Mapping the Orthosteric Binding Site of the Human 5-HT₃ Receptor Using Photo-cross-linking Antagonists

The serotonin-gated 5-HT₃ receptor is a ligand-gated ion channel. Its location at the synapse in the central and peripheral nervous system has rendered it a prime pharmacological target, for example, for antiemetic drugs that bind with high affinity to the neurotransmitter binding site and prevent the opening of the channel. Advances in structural biology techniques have led to a surge of disclosed three-dimensional receptor structures; however, solving ligand-bound high-resolution 5-HT₃ receptor structures has not been achieved to date. Ligand binding poses in the orthosteric binding site have been largely predicted from mutagenesis and docking studies. We report the synthesis of a series of photo-cross-linking compounds whose structures are based on the clinically used antiemetic drug granisetron (Kytril). These displaced [³H]granisetron from the orthosteric binding site with low nanomolar affinities and showed specific photo-cross-linking with the human 5-HT₃ receptor. Detailed analysis by protein-MS/MS identified a residue (Met-228) near the tip of binding loop C as the covalent modification site.