Development of Covalent Ligands for G Protein-Coupled Receptors: A Case for the Human Adenosine A(3) Receptor

[Image: see text] The development of covalent ligands for G protein-coupled receptors (GPCRs) is not a trivial process. Here, we report a streamlined workflow thereto from synthesis to validation, exemplified by the discovery of a covalent antagonist for the human adenosine A(3) receptor (hA(3)AR). Based on the 1H,3H-pyrido[2,1-f]purine-2,4-dione scaffold, a series of ligands bearing a fluorosulfonyl warhead and a varying linker was synthesized. This series was subjected to an affinity screen, revealing compound 17b as the most potent antagonist. In addition, a nonreactive methylsulfonyl derivative 19 was developed as a reversible control compound. A series of assays, comprising time-dependent affinity determination, washout experiments, and [(35)S]GTPγS binding assays, then validated 17b as the covalent antagonist. A combined in silico hA(3)AR-homology model and site-directed mutagenesis study was performed to demonstrate that amino acid residue Y265(7.36) was the unique anchor point of the covalent interaction. This workflow might be applied to other GPCRs to guide the discovery of covalent ligands.