Transmembranal G Protein-Coupled Receptors (GPCRs) transduce extracellular chemical signals to the cell, via conformational change from a resting (inactive) to an active (canonically bound to a G... Show moreTransmembranal G Protein-Coupled Receptors (GPCRs) transduce extracellular chemical signals to the cell, via conformational change from a resting (inactive) to an active (canonically bound to a G-protein) conformation. Receptor activation is normally modulated by extracellular ligand binding, but mutations in the receptor can also shift this equilibrium by stabilizing different conformational states. In this work, we built structure-energetic relationships of receptor activation based on original thermodynamic cycles that represent the conformational equilibrium of the prototypical A2A adenosine receptor (AR). These cycles were solved with efficient free energy perturbation (FEP) protocols, allowing to distinguish the pharmacological profile of different series of A2AAR agonists with different efficacies. The modulatory effects of point mutations on the basal activity of the receptor or on ligand efficacies could also be detected. This methodology can guide GPCR ligand design with tailored pharmacological properties, or allow the identification of mutations that modulate receptor activation with potential clinical implications. Show less
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,... Show moreThe 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. Show less
TrendsRecent technological advances in membrane protein crystallization have resulted in a nearly exponential increase of available receptor structures. The AR family is an important example in... Show moreTrendsRecent technological advances in membrane protein crystallization have resulted in a nearly exponential increase of available receptor structures. The AR family is an important example in this respect. Crystal structures of antagonist- and agonist-bound adenosine A2A receptor have recently been supplemented by a fully activated conformation in complex with a G-protein mimic, and by antagonist bound structures of the A1 receptor.SDM experiments have been essential to identify residues involved in molecular interactions between ARs and their ligands. Leveraging on recent crystal structures, this vast amount of data can now be systematically classified and interconnected with chemical and structural information of ligands and receptors.The mapping of mutational data onto crystal structures provides new understanding of molecular interactions involved in ligand recognition. Together with computational modeling, this can be used as a roadmap to create novel hypotheses and assist in the design of more systematic mutagenesis studies to answer remaining structural and functional questions.The four adenosine receptors (ARs), A1, A2A, A2B, and A3, constitute a subfamily of G protein-coupled receptors (GPCRs) with exceptional foundations for structure-based ligand design. The vast amount of mutagenesis data, accumulated in the literature since the 1990s, has been recently supplemented with structural information, currently consisting of several inactive and active structures of the A2A and inactive conformations of the A1 ARs. We provide the first integrated view of the pharmacological, biochemical, and structural data available for this receptor family, by mapping onto the relevant crystal structures all site-directed mutagenesis data, curated and deposited at the GPCR database (available through http://www.gpcrdb.org). This analysis provides novel insights into ligand binding, allosteric modulation, and signaling of the AR family.Keywords: G protein-coupled receptor, adenosine receptor, mutagenesis, chemical modulationShow less
