1a:I[76865,["/_next/static/chunks/d07a36b34c614bac.js","/_next/static/chunks/a439560253821848.js","/_next/static/chunks/992d80515d7f780d.js","/_next/static/chunks/3adfb55b15c8d19c.js","/_next/static/chunks/b642b3e65e2c6231.js","/_next/static/chunks/fc50eca1ad01dc11.js","/_next/static/chunks/afc6bdd59b458409.js","/_next/static/chunks/6448cccc5d968256.js"],"Grid"] 1b:I[17291,["/_next/static/chunks/d07a36b34c614bac.js","/_next/static/chunks/a439560253821848.js","/_next/static/chunks/992d80515d7f780d.js","/_next/static/chunks/3adfb55b15c8d19c.js","/_next/static/chunks/b642b3e65e2c6231.js","/_next/static/chunks/fc50eca1ad01dc11.js","/_next/static/chunks/afc6bdd59b458409.js","/_next/static/chunks/6448cccc5d968256.js"],"GridCol"] 1c:I[34616,["/_next/static/chunks/d07a36b34c614bac.js","/_next/static/chunks/a439560253821848.js","/_next/static/chunks/992d80515d7f780d.js","/_next/static/chunks/3adfb55b15c8d19c.js","/_next/static/chunks/b642b3e65e2c6231.js","/_next/static/chunks/fc50eca1ad01dc11.js","/_next/static/chunks/afc6bdd59b458409.js","/_next/static/chunks/6448cccc5d968256.js"],"Avatar"] 1d:I[32972,["/_next/static/chunks/d07a36b34c614bac.js","/_next/static/chunks/a439560253821848.js","/_next/static/chunks/992d80515d7f780d.js","/_next/static/chunks/3adfb55b15c8d19c.js","/_next/static/chunks/b642b3e65e2c6231.js","/_next/static/chunks/fc50eca1ad01dc11.js","/_next/static/chunks/afc6bdd59b458409.js","/_next/static/chunks/6448cccc5d968256.js"],"default"] 1e:T47d,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 A₃ receptor (hA₃AR). Based on the 1 H,3 H-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 [³⁵S]GTPγS binding assays, then validated 17b as the covalent antagonist. A combined in silico hA₃AR-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.

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