Intracellular Allosteric Antagonist of the Olfactory Receptor OR51E2

Olfactory receptors are members of Class A (rhodopsin-like) family of G protein-coupled receptors (GPCRs). Their expression and function have been increasingly studied in nonolfactory tissues, and many have been identified as potential therapeutic targets. In this manuscript, we focus on discovery of novel ligands for the olfactory receptor OR51E2. We performed an artificial-intelligence-based virtual drug screen of a ~2.2 million small molecule library. Cell-based functional assay identified compound 80 (C80) as an antagonist and inverse agonist, and detailed pharmacological analysis revealed C80 acts as a negative allosteric modulator (NAM) by significantly decreasing the agonist efficacy, while having a minimal effect on receptor affinity for agonist. C80 binds to an allosteric binding site formed by a network of 9 residues localized in the intracellular parts of TM 3, 5, 6, 7 and H8, which also partially overlaps with a G-protein binding site. Mutational experiments of residues involved in C80 binding uncovered the significance of C240^6.37 position in blocking the activation-related conformational change and keeping the receptor in the inactive form. Our study provides a mechanistic understanding for a negative allosteric action of C80 on agonist activated OR51E2. We believe identification of antagonist of OR51E2 will enable multitude studies aiming to determine the functional role of this receptor in specific biological process. Significance Statement Olfactory receptor 51E2 has been implicated in various biological processes and the modulators os its activity have therapeutic potential. Here, we report the discovery of a negative allosteric modulator (NAM) of OR51E2 and provide a mechanistic understanding of its action. We demonstrate that this modulator has a significant inhibitory effect on the efficacy of agonist for the receptor and reveal a network of 9 residues that constitute its binding pocket which also partially overlaps with the G-protein binding site.