Identification of V6.51L as a selectivity hotspot in stereoselective A2B adenosine receptor antagonist recognition

Xuesong Wang,Willem Jespers,Eddy Sotelo,Hugo Gutiérrez-De-Terán,Laura H Heitman,Maria Majellaro,Adriaan P Ijzerman,Rubén Prieto-Díaz,Gerard J P Van Westen

doi:10.1038/s41598-021-93419-x

Abstract

The four adenosine receptors (ARs) A1AR, A2AAR, A2BAR, and A3AR are G protein-coupled receptors (GPCRs) for which an exceptional amount of experimental and structural data is available. Still, limited success has been achieved in getting new chemical modulators on the market. As such, there is a clear interest in the design of novel selective chemical entities for this family of receptors. In this work, we investigate the selective recognition of ISAM-140, a recently reported A2BAR reference antagonist. A combination of semipreparative chiral HPLC, circular dichroism and X-ray crystallography was used to separate and unequivocally assign the configuration of each enantiomer. Subsequently affinity evaluation for both A2A and A2B receptors demonstrate the stereospecific and selective recognition of (S)-ISAM140 to the A2BAR. The molecular modeling suggested that the structural determinants of this selectivity profile would be residue V2506.51 in A2BAR, which is a leucine in all other ARs including the closely related A2AAR. This was herein confirmed by radioligand binding assays and rigorous free energy perturbation (FEP) calculations performed on the L249V6.51 mutant A2AAR receptor. Taken together, this study provides further insights in the binding mode of these A2BAR antagonists, paving the way for future ligand optimization.

Highlights

Adenosine receptors (ARs) are a family of G protein-coupled receptors (GPCR) for which an exceptional amount of structural and experimental data is available[1,2]
The binding mode of (S)-ISAM-140 was obtained by superposition of the previously published complex of this molecule with our A2BAR homology-based model[11] onto a modeled L249V6.51 A2AAR mutant, i.e. introducing the A2BAR sidechain in this position
We investigated the role of position 6.51 in determining the specificity for A 2BAR binding of a series of chiral antagonists recently developed for this receptor

Summary

Introduction

Adenosine receptors (ARs) are a family of G protein-coupled receptors (GPCR) for which an exceptional amount of structural and experimental data is available[1,2]. This binding model proposed that the stereospecific complementarity to the A 2BAR cavity was due to the optimal accommodation of the thiophene/furan ring around the chiral center of the core scaffold (Fig. 1), with the A 2BAR specific residue V 2506.51 (Ballesteros Weinstein numbering in superscripts)[15] This valine is replaced by a leucine in all other AR subtypes, which could explain the highly selective profile of these series of non-planar antagonists towards the A2BAR. An A 2AAR construct was designed to include the corresponding A2BAR valine sidechain (L249V6.51 A2AAR mutant), which in line with the starting hypothesis partially recovered the affinity for ISAM-140 This effect was observed for both stereoisomers of the antagonist, and is explained on the basis of structure-energetic modeling via rigorous free energy perturbation (FEP) calculations. These results validate the proposed role of V 2506.51 in the A 2BAR subtype selectivity of these stereospecific chemotype, and paves the road for further design of selective antagonists as well as dual A2AR ligands

Methods

Results

Conclusion