1b:I[76749,["/_next/static/chunks/bf18fea59be6d5b1.js","/_next/static/chunks/1ba21a8e081e29d1.js","/_next/static/chunks/0383663887945ae7.js","/_next/static/chunks/2060b26370dc6c0a.js","/_next/static/chunks/c89b4435b8c37fcf.js","/_next/static/chunks/8ddf2550f38c2f2a.js","/_next/static/chunks/5bf36002c846d2d8.js"],"default"] 1c:I[3486,["/_next/static/chunks/bf18fea59be6d5b1.js","/_next/static/chunks/1ba21a8e081e29d1.js","/_next/static/chunks/0383663887945ae7.js","/_next/static/chunks/2060b26370dc6c0a.js","/_next/static/chunks/c89b4435b8c37fcf.js","/_next/static/chunks/8ddf2550f38c2f2a.js","/_next/static/chunks/5bf36002c846d2d8.js"],"default"] 1d:I[17707,["/_next/static/chunks/bf18fea59be6d5b1.js","/_next/static/chunks/1ba21a8e081e29d1.js","/_next/static/chunks/0383663887945ae7.js","/_next/static/chunks/2060b26370dc6c0a.js","/_next/static/chunks/c89b4435b8c37fcf.js","/_next/static/chunks/8ddf2550f38c2f2a.js","/_next/static/chunks/5bf36002c846d2d8.js"],"default"] 1e:I[66528,["/_next/static/chunks/bf18fea59be6d5b1.js","/_next/static/chunks/1ba21a8e081e29d1.js","/_next/static/chunks/0383663887945ae7.js","/_next/static/chunks/2060b26370dc6c0a.js","/_next/static/chunks/c89b4435b8c37fcf.js","/_next/static/chunks/8ddf2550f38c2f2a.js","/_next/static/chunks/5bf36002c846d2d8.js"],"default"] 1f:I[63176,["/_next/static/chunks/bf18fea59be6d5b1.js","/_next/static/chunks/1ba21a8e081e29d1.js","/_next/static/chunks/0383663887945ae7.js","/_next/static/chunks/2060b26370dc6c0a.js","/_next/static/chunks/c89b4435b8c37fcf.js","/_next/static/chunks/8ddf2550f38c2f2a.js","/_next/static/chunks/5bf36002c846d2d8.js"],"default"] 20:I[87099,["/_next/static/chunks/bf18fea59be6d5b1.js","/_next/static/chunks/1ba21a8e081e29d1.js","/_next/static/chunks/0383663887945ae7.js","/_next/static/chunks/2060b26370dc6c0a.js","/_next/static/chunks/c89b4435b8c37fcf.js","/_next/static/chunks/8ddf2550f38c2f2a.js","/_next/static/chunks/5bf36002c846d2d8.js"],"default"] 21:I[26200,["/_next/static/chunks/bf18fea59be6d5b1.js","/_next/static/chunks/1ba21a8e081e29d1.js","/_next/static/chunks/0383663887945ae7.js","/_next/static/chunks/2060b26370dc6c0a.js","/_next/static/chunks/c89b4435b8c37fcf.js","/_next/static/chunks/8ddf2550f38c2f2a.js","/_next/static/chunks/5bf36002c846d2d8.js"],"Group"] 22:I[23365,["/_next/static/chunks/bf18fea59be6d5b1.js","/_next/static/chunks/1ba21a8e081e29d1.js","/_next/static/chunks/0383663887945ae7.js","/_next/static/chunks/2060b26370dc6c0a.js","/_next/static/chunks/c89b4435b8c37fcf.js","/_next/static/chunks/8ddf2550f38c2f2a.js","/_next/static/chunks/5bf36002c846d2d8.js"],"default"] 23: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.

Jelle Offringa

Publications