and nonsubstituted agonists fit perfectly

in the pocket of the 4−A_2AAR and 14−A_2AAR structures with

the EL3 conformation closer to the binding pocket and the

EL2. Another novel structural feature of the A_2AAR appearing

in the 4−A_2AAR and 14−A_2AAR structures is the presence of

the entire EL2 domain, including residues 149−157 missing in

both the 1−A_2AAR and 2−A_2AAR structures. The closer

conformation of EL3 and the complete EL2 domain in the 4−

A_2AAR and 14−A_2AAR structures led to a different shaped

opening of the pocket compared to the one observed in 1−

A_2AAR.

Diphenyl derivative 13 was similar to compound 1 with its

C2-substituent containing an amide group and a piperidine

ring.³⁶The docking poses of compound 13 in the binding

cavity of the receptor showed the orientation of the diphenyl

substituent at the N⁶position comparable to the orientation

assumed by the best poses of compound 11 and 12. The C2-

substituent was pointing toward the upper part to orient the

piperidine ring between Tyr271 (7.36) and Leu167 (EL2),

stabilized by hydrophobic interactions with these two residues

and also with Met270 (7.35) and one of the phenyl rings of the

N⁶-diphenyl moiety of the ligand itself.

Carbocyclic derivative 20 shows a different core at the N⁶

substituent with a chiral chain and a thiophene ring.⁴⁴In the

best docking pose, the thiophene ring was located below

Met270 (7.35) with the edge of the aromatic ring close to

Tyr271 (7.35) and the chlorine atom pointing toward the

interface between TM6 and TM7 close to Ile252 (6.54) and

Ala273 (7.38). The ethyl group attached to the chiral center

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Figure 5. Docking poses of C2 extended chain derivatives 16 ((a) with carbon atoms colored in green), 17 ((b) with carbons in orange), and 18

((c) with carbons colored in green) in the binding site of the 14−A_2AAR structure. The key H-bond interactions between the compounds and the

residues of the binding pocket are highlighted as dotted lines. The proximity of the isothiocyanate moiety of 18 to the positively charged amine of

Lys150 suggested the involvement of this residue in the irreversible binding of 18 to the receptor.

Table 3. Binding Affinity of a Series of Adenosine C2 Long Chain Amide Derivatives of 16 at Three Subtypes of Human ARs

a

affinity K_i, nM or (% inhibition)

b

compd

R =

A₁

380

A_2A

A₃

570

efficacy (A_2A), % of maximal

16

27

28

29

30

33

34

35

36

37

38

39

40

41

42

OH

70

100

L-Phe-OMe

D-Phe-OMe

L-Trp-OMe

D-Trp-OMe

L-Asp-OH

D-Asp-OH

L-Arg-OH

D-Arg-OH

L-Phe-OH

D-Phe-OH

L-Trp-OH

D-Trp-OH

L-His-OH

D-His-OH

1080 210

1230 180

1670 260

1610 100

1900 660

1180 360

1110 30

990 320

640 170

550 100

1060 310

2160 280

1000 160

350 40

160 50

84.3 3.0

87.2 3.8

130 40

160 80

140 10

250 90

1460 600

790 160

620 250

220 60

260 140

140 30

200 50

520 60

830 290

320 150

110.9 2.5

92.6 12.4

104.5 13.6

118.6 5.7

94.8 6.7

94.3 7.3

94.5 5.8

94.7 6.7

103.3 13.8

93.4 12.4

95.8 7.4

113.3 5.0

105.2 4.9

105.9 6.5

130

4

180 60

110 10

100

50

4

5

63.7 13.1

34.0 3.2

71.7 16.9

130 30

110 30

40

4

a

Using CHO or HEK293 (A_2Aonly) cells stably expressing a human AR (Supporting Information); affinity was expressed as K_ivalue (n = 3−5) or

percent inhibition of radioligand binding at 10 μM. Compounds 31 and 32 are conjugates of ^L- and D-His-OMe, respectively, and were not tested

biologically. Maximal efficacy (at 10 μM) in an A_2AAR functional assay, determined by stimulation of cyclic AMP production in stably transfected

b

CHO cells, expressed as percent (mean standard error, n = 3−5) in comparison to effect (100%) of full agonist 16 at 10 μM.

The binding sites of the 4−A_2AAR or 14−A_2AAR structures

are not optimal for docking the adenosine moiety because of

the substitution of the binding site residue Gln89 (3.37) with

Ala in the thermostabilized constructs. Gln89 is not involved in

direct interactions with the agonists, as shown in the 1−A_2AAR

structure, but its mutation could affect the binding of ligands

and the activity of the A_2AAR (Supporting Information Table

S1). Nevertheless, the 4−A_2AAR and 14−A_2AAR structures,

compared to the 1−A_2AAR structure, have the advantages of

the presence of the density of the whole EL2 domain and the

proximity of EL3 to the binding cavity. In the 1−A_2AAR, the

residues 149−157 are missing, and within this region are the

positively charged residues Lys150 and Lys153, which in the

14−A_2AAR complex are located in proximity of the binding site

above Glu169 and Asp170.

The docking poses of 16, 17, and 18 in the 14−A_2AAR

structure were overall similar to the ones in the 1−A_2AAR

complex but with different orientations and different

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Figure 6. Predicted docking orientations of D-amino acid conjugates 36 ((a) with carbons colored in yellow), 38 ((b) with carbons in orange), and

42 ((c) with carbons colored in orange). The guanidinium group of 36 was engaged in H-bond interactions with Glu169 (EL2) and Thr256 (6.58).

The phenyl ring of 38 and the imidazole ring of 42 were located between the positively charged amino group of Lys150 (EL2) and the aromatic side

chain of His264 (EL3).

interactions of the C2 substituents. The binding pose of 16 in

the 14−A_2AAR binding site showed the carboxyl group

anchored to Lys153 by an ionic interaction (Figure 5a). The

positively charged amino group of compound 17 docked in the

14−A_2AAR structure was engaged in an ionic interaction with

Glu169, while the amide carbonyl group was anchored to

Lys153. Those interactions oriented the long C2 chain of

amino congener 17 in the groove formed by residues of EL2

and EL3 at the top of the opening of the receptor binding

cavity (Figure 5b).

After an MCMM conformational search of the isothiocyanate

derivative in the binding site of 14−A_2AAR, the long C2-chain

of the affinity label 18 was found in the EL2−EL3 groove with

the thiourea anchored to the carboxyl group of Glu169 and the

isothiocyanate group in close proximity to the positively

charged amino group of Lys150 (Figure 5c), identifying this

residue as a possible irreversible anchoring site to the A_2AAR by

18 and perhaps other electrophilic affinity labels.

Molecular Docking of Novel Agonists to Agonist-Bound

A_2AAR Crystal Structures and Their Synthesis and Pharma-

cological Characterization. The terminal carboxylate of 16

was selected as the site for modification in new derivatives to be

synthesized (Table 3). Amide derivatives were prepared by

condensing the α-amino group with various charged or

aromatic amino acids in protected form with 16, followed by

saponification of the ester protecting groups. The amino acid

conjugates were tested in standard binding assays at AR

subtypes and a functional assay at the A_2AAR. The

pharmacological properties of ^L- and D- stereoisomers were

compared. The corresponding methyl esters of the Phe and Trp

derivatives, which were synthetic intermediates, were also

included in the biological assays.

D-Amino acid conjugates were generally more potent than L-

and free terminal carboxylates more potent than the

corresponding methyl esters (Table 3). Compounds 38 and

42, ^D-Phe and D-His conjugates, respectively, were the most

potent at the A_2AAR with slightly greater affinities than the

parent 16. The selectivity for the A_2AAR in comparison to the

A₁AR was generally greater than with respect to the A₃AR. The

differences in A_2AAR affinity depending on the attached amino

acid were relatively small, indicating that this region of the

nucleoside is not subject to precise constraints imposed on the

adenosine moiety deeper in the binding site. The functional

assay of stimulation of cAMP production indicated that all of

the analogues were full agonists of the A_2AAR.

Upon receptor docking, the amino acid moieties were

coordinated in a subpocket close to the exofacial surface

(Figure 6). This subpocket was previously defined in the

binding site of the terminal portion of the C2 chain of 16−18.

In the predicted binding modes, the free carboxyl terminal of all

the amino acid derivatives was engaged in an ionic interaction

with one of the lysine residues in EL2, mostly Lys153, as was

the carboxyl group of 16. Doubling the negative charge with

aspartic side chains in derivatives 33 and 34 led to another

predicted ionic interaction with also Lys150 but not to a gain in

binding affinity, possibly because of the repulsive proximity of

Glu169, located just below the two lysine residues, which kept

the aspartic side chain of 33 and 34 away from the EL2−EL3

groove. We tried the addition of a positive charge with the

guanidinium group of an arginine with the aim to create an

interaction with Glu169. In the predicted docking modes of 36,

the long arginine chain not only interacted with Glu169 but was

able to reach to Thr256 (6.58).

We used some aromatic side chains to test the engagement of

His264 (EL3) in the binding affinity of these agonist

derivatives. The phenylalanine ring, in the docking poses of

37 and 38, was located between the positive charge of Lys150

(EL2) and the aromatic imidazole of His264 gaining favorable

interactions and binding affinity. The same involvement of

Lys150 and His264 to sandwich the aromatic moiety of the

agonist substituent was observed in the predicted binding

modes of 40 and 42. The tryptophan indole moiety was,

evidently, too bulky to fit properly in the EL2−EL3 groove,

particularly for the ^L-enantiomer 39, while the small imidazole

ring of the histidine substituent, studied in both its protonated

and nonprotonated states, could be easily located between

Lys150 and His264 with also an H-bond anchoring to Glu169.

DISCUSSION

■

We have found that docking of known adenosine agonists to

the agonist-bound X-ray crystallographic structures of the

A_2AAR provides a consistent set of interactions between ligand

and receptor. These docking models are closely tied to

physically determined structures and are expected to be more

accurate than previous agonist binding to homology models

based on the inactive state of bovine rhodopsin or other

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receptors. Nevertheless, many of the agonist binding features

determined in prior modeling to inactive states of GPCRs were

confirmed in the subsequent crystallographic studies.

ethylcarboxamide substituent than in the case of the smaller

OH group, as shown by the energy components in Table 2.

The docking results of compound 22, with the bulky and rigid

tetrazole moiety, suggested that the 5′ subpocket was spacious

and easily adaptable to accept substituents of different size and

nature. The 5′ subpocket is a potential site for the development

of new substituted agonists in order to analyze the SARs

needed to improve the binding and the selectivity of the

adenosine derivatives as agonists for the A_2AAR (Tosh et al., in

preparation).

It is anticipated that the new structures will guide the

structure-based discovery of new GPCR agonists and,

specifically, AR agonists. We have tested the approach of

using the docking of new compounds with a series of amino

acid derivatives. The amino acids were appended at the same

site, located at the terminal position of a long C2 chain, which

was largely exposed to the extracellular space in the crystal

structure of the 1−A_2AAR complex. Thus, the interaction with

the receptor was expected to be less constrained than if similar

changes were made in other parts of the molecule closer to the

minimal nucleoside pharmacophore.

Adenosine Core Subpocket of A_2AAR. The adenosine

pharmacophore of the agonists is bound in a cavity formed

among TM3, TM6, and TM7. In the pocket, the adenine ring

oriented vertically, anchored by strong π−π stacking

interactions with Phe168 (EL2), H-bond interactions with

Asn253 (6.55), and favorable vdW contacts with Leu249 (6.51)

and Ile274 (7.39). The ribose moiety was located deeper in the

pocket, anchored by H-bond interactions with Ser277 (7.42)

and His278 (7.43) through the 2′- and 3′-OH, and embedded

by hydrophobic residues Val84 (3.32), Leu274 (7.39), and

Leu85 (3.33). More H-bond interactions are possible with

His250 (6.52) and Thr88 (3.36) through the 5′-substituents of

the ligands. The interactions between the agonists and the

residues in this subpocket are expected to be conserved for all

the agonists sharing the adenosine moiety. The poses of 1

redocked into the 1−A_2AAR structure were retrieved with a

docking procedure based on the knowledge of those

interactions between the agonist and the receptor, i.e. the H-

bond interactions with Asn253 (6.55), Ser277 (7.42), and

His278 (7.43). For the agonists in our list, those main

interactions between the adenosine core and the residues of this

subpocket were all retrieved by our docking approach in 1−

A_2AAR or 14−A_2AAR.

Ribose 5′ Subpocket of A_2AAR. The biological activity of

truncated nucleosides lacking 5′-substitution at the furanose

ring, e.g. compound 3, suggests that the occupation of the 5′

subpocket is not essential for the binding to the A_2AAR,

although the favorable interactions with this pocket of a 5′-

uronamide, as in 14, tend to enhance the activity and the

selectivity of the agonists for the receptor. From the docking

results, the 5′-subpocket could adjust to different substituents,

from the small and flexible hydroxyl group to the bulky and

rigid tetrazole moiety, in order to create favorable interactions

between the residues and the substituents. The pocket of

A_2AAR accommodating the agonist 5′-substituent was formed

by both hydrophilic residues, such as His250 (6.52), Thr88

(3.36), Gln89 (3.37), and Asn181 (5.42), and hydrophobic and

bulky residues, like Leu85 (3.33), Trp246 (6.48), Ile92 (3.40),

Cys185 (5.46), and Val186 (5.47). The OH-group of agonists

4−13 was small enough to assume different orientations in the

pocket, preferring the orientation toward His250 (6.52) and, as

shown by the adenosine-bound A_2AAR complex,¹⁷the

interposition of a water molecule. The ethyl- and cyclo-

propylcarboxamide groups of agonists 14−21, instead, were

firmly locked in the 5′ subpocket by H-bond interactions with

His250 (6.52) and Thr88 (3.36) and further stabilized by

favorable vdW contacts through their hydrophobic terminal. In

particular, the vdW contributions to the binding energy of

Leu85 (3.33) was more conspicuous in the case of the

Adenine N⁶Subpocket of A_2AAR. The region of 1−

A_2AAR that accommodates the bulky N⁶-diphenylethyl group of

1 is located between the extracellular termini of two helices

(TM6, TM7) and two loops (EL2, EL3) and is formed

predominantly by hydrophobic residues. The same subpocket

was occupied by the N⁶substituents of compounds 11−13 and

20, according to their predicted binding modes. The subpocket

that holds the N⁶substituent in the A_2AAR is quite flexible due

to the easy movements of some of the residues in TM7 and

EL3. In fact, comparing the X-ray structures of 1−A_2AAR and

14−A_2AAR or 4−A_2AAR, the pocket for the N⁶substituent is

wide open in the 1−A_2AAR but obstructed in the 14−A_2AAR

structure. The specific rotamer of Met270 (7.35) and the

outward folding of EL3 in 1−A_2AAR allowed the formation of a

subpocket for the N⁶substituent of 1. The space created in

TM7 between Met270 (7.35) and Ile274 (7.39) by the

movement of Met270 side chain toward the extracellular part of

the cavity of 1−A_2AAR, according to the predicted binding

modes of 11−13 and 20, could be occupied by one of the

aromatic rings of the N⁶substituents, a phenyl ring for 11 and

13, the methylphenyl ring for 12, and the thiophene moiety for

20.

Other hydrophobic residues embedding this aromatic ring

were Leu249 (6.51), Ile252 (6.54), and Ala273 (7.38). In the

predicted binding poses, the other phenyl ring of the N⁶

substituent of 11−13 was always pointing toward the solvent

in the extracellular medium, exiting the binding cavity in a

vertical way between Met270 (7.35) and Thr256 (6.58). The

side chains of Thr256 (6.58), Glu169 (EL2), Met270 (7.35),

Met174 (5.35), and Ile252 (6.54) also contributed with

favorable vdW contacts to the stabilization of the N⁶

substituent. In the predicted binding modes of the N⁶-

substituted agonists, Glu169 in EL2 had to assume a rotamer

similar to the one in 1−A_2AAR in order to allow the bulky

substituents of 11−13 and 20 to adjust in the pocket and to

contribute with favorable vdW contacts through the aliphatic

part of the side chain, pointing the charged carboxyl group away

even in the absence of a polar substituent at the C2 position of

the agonists, as in the case of 11, 12, and 20.

Obviously, Met270 (7.35) was a key residue in the binding of

the N⁶substituted agonists, not only contributing with

favorable interactions to the recognition of the N⁶group but

also with its flexibility that allowed the formation of the

subpocket of the substituent. The nonconserved nature of the

residue at position 7.35 among the adenosine receptors

subtypes could also be a discriminating residue for the

selectivity of N⁶-substituted agonists. The corresponding

residues of Met270 in A_2AAR are the small Thr270 in A₁AR

and the bulky Leu264 in A₃AR.

Another residue located in the N⁶subpocket and involved in

the anchoring of the N⁶substituent of the A_2AAR agonists, but

nonconserved among the adenosine receptors, is the Thr256

(6.58), which becomes a bulky leucine in A₃AR.

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Adenine C2 Subpocket of A_2AAR. The C2-subpocket of

A_2AAR is formed between TM7, EL2, and, marginally, TM2. A

key residue in the C2-subpocket of A_2AAR is Tyr271 (7.36), at

the extracellular terminus of TM7. The polar and aromatic

nature of Tyr271 could contribute in different ways to the

anchoring of the agonists. In the 2−A_2AAR structure, the

binding contribution from Tyr271 was minimal and predom-

inantly from vdW contacts. In the 1−A_2AAR structure, instead,

the contribution of Tyr271 to the binding of 1 became

prominent due to the strong H-bond interaction between the

OH group of the tyrosine and the carbonyl group of the urea

moiety in 1. Obviously, the involvement to the ligand binding

by Tyr271 was less relevant for small agonists not-substituted at

the C2 position, like 4 or 14. From the predicted binding

modes of the C2-substituted agonists in 1−A_2AAR and 14−

A_2AAR, the bivalent role of Tyr271, able to adjust to

substituents of a different nature, was strategic for the creation

of favorable contacts with the ligands. For agonists with

hydrophobic C2 side chains, compounds 3 and 5 among the

others, the main contribution to the stabilization of agonists by

Tyr271 was through vdW contacts. For compounds with a H-

bond acceptor group on the C2 chain, as in the case of 10 and

13, Tyr271 could form strong H-bond interactions to anchor

the molecule in the binding site. Other residues in the C2

subpocket are hydrophobic, like Ile274 (7.39), Ile66 (2.66),

Leu167 (EL2), Met270 (7.35), and Leu267 (7.32), favoring

hydrophobic chains at the C2 position of the agonists. Longer

C2 side chains, as for compounds 17 and 18, once reached the

top of the cavity preferred to turn toward TM6 occupying the

groove formed between EL2 and EL3, in order to interact with

the residues of the ELs, such as Glu169, Lys150, and Ly153.

The binding region on the receptor for long and flexible C2

substituents extended above the principal adenosine binding

cavity, following the groove between EL3 and EL2 but mostly

exposed to the solvent. Compared to 1−A_2AAR, this EL2-EL3

groove in 14−A_2AAR is narrow due to the proximity of EL3 to

EL2 through the H-bond interaction between His264 and

Glu169. The main functional side chains of the receptor

exposed to the agonist C2 substituents are the two positively

charged lysine residues, Lys150 and Ly153, the negative

carboxylate group of Glu169, and the indole ring of His264.

In the predicted binding poses, the carboxyl group of 16 was

engaged in an ionic interaction with Lys153, while the long

chain of agonist 17 interacted with the Lys153 through its

amide carbonyl group and with Glu169 through its charged

amine group. The longer and specific side chain of 18 was

locked in the EL2-EL3 groove of the 14−A_2AAR structure by

strong interactions between the thiourea moiety and Glu169

and the possible attack by the thiocyanate group to the amine

group of Lys150.

comparison between the antagonist-bound and the agonist-

bound structures of A_2AAR, making this region easily adjustable

to various substituents.

The ^D-derivatives of this amino acid series of compounds

showed higher binding affinities compared to the ^L-derivatives.

From the predicted poses, the ^L-side chains were located very

close to the EL2 wall, while the ^D-side chains were more in the

middle of the groove. EL3 is more flexible than EL2 and can

adjust more easily to the shape of the various ligand

substituents, as shown by the outfolding of EL3 in the 1−

A_2AAR structure. Besides the highly flexible portion with

Lys150 and Lys153, present only in the 14−A_2AAR and 4−

A_2AAR structures, EL2 contains a α-helical portion with Phe168

and Glu169 that is quite fixed in the binding cavity of A_2AAR

and less adjustable to bulky ligand substituents. Among the

amino acids derivatives tested, some gain in affinity at the

A_2AAR was possible with the positive charged side chain of 36

and the aromatic side chains of 38 and 42. In the predicted

binding pose of 36 in the 14−A_2AAR binding cavity, the

arginine side chain was anchored to Glu169 (EL2) and Thr256

(6.58), while the aromatic rings of 38 and 42 were located

between Lys150 (EL2) and His264 (EL3). Those docking

poses suggested an involvement of residues in the ELs in the

coordination of long C2 substituents of nucleoside agonists.

CONCLUSION

■

In conclusion, molecular modeling of GPCRs is shown to be a

useful technique in probing the determinants of agonist

recognition, especially when the modeling takes into account

all of the available supporting data (multiple X-ray structures,

mutagenesis, and SAR). The position of the adenosine moiety

and its overlay with a bound antagonist were well approximated

using modeling based on the inactive A_2AAR structure, and now

the agonist-bound structures provide detailed information. By

examining the molecular recognition of 20 known A_2AAR

agonists, a structural basis for previous empirical SARs

consistent with previous mutagenesis results is provided. We

are just at the beginning of learning how to effectively utilize

the new agonist-bound X-ray structures of GPCRs in drug

discovery, but it is clear that the insights gained can be used to

guide the rational design of new synthetic analogues. Docking

predictions made for these agonists can now be used as an aid

in extending the SAR in this chemical series to predict the

likelihood in increasing A_2AAR affinity. We have prepared a

series of amino acid derivatives at the terminal position of a C2

chain that maintain moderate affinity and the putative site of

binding of the added moieties associated with EL2 and EL3 has

been predicted. This approach can potentially be extended to

other regions of the nucleoside structure.

To understand if interactions with the residues in the EL2−

EL3 groove could bring advantage to the binding affinity of the

agonists, we designed some amino acid derivatives of 16 with

different functional groups that, from the predicted binding

poses, could fit the C2 subpocket. In predicted binding poses of

nucleoside derivatives with long C2 side chains, the C2

substituents were mostly directed toward the flexible and

solvent exposed region at the opening of the binding pocket

formed by residues of EL2 and EL3. This region of the receptor

is the most variable in amino acid nature within the ARs,

possibly granting some selectivity toward specific subtypes of

the ARs. Moreover, upon agonist binding, this region

undergoes many conformational changes as shown by the

EXPERIMENTAL SECTION

■

Chemical Synthesis. Briefly, commercially available nucleoside

carboxylic acid 16 was coupled to various carboxylate-protected amino

acids using either EDC·HCl (1-ethyl-3-[3-dimethylaminopropyl]-

carbodiimide hydrochloride) or HATU (2-(1H-7-azabenzotriazol-1-

yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate methanami-

nium) as coupling agent in anhydrous DMF to obtain the

corresponding amino acid coupled methyl ester derivatives (Support-

ing Information). Subsequently, the methyl ester was hydrolyzed using

0.1 M aqueous NaOH to give the target carboxylic acid derivatives

33−42.

Purification of the nucleoside derivatives for biological testing was

performed by HPLC with a Luna 5 μ RP-C18(2) semipreparative

column (250 mm × 10.0 mm; Phenomenex, Torrance, CA) under the

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Journal of Medicinal Chemistry

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following conditions: flow rate of 2 mL/min, 0.5% trifluoroacetic acid

in H₂O−CH₃CN from 100:0 (v/v) to 50:50 (v/v) in 32 min.

Analytical purity of compounds was checked using a Hewlett-Packard

1100 HPLC equipped with Zorbax SB-Aq 5 μm analytical column (50

mm × 4.6 mm; Agilent Technologies Inc., Palo Alto, CA). Mobile

phase: linear gradient solvent system, 5 mM TBAP (tetrabutylammo-

nium dihydrogenphosphate)−CH₃CN from 80:20 to 40:60 in 13 min;

the flow rate was 0.5 mL/min. Peaks were detected by UV absorption

with a diode array detector at 254, 275, and 280 nm. All derivatives

tested for biological activity showed >99% purity by HPLC analysis

(detection at 254 nm).

structure. The best docking pose of 1 was chosen based on the

docking energies and the best rmsd value compared with the X-ray

pose of 1.

Molecular Docking of Agonists 11−13 and 20. The molecular

docking of 11−13 and 18 was performed in the binding site of 1−

A_2AAR with the Glide SP protocol without distances restraints or with

MOE docking and the pharmacophore query, as described for agonist

1. The best scored docking poses showing the crucial H-bond

interactions between the adenosine core of the agonists and the

important residues Asn253, Ser277, and His278 of the receptor were

retained and subjected to MCMM conformational analysis by means

of MacroModel^31,47,48for further structural optimization and energy

minimization of the complexes. During the conformational search, full

flexibility was granted to the ligand and the residues of the receptor

within a radius of 4 Å from the ligand. All the other residues were

considered conformationally frozen during the calculations. One

thousand steps of MCMM were performed with the MMFFs force

field and the water GB/SA model as implicit solvent. Polak−Ribier

conjugate gradient (PRCG) minimization method with a convergence

threshold on the gradient of 0.05 kJ/mol·Å was used.

Molecular Docking of Agonists 3−10, 14−19, and 21−22 in the

Optimized Binding site of 1−A_2AAR. The automated docking of

agonists 3−10, 14−19, and 21−22 was performed in the optimized

binding sites of 1−A_2AA with a different rotameric state of the Glu169

side chain from the crystal structure. A rotamer of Glu169 with the

carboxyl group oriented toward the binding site was chosen after

MCMM conformational search of the binding site in order to recreate

the H-bond interaction between this residue and the primary N⁶

exocyclic amine of these agonists, in a similar way as in the 2−A_2AAR,

14−A_2AAR, and 4−A_2AAR crystal structures. The same molecular

docking protocols and parameters describe above for agonists 11−13

and 18 were used. After the automated docking the best scored poses

of the agonists were optimized by means of MCMM conformational

search as described in the previous section.

Molecular Docking of Agonists 16−18, and 27−42 in the

Optimized Binding Site of 14−A_2AAR. The binding site of 14−A_2AAR

was considered optimal for the docking study of agonists with long C2

substituents due to the presence of the entire EL2 of the receptor,

missing in the 2−A_2AAR structure. The automated docking and

MCMM conformational search for agonists 16−18 and 27−42,

characterized by long C2 substituents, were conducted in the binding

site of 14−A_2AAR using the same protocols described above.

The graphical pictures were made with the Pymol program (Delano

Scientific LLC, CA, USA) and MOE.

Pharmacological Characterization. Binding assays were per-

formed using membranes of mammalian cells stably expressing

recombinant A₁AR or A₃AR (CHO, Chinese hamster ovary) cells or

the A_2AAR (HEK-293, human embryonic kidney cells)⁴⁹⁻⁵¹and

radioligands [¹²⁵I]N⁶-(4-amino-3-iodobenzyl)adenosine-5′-N-methyl-

uronamide ([¹²⁵I]I-AB-MECA; 2000 Ci/mmol), [³H]R-N⁶-phenyl-

isopropyladenosine ([³H]R-PIA, 63 Ci/mmol), [³H]16 (47 Ci/

mmol), as described (Supporting Information).^50,51IC₅₀values

obtained from competition curves were converted to K_ivalues using

the Cheng−Prusoff equation.⁵²Data are expressed as mean standard

error.

For structural confirmation, ¹H NMR spectra were recorded at 300

Mz. Chemical shifts are reported in parts per million (ppm) relative to

deuterated solvent as the internal standard. ESI−high resolution mass

spectroscopic measurements were performed on a proteomics

optimized Q-TOF-2 (Micromass-Waters) using external calibration

with polyalanine.

Computational Methods. Receptor and Agonists Structures.

The crystal structures of the A_2Areceptor in complex with 1, 4, and 14

were obtained from the Protein Data Bank (PDB entry codes:

3QAK,¹⁶2YDO,¹⁷2YDV¹⁷). All the proteins were cleaned from water

molecules and ions, and hydrogen atoms were added to the receptors

and minimized by means of the Protein Preparation Wizard in

Maestro.³¹The T4-lysozyme insertion replacing the third intracellular

loop (IL3) in the 3QAK crystal structure was removed. The IL3,

which was far from the binding cavity, was not replaced. The

protonation states of the histidine residues in the binding cavity were

based on hydrogen bonding networks present in the crystal structures.

The previously published complexes of the docked agonists 4 and 14

to the antagonist-bound A_2AAR crystal structure¹⁹were also used. The

structures of the agonists were sketched in Maestro and minimized

using the Polak−Ribiere conjugated gradient (PRCG) method with a

convergence gradient of 0.001 kJ/mol·Å.

Molecular Docking of Agonist 1 to the 1−A_2AAR Structure. The

automated docking of 1 was performed in a rigid binding site of the

A_2AAR with different docking protocols to test the docking ability to

reproduce the cocrystallized orientation. The binding pocket of 1 in

the crystal structure of the 1−A_2AAR complex was considered for all

the docking approaches. The best scored docking poses from each

protocol were compared to the cocrystallized orientation of 1, and the

root-mean-square deviation (rmsd) was calculated as a measure of

docking reliability. Glide³¹was used to dock 1 using both the SP

(standard precision) and XP (extra precision) procedures, with default

parameters or with hydrogen bond constraints. The H-bond

constraints in Glide were applied during the generation of the docking

grid to enforce the H-bond formation between the adenosine core of 1

and the crucial residues in the binding site, i.e. between the amide

group of Asn253 and the N⁶amino group and N⁷of 1, between the N^ε

of His278 and the 2′-OH group of the agonist, and between the OH

group of Ser277 and the 3′-OH group of 1.

MOE³²docking was performed with Alpha PMI, Alpha Triangle,

Proxy Triangle, and Triangle Matcher as docking placement functions.

The cocrystallized orientation of 1 in the binding site of 1−A_2AAR

with a rmsd lower than 2 Å was reproduced using the “Pharmacophore

consensus” tool implemented in MOE. A receptor-based pharmaco-

phore model was build based on the crucial interactions between the

adenosine moiety of 1 and the binding site residues, and the

pharmacophore model was then used to filter the docking placements

in order to retrieve only the docking poses that could reproduce those

relevant agonist−protein interactions. The MOE pharmacophore

query (Supporting Information Figure S2) was defined on the crystal

structure of the 1−A_2AAR complex, and four pharmacophore features

were used: the H-bond acceptor/donor interactions between

Asn253(6.55) and the exocyclic amino group and N⁷of 1, and the

H-bond acceptor interactions between Ser277 and His278 and the 2′-

OH and 3′-OH of 1. The radii of the features were not modified.

Agonist 1 was docked to the binding site of the 1−A_2AAR structure

also with ICM-Dock module in the MolSoft LLC suite,^47,48using

mmff charges and the default setup. The binding cavity of the receptor

was defined as being within 5 Å of the cocrystallized 1 in the 1−A_2AAR

Functional assays of cAMP accumulation were performed using

membranes of CHO cells stably expressing recombinant A_2AAR, as

described.^53,54Statistical analysis was performed using Prism 4.0

(GraphPad, San Diego,CA).

ASSOCIATED CONTENT

■

S

* Supporting Information

Comparison of the predicted role in agonist recognition of

selected residues of the human A_2AAR and relation to the

agonist binding interactions in the X-ray structure, docking

figures for selected compounds, procedures for synthesis, and

biological assays for novel nucleoside derivatives, and

coordinates of complexes with 7 and 19 (PDF, PDB). This

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opsin in its G-protein-interacting conformation. Nature 2008, 455,

497−502.

material is available free of charge via the Internet at http://

pubs.acs.org.

(11) Standfuss, J.; Edwards, P.; D’Antona, A.; Fransen, M.; Xie, G.;

Oprian, D. D.; Shertler, G. F. X. The structural basis of agonist-

induced activation in constitutively active rhodopsin. Nature 2011,

471, 656−660.

AUTHOR INFORMATION

■

Corresponding Author

*Phone: (301) 496-9024. Fax: (301) 480-8422. E-mail:

kajacobs@helix.nih.gov.

(12) Choe, H. W.; Kim, Y. J.; Park, J. H.; Morizumi, T.; Pai, E. F.;

Krauss, N.; Hofmann, K. P.; Scheerer, P.; Ernst, O. P. Crystal structure

of metarhodopsin II. Nature 2011, 471, 651−655.

(13) Rasmussen, S. G.; Choi, H. J.; Fung, J. J.; Pardon, E.; Casarosa,

P.; Chae, P. S.; Devree, B. T.; Rosenbaum, D. M.; Thian, F. S.;

Kobilka, T. S.; Schnapp, A.; Konetzki, I.; Sunahara, R. K.; Gellman, S.

H.; Pautsch, A.; Steyaert, J.; Weis, W. I.; Kobilka, B. K. Structure of a

nanobody-stabilized active state of the β₂adrenoceptor. Nature 2011,

469, 175−180.

ACKNOWLEDGMENTS

■

This research was supported by the Intramural Research

Program of the NIH, National Institute of Diabetes and

Digestive and Kidney Diseases and the PSI:Biology program,

National Institute of General Medical Sciences U54

GM094618. We thank Dr. Stefano Costanzi (NIDDK) and

Dr. Soo-Kyung Kim (California Institute of Technology) for

helpful discussion.

(14) Rasmussen, S. G. F.; DeVree, B. T.; Zou, Y.; Kruse, A. C.;

Chung, K. Y.; Kobilka, T. S.; Thian, F. S.; Chae, P. S.; Pardon, E.;

Calinski, D.; Mathiesen, J. M.; Shah, S. T. A.; Lyons, J. A.; Caffrey, M.;

Gellman, S. H.; Steyaert, J.; Skiniotis, G.; Weis, W. I.; Sunihara, R. K.;

Kobilka, B. K. Crystal structure of the β₂adrenergic receptor−Gs

protein complex. Nature 2011, 477, 549−555.

ABBREVIATIONS USED

■

AR, adenosine receptor; CHO, Chinese hamster ovary; EL,

extracellular loop; GPCR, G protein-coupled receptor; HEK,

human embryonic kidney; HPLC, high performance liquid

chromatography; IL, intracellular loop; MCMM, Monte Carlo

multiple minimum; MOE, Molecular Operating Environment;

NECA, 5′-N-ethylcarboxamidoadenosine; rmsd, root-mean-

square deviation; TM, transmembrane α-helix; vdW, van der

Waals

́

(15) Warne, T.; Moukhametzianov, R.; Baker, J. G.; Nehme, R.;

Edwards, P. C.; Leslie, A. G.; Schertler, G. F.; Tate, C. G. The

structural basis for agonist and partial agonist action on a β₁-adrenergic

receptor. Nature 2011, 469, 241−244.

(16) Xu, F.; Wu, H.; Katritch, V.; Han, G. W.; Jacobson, K. A.; Gao,

Z. G.; Cherezov, V.; Stevens, R. C. Structure of an agonist-bound

human A_2Aadenosine receptor. Science 2011, 332, 322−327.

(17) Lebon, G.; Warne, T.; Edwards, P. C.; Bennett, K.; Langmead,

C. J.; Leslie, A. G. W.; Tate, C. G. Agonist-bound adenosine A_2A

receptor structures reveal common features of GPCR activation.

Nature 2011, 474, 521−525.

REFERENCES

■

(1) Fredriksson, R.; Lagerstrom, M. C.; Lundin, L. G.; Schioth, H. B.

The G-protein-coupled receptors in the human genome form five

main families. Phylogenetic analysis, paralogon groups, and

fingerprints. Mol. Pharmacol. 2003, 63, 1256−1272.

(2) Lagerstrom, M. C.; Schioth, H. B. Structural diversity of G

protein-coupled receptors and significance for drug discovery. Nature

Rev. Drug Discovery 2008, 7, 339−357.

(3) Tyndall, J. D.; Sandilya, R. GPCR agonists and antagonists in the

clinic. Med. Chem. 2005, 1, 405−421.

(4) Jaakola, V. P.; Griffith, M. T.; Hanson, M. A.; Cherezov, V.;

Chien, E. Y.; Lane, J. R.; IJzerman, A. P.; Stevens, R. C. The 2.6

angstrom crystal structure of a human A_2Aadenosine receptor bound

to an antagonist. Science 2008, 322, 1211−1217.

(5) Shimamura, T.; Shiroishi, M.; Weyand, S.; Tsujimoto, H.; Winter,

G.; Katritch, V.; Abagyan, R.; Cherezov, V.; Liu, W.; Han, G. W.;

Kobayashi, T.; Stevens, R. C.; Iwata, S. Structure of the human

histamine H₁receptor complex with doxepin. Nature 2011, 475, 65−

70.

(6) Wu, B.; Chien, E. Y.; Mol, C. D.; Fenalti, G.; Liu, W.; Katritch,

V.; Abagyan, R.; Brooun, A.; Wells, P.; Bi, F. C.; Hamel, D. J.; Kuhn,

P.; Handel, T. M.; Cherezov, V.; Stevens, R. C. Structures of the

CXCR4 chemokine GPCR with small-molecule and cyclic peptide

antagonists. Science 2010, 330, 1066−1071.

(7) Warne, T.; Serrano-Vega, M. J.; Baker, J. G.; Moukhametzianov,

R.; Edwards, P. C.; Henderson, R.; Leslie, A. G.; Tate, C. G.; Schertler,

G. F. Structure of a beta₁-adrenergic G-protein-coupled receptor.

Nature 2008, 454, 486−491.

(18) Cherezov, V. Lipidic cubic phase technologies for membrane

protein structural studies. Curr. Opin. Struct. Biol. 2011, 21, 559−566.

(19) Ivanov, A. A.; Barak, D.; Jacobson, K. A. Evaluation of homology

modeling of G protein-coupled receptors in light of the A_2Aadenosine

receptor crystallographic structure. J. Med. Chem. 2009, 52, 3284−

3292.

(20) Jaakola, V. P.; Lane, J. R.; Lin, J. Y.; Katritch, V.; IJzerman, A. P.;

Stevens, R. C. Ligand binding and subtype selectivity of the human A_2A

adenosine receptor: identification and characterization of essential

amino acid residues. J. Biol. Chem. 2010, 285, 13032−13044.

(21) Katritch, V.; Abagyan, R. GPCR agonist binding revealed by

modeling and crystallography. Trends Pharmacol. Sci. 2011, 32, 637−

643.

(22) Zablocki, J.; Palle, V.; Blackburn, B.; Elzein, E.; Nudelman, G.;

Gothe, S.; Gao, Z.; Li, Z.; Meyer, S.; Belardinelli, L. 2-Substituted pi

system derivatives of adenosine that are coronary vasodilators acting

via the A_2Aadenosine receptor. Nucleosides, Nucleotides Nucleic Acids

2001, 20, 343−360.

(23) Kobilka, B.; Schertler, G. F. New G-protein-coupled receptor

crystal structures: insights and limitations. Trends Pharmacol. Sci. 2008,

29, 79−83.

(24) Nygaard, R.; Frimurer, T. M.; Holst, B.; Rosenkilde, M. M.;

Schwartz, T. W. Ligand binding and micro-switches in 7TM receptor

structures. Trends Pharmacol. Sci. 2009, 30, 249−259.

(25) Ballesteros, J.; Weinstein, H. Integrated methods for the

construction of three-dimensional models of structure−function

relations in G protein-coupled receptors. Methods Neurosci. 1995, 25,

366−428.

(8) Katritch, V.; Reynolds, K. A.; Cherezov, V.; Hanson, M. A.; Roth,

C. B.; Yeager, M.; Abagyan, R. Analysis of full and partial agonists

binding to beta₂-adrenergic receptor suggests a role of transmembrane

helix V in agonist-specific conformational changes. J. Mol. Recognit.

2009, 22, 307−318.

(9) Park, J. H.; Scheerer, P.; Hofmann, K. P.; Choe, H. W.; Ernst, O.

P. Crystal structure of the ligand-free G-protein-coupled receptor

opsin. Nature 2008, 454, 183−187.

(10) Scheerer, P.; Park, J. H.; Hildebrand, P. W.; Kim, Y. J.; Krauss,

N.; Choe, H. W.; Hofmann, K. P.; Ernst, O. P. Crystal structure of

(26) Kim, J.; Wess, J.; van Rhee, M.; Schoneberg, T.; Jacobson, K. A.

̈

Site-directed mutagenesis identifies residues involved in ligand

recognition in the human A_2Aadenosine receptor. J. Biol. Chem.

1995, 270, 13987−13997.

(27) Olah, M. E.; Jacobson, K. A.; Stiles, G. L. Role of the second

extracellular loop of adenosine receptors in agonist and antagonist

binding: Analysis of chimeric A₁/A₃adenosine receptors. J. Biol. Chem.

1994, 269, 24692−24698.

551

dx.doi.org/10.1021/jm201461q | J. Med. Chem. 2012, 55, 538−552

Journal of Medicinal Chemistry

Article

(28) Gao, Z. G.; Chen, A.; Barak, D.; Kim, S. K.; Muller, C. E.;

4′-aza-carbocyclic nucleosides as adenosine A_2Areceptor agonists.

Bioorg. Med. Chem. Lett. 2010, 20, 1219−1224.

̈

Jacobson, K. A. Identification by site-directed mutagenesis of residues

involved in ligand recognition and activation of the human A₃

adenosine receptor. J. Biol. Chem. 2002, 277, 19056−19063.

(29) Mantell, S. J.; Stephenson, P. T.; Monaghan, S. M.; Maw, G. N.;

Trevethick, M. A.; Yeadon, M.; Walker, D. K.; Selby, M. D.; Batchelor,

D. V.; Rozze, S.; Chavaroche, H.; Lemaitre, A.; Wright, K. N.;

Whitlock, L.; Stuart, E. F.; Wright, P. A.; Macintyre, F. SAR of a series

of inhaled A_2Aagonists and comparison of inhaled pharmacokinetics in

a preclinical model with clinical pharmacokinetic data. Bioorg. Med.

Chem. Lett. 2009, 19, 4471−4475.

(30) Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J.

Experimental and computational approaches to estimate solubility and

permeability in drug discovery and development settings. Adv. Drug

Delivery Rev. 2001, 46, 3−26.

(31) Kellenberger, E.; Rodrigo, J.; Muller, P.; Rognan, D.

Comparative evaluation of eight docking tools for docking and

virtual screening accuracy. Proteins 2004, 57, 225−242.

(46) Trivedi, B. K.; Blankley, C. J.; Bristol, J. A.; Hamilton, H. W.;

Patt, W. C.; Kramer, W. J.; Johnson, S. A.; Bruns, R. F.; Cohen, D. M.;

Ryan, M. J. N⁶-Substituted adenosine receptor agonists: potential

antihypertensive agents. J. Med. Chem. 1991, 34, 1043−1049.

(47) Chang, G.; Guida, W. C.; Still, W. C. An internal coordinate

Monte Carlo method for searching conformational space. J. Am. Chem.

Soc. 1998, 111, 4379−4386.

(48) Mohamadi, F.; Richards, N. G. J.; Guida, W. C.; Liskamp, R.;

Lipton, M.; Caufield, C.; Chang, G.; Hendrickson, T.; Still, W. C.

Macromodel (1990) An integrated software system for modelling

organic and bioorganic molecules using molecular mechanics. J.

Comput. Chem. 1990, 11, 440−467.

(49) Klotz, K.-N.; Hessling, J.; Hegler, J.; Owman, C.; Kull, B.;

Fredholm, B. B.; Lohse, M. J. Comparative pharmacology of human

adenosine receptor subtypescharacterization of stably transfected

receptors in CHO cells. Naunyn-Schmiedeberg’s Arch. Pharmacol. 1998,

357, 1−9.

(32) Prime, version 2.2; Glide, version 5.6; MacroModel, version 9.8;

Maestro, version 9.1; Schrodinger, LLC: New York, NY, 2010.

(33) Molecular Operating Environment (MOE), version 2009.10;

Chemical Computing Group Inc.: 1255 University Street, Suite

1600, Montreal, QC, H3B 3X3, Canada, 2009.

(50) Gao, Z. G.; Mamedova, L.; Chen, P.; Jacobson, K. A. 2-

Substituted adenosine derivatives: affinity and efficacy at four subtypes

of human adenosine receptors. Biochem. Pharmacol. 2004, 68, 1985−

1993.

(51) Kim, Y. C.; Ji, X.-D.; Melman, N.; Linden, J.; Jacobson, K. A.

Anilide derivatives of an 8-phenylxanthine carboxylic congener are

highly potent and selective antagonists at human A_2Badenosine

receptors. J. Med. Chem. 2000, 43, 1165−1172.

(52) Cheng, Y.-C.; Prusoff, W. H. Relationship between the

inhibition constant (K_i) and the concentration of inhibitor which

causes 50% inhibition (I₅₀) of an enzymatic reaction. Biochem.

Pharmacol. 1973, 22, 3099−3108.

(53) Nordstedt, C.; Fredholm, B. B. A modification of a protein-

binding method for rapid quantification of cAMP in cell-culture

supernatants and body fluid. Anal. Biochem. 1990, 189, 231−234.

(54) Post, S. R.; Ostrom, R. S.; Insel, P. A. Biochemical methods for

detection and measurement of cyclic AMP and adenylyl cyclase

activity. Methods Mol. Biol. 2000, 126, 363−374.

(55) Valls, M. D.; Cronstein, B. N.; Montesinos, M. C. Adenosine

receptor agonists for promotion of dermal wound healing. Biochem.

Pharmacol. 2009, 77, 1117−1124.

(56) Udelson, J. E.; Heller, G. V.; Wackers, F. J.; Chai, A.; Hinchman,

D.; Coleman, P. S.; Dilsizian, V.; DiCarli, M.; Hachamovitch, R.;

Johnson, J. R.; Barrett, R. J.; Gibbons, R. J. Randomized, controlled

dose-ranging study of the selective adenosine A_2Areceptor agonist

binodenoson for pharmacological stress as an adjunct to myocardial

perfusion imaging. Circulation 2004, 109, 457−464.

(57) Bevan, N.; Butchers, P. R.; Cousins, R.; Coates, J.; Edgar, E. V.;

Morrison, V.; Sheehan, M. J.; Reeves, J.; Wilson, D. J. Pharmacological

characterisation and inhibitory effects of (2R,3R,4S,5R)-2-(6-amino-2-

{[(1S)-2-hydroxy-1-(phenylmethyl)ethyl]amino}-9H-purin-9-yl)-5-(2-

ethyl-2H-tetrazol-5-yl)tetrahydro-3,4-furandiol, a novel ligand that

demonstrates both adenosine A_2Areceptor agonist and adenosine A₃

receptor antagonist activity. Eur. J. Pharmacol. 2007, 564, 219−225.

(34) Totrov, M.; Abagyan., R. Flexible protein−ligand docking by

global energy optimization in internal coordinates. Proteins 1997,

No. Suppl 1, 215−220.

(35) Jacobson, K. A.; Gao, Z. G. Adenosine receptors as therapeutic

targets. Nature Rev. Drug Discovery 2006, 5, 247−264.

(36) Barnard, A.; Keir, R.; Salmon, G.; Stuart, E.; Trevethick, M.;

Yeadon, M.New insights into the anti-inflammatory effects of

adenosine A_2Areceptor agonists. UK-371,104, a novel adenosine A_2A

receptor agonist, inhibits acute mediator release in the human

neutrophil: comparison with CGS-21,680 and the phosphodiesterase

4 inhibitor, cilomilast. Proc. Life Sci. 2007, C70

(37) El-Tayeb, A.; Iqbal, J.; Behrenswert, A.; Romio, M.; Schneider,

M.; Zimmermann, H.; Schrader, J.; Muller, C. E. Nucleoside-5′-

̈

monophosphates as prodrugs of adenosine A_2Areceptor agonists

activated by ecto-5′-nucleotidase. J. Med. Chem. 2009, 52, 7669−7677.

(38) Hou, X.; Kim, H. O.; Alexander, V.; Kim, K.; Choi, S.; Park, S.

G.; Lee, J. H.; Yoo, L. S.; Gao, Z. G.; Jacobson, K. A.; Jeong, L. S.

Discovery of a new human A_2Aadenosine receptor agonist, truncated

2-hexynyl 4′-thioadenosine. ACS Med. Chem. Lett. 2010, 1, 516−520.

(39) Rieger, J. M.; Brown, M. L.; Sullivan, G. W.; Linden, J.;

Macdonald, T. L. Design, synthesis, and evaluation of novel A_2A

adenosine receptor agonists. J. Med. Chem. 2001, 44, 531−539.

(40) Cristalli, G.; Muller, C. E.; Volpini, R. Recent developments in

̈

adenosine A_2Areceptor ligands. Handb. Exp. Pharmacol. 2009, 193,

59−98.

(41) Daly, J. W.; Padgett, W. L.; Secunda, S. I.; Thompson, R. D.;

Olsson, R. A. Structure-activity relationships for 2-substituted

adenosines at A₁and A₂adenosine receptors. Pharmacol. 1993, 46,

91−100.

(42) Tchilibon, S.; Kim, S. K.; Gao, Z. G.; Harris, B. A.; Blaustein, J.;

Gross, A. S.; Melman, N.; Jacobson, K. A. Exploring distal regions of

the A₃adenosine receptor binding site: Sterically-constrained N⁶-(2-

phenylethyl)adenosine derivatives as potent ligands. Bioorg. Med.

Chem. 2004, 12, 2021−2034.

(43) Jacobson, K. A.; Barrington, W. W.; Pannell, L. K.; Jarvis, M. F.;

Ji, X.-D.; Williams, M.; Hutchison, A. J.; Stiles, G. L. Agonist-derived

molecular probes for A₂-adenosine receptors. J. Mol. Recognit. 1989, 2,

170−178.

(44) Smits, G. J.; McVey, M.; Cox, B. F.; Perrone, M. H.; Clark, K. L.

Cardioprotective effects of the novel adenosine A₁/A₂receptor agonist

AMP 579 in a porcine model of myocardial infarction. J. Pharmacol.

Exp. Ther. 1998, 286, 611−618.

(45) Beattie, D.; Brearley, A.; Brown, Z.; Charlton, S. J.; Cox, B.;

Fairhurst, R. A.; Fozard, J. R.; Gedeck, P.; Kirkham, P.; Meja, K.;

Nanson, L.; Neef, J.; Oakman, H.; Spooner, G.; Taylor, R. J.; Turner,

R. J.; West, R.; Woodward, H. Synthesis and evaluation of two series of

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Evaluation of molecular modeling of agonist binding in light of the crystallographic structure of an agonist-bound A2A adenosine receptor

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