X‐Ray Crystallography and Free Energy Calculations Reveal the Binding Mechanism of A_2A Adenosine Receptor Antagonists

Abstract

We present a robust protocol based on iterations of free energy perturbation (FEP) calculations, chemical synthesis, biophysical mapping and X‐ray crystallography to reveal the binding mode of an antagonist series to the A2A adenosine receptor (AR). Eight A2AAR binding site mutations from biophysical mapping experiments were initially analyzed with sidechain FEP simulations, performed on alternate binding modes. The results distinctively supported one binding mode, which was subsequently used to design new chromone derivatives. Their affinities for the A2AAR were experimentally determined and investigated through a cycle of ligand‐FEP calculations, validating the binding orientation of the different chemical substituents proposed. Subsequent X‐ray crystallography of the A2AAR with a low and a high affinity chromone derivative confirmed the predicted binding orientation. The new molecules and structures here reported were driven by free energy calculations, and provide new insights on antagonist binding to the A2AAR, an emerging target in immuno‐oncology.

Pathways and Mechanism of Caffeine Binding to Human Adenosine A2A Receptor

“…In the inactive state, the distances between residues R3.50 and E6.30 and R3.50 and Y7.53 were 4.4 and 12.4 Å, respectively. In this context, the average distances between residues R3.50 and E6.30 and R3.50 and Y7.53 in 46 experimental structures of the inactive A 2A AR ( Supplementary Table 2 ; Jaakola et al, 2008 ; Doré et al, 2011 ; Congreve et al, 2012 ; Hino et al, 2012 ; Liu et al, 2012 ; Batyuk et al, 2016 ; Segala et al, 2016 ; Cheng et al, 2017 ; Martin-Garcia et al, 2017 , 2019 ; Melnikov et al, 2017 ; Sun et al, 2017 ; Weinert et al, 2017 ; Broecker et al, 2018 ; Eddy et al, 2018 ; Rucktooa et al, 2018 ; Ishchenko et al, 2019 ; Shimazu et al, 2019 ; Borodovsky et al, 2020 ; Ihara et al, 2020 ; Jespers et al, 2020 ; Lee et al, 2020 ; Nass et al, 2020 ) were calculated to be 6.5 ± 1.0 Å and 12.7 ± 0.4 Å , respectively. Therefore, the highly conserved residues R3.50 and E6.30 ionic lock became fully closed in the GaMD simulations of the inactive A 2A AR during binding of the CFF antagonist.…”

Structural Basis of Inhibition of Human Insulin-Regulated Aminopeptidase (IRAP) by Benzopyran-Based Inhibitors

“…For this reason, we investigated the 6–9 series and the mutagenesis effects with FEP simulations. The use of complementary FEP transformations (i.e., ligand perturbations on different enzyme forms, and receptor mutation upon binding of different ligands) provides “two sides of the same coin” that provide a comprehensive perspective of the ligand binding process, as recently showed for the elucidation of the binding mode of A 2A adenosine receptor antagonists ( Jespers et al, 2020 ). In the IRAP system, these simulations allowed to identify interactions that were selectively lost on the mutant versions of the enzyme (i.e., between the ligand and Arg439), or differences in ligand binding consistently observed within wt and mutant versions of the enzyme, as the case of the interactions of compound 9 with the network of glutamic acid residues ( Figure 6 ).…”

Deciphering conformational selectivity in the A2A adenosine G protein-coupled receptor by free energy simulations

“…In this scenario, we have recently developed robust FEP protocols that were thoroughly applied in the context of GPCR ligand-binding investigations: QligFEP [ 25 ] allows to systematically compute relative binding affinity changes between a series of ligands, while QresFEP [ 26 ] was designed to evaluate the binding affinity shifts due to single point mutations. The synergistic combination of both approaches recently led to the elucidation of the binding mode of a series of A 2A AR antagonists [ 27 ]. In this work, we extend the applicability of these methods to study the effects of chemical modifications of ligands, as well as receptor mutations, on the conformational equilibrium of the receptor, which we relate to efficiency of the ligand as an agonist modulator.…”