Identification of V6.51L as a selectivity hotspot in stereoselective A(2B) adenosine receptor antagonist recognition

The four adenosine receptors (ARs) A(1)AR, A(2A)AR, A(2B)AR(,) and A(3)AR 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 A(2B)AR 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 A(2A) and A(2B) receptors demonstrate the stereospecific and selective recognition of (S)-ISAM140 to the A(2B)AR. The molecular modeling suggested that the structural determinants of this selectivity profile would be residue V250(6.51) in A(2B)AR, which is a leucine in all other ARs including the closely related A(2A)AR. This was herein confirmed by radioligand binding assays and rigorous free energy perturbation (FEP) calculations performed on the L249V(6.51) mutant A(2A)AR receptor. Taken together, this study provides further insights in the binding mode of these A(2B)AR antagonists, paving the way for future ligand optimization.