Identification of a Novel Small Molecule Antagonist of the Ectopic Olfactory Receptor Olfr78/OR51E2

Olfactory receptors are a large family of G protein-coupled receptors used in the olfactory system for detection of odorants. Interestingly, a subset of olfactory receptors is also expressed outside of the olfactory system. The ectopic olfactory receptor mouse Olfr78/human OR51E2 is expressed in the carotid body, kidney, prostate, and gut, and its activity regulates diverse physiological processes such as oxygen sensing, blood pressure regulation, and cancer progression. Several small molecule agonists for Olfr78/OR51E2 have been identified, but antagonists for Olfr78/OR51E2 are lacking to determine the effect of pharmacological inhibition of Olfr78/OR51E2 activity and the therapeutic potential of targeting this receptor. To identify new small molecule antagonists, we conducted a high throughput screen of 2,177 FDA-approved drugs and 2,541 drug-like fragments on Olfr78 expressed in heterologous HEK293T cells using a dual luciferase reporter system. Our criteria for selection of candidate antagonists were (1) dose-dependent inhibition of Olfr78 activity, (2) receptor dependence, and (3) minimal cytotoxicity. All three parameters were incorporated into the primary screen by using both an inducible Firefly luciferase and a constitutively expressed Renilla luciferase and comparing the effects of compounds on cells expressing Olfr78 to cells transfected with reporters but without receptor. Our screen identified two candidate Olfr78/OR51E2 antagonists that met these criteria. To assay for acute receptor regulation, we coupled receptor activity to calcium response in HEK293T cells and found that one of these candidates antagonizes Olfr78/OR51E2 receptor activation at 1-10 uM. We are currently testing this small molecule for regulation of Olfr78/OR51E2 activity in its endogenous setting in the carotid body, where the receptor mediates oxygen sensing, and in prostate cancer.