Crystal structure of adenosine A2A receptor in complex with clinical candidate Etrumadenant reveals unprecedented antagonist interaction

Abstract

The Gs protein-coupled adenosine A2A receptor (A2AAR) represents an emerging drug target for cancer immunotherapy. The clinical candidate Etrumadenant was developed as an A2AAR antagonist with ancillary blockade of the A2BAR subtype. It constitutes a unique chemotype featuring a poly-substituted 2-amino-4-phenyl-6-triazolylpyrimidine core structure. Herein, we report two crystal structures of the A2AAR in complex with Etrumadenant, obtained with differently thermostabilized A2AAR constructs. This led to the discovery of an unprecedented interaction, a hydrogen bond of T883.36 with the cyano group of Etrumadenant. T883.36 is mutated in most A2AAR constructs used for crystallization, which has prevented the discovery of its interactions. In-vitro characterization of Etrumadenant indicated low selectivity versus the A1AR subtype, which can be rationalized by the structural data. These results will facilitate the future design of AR antagonists with desired selectivity. Moreover, they highlight the advantages of the employed A2AAR crystallization construct that is devoid of ligand binding site mutations.

Subtle Structural Changes across the Boundary between A2AR/A2BR Dual Antagonism and A2BR Antagonism: A Novel Class of 2-Aminopyrimidine-Based Derivatives

“…The GloSensor cAMP assay showed that compound 7ai exhibited robust antagonistic activity against human A 2A R and A 2B R with IC 50 values of 11.2 ± 0.2 and 6.4 ± 0.3 nM, respectively, with no antagonistic activity against human A 1 R and A 3 R (IC 50 > 1000 nM) (Figure A). In contrast, we found that the control AB928 exhibited potent antagonistic activity against human A 1 R (IC 50 = 87.3 ± 6.3 nM) (Figure S9A), which is consistent with a recent study . To explore the vast difference in A 1 R antagonistic activity of compounds 7ai and AB928, the top-scored binding pose of 7ai was superimposed to the crystal structure of the DU172-A 1 R complex (PDB: 5UEN) by A 1 R–A 2A R alignment.…”

“…Hence, we postulated the involvement of three potentially conserved water molecules in facilitating the interaction between the A 2A R receptor and the two small-molecule ligands. Within this context, W1 likely acts as a bridge linking residues N253 and E169, while W2 functions as a bridge between the nitrogen atom on the pyrimidine ring with E169, as documented in the prior literature. , On the other hand, W3 emerges as a novel bridge between the cyano group on the indole ring and the backbone amino group of residues E169 or F168. These conserved water molecules were also observed in the principal binding conformations of 7ai , 7aj , and A 2A R, as established through cluster analysis (Figure ).…”

“…In contrast, we found that the control AB928 exhibited potent antagonistic activity against human A 1 R (IC 50 = 87.3 ± 6.3 nM) (Figure S9A), which is consistent with a recent study. 31 To explore the vast difference in A 1 R antagonistic activity of compounds 7ai and AB928, the top-scored binding pose of 7ai was superimposed to the crystal structure of the DU172-A 1 R complex (PDB: 5UEN) by A 1 R−A 2A R alignment. As shown in Figure S9B,C, the cyano-substituted indole ring of 7ai is in steric conflict with Glu170 and Glu172 of A 1 R, while the hydroxyl isopropyl structure of AB928 is just accommodated in the protein cavity.…”

“…13 Molecular Docking. The crystal complex involving A 2A R and the antagonist Etrumadenant (PDB code: 8C9W) 31 was selected as the fundamental model for our molecular docking studies since our compounds have the most similar scaffold to Etrumadenant. To prepare the protein structure, we removed the solvent molecules, ions, and PSB2-bRIL and mutated two residues K91 and A154 back to native amino acids.…”

Structural Insights into Partial Activation of the Prototypic G Protein-Coupled Adenosine A2A Receptor

“…Although the binding affinity (determined vs. the antagonist radioligand [ 3 H]ZM241385) of the hydroxyphenyl derivative LUF5834 was reported to be unaffected by the mutations, the S277 7.42 A mutation was found to increase the compound's efficacy. 22 In the present study, we determined the crystal structure of LUF5834 in complex with the recently developed optimized crystallization construct A 2A -PSB2-bRIL 23 (PSB refers to Pharmaceutical Sciences Bonn) that contains less mutations than other frequently used A 2A AR crystallization constructs and does not have any mutation in the orthosteric binding site.…”