Structure-based design of novel pharmacological tools for the A3 adenosine G protein-coupled receptor

Adenosine receptors (ARs) are a family belonging to the GPCR superfamily, involved with multiple physiological processes and widely distributed throughout the body. In this study, we focus on the A3 AR due to its vast scale applications in various pathophysiological conditions such as inflammation, pain, allergic asthma, and cancer chemotherapy. We report the validation of the binding mode of A3 AR antagonists, through a comprehensive in-silico mutagenesis study using free energy perturbations, reproducing prior experimental site-directed mutagenesis data for A3 AR antagonists. After validating the antagonist binding mode, we performed an extensive in-silico site-directed mutagenesis study on the hA3 AR using potent, selective, and structurally simple A3 AR antagonist based on the N-(2,6-diarylpyrimidin-4- yl)acetamide scaffold (pyrimidine core). The results of this study will be used to design in- vitro site-directed mutagenesis performed by collaborators (University of Barcelona). Once the binding mode of this scaffold is validated, it will be the basis for the design of compounds with two well-defined functionalities: a fluorophore moiety and bivalent ligands that target A3 dimers. The discovery of novel mutations on the hA3 AR is a step forward in the development of both chemically simple, potent, and selective A3 AR antagonists as well as in the characterization and crystallization of the A3 AR.