Small molecule discovery for modulation of Otopetrin proton channels.

Abstract

Proton conductance across the plasma membrane is involved in many biological roles, ranging from sour taste reception to regulation of intracellular and extracellular pH. Recently, a eukaryotic family of membrane-bound proteins, named Otopetrins, were characterized as proton channels. Otopetrins elicit inward conducting proton currents in response to low extracellular pH levels. Cryo-electron microscopy structures of Otopetrins revealed that they bear no structural similarity to other proton channels, like the voltage-gated proton channel Hv1 or the influenza proton channel M2, marking them a new architecture for proton channels. Since their discovery, OTOP1 was shown to be the sour-taste receptor for vertebrates; however, the biological roles of other members, OTOP2 and OTOP3, remain uninvestigated. Moreover, current research on Otopetrins falls short of elucidating their mechanisms of proton conductance and gating and there are no known molecular modulators of these channels - agonistic or antagonistic. Here, using molecular docking with AutoDock Vina, we virtually screened a diversity set of the Chembridge small molecule library against the available cryo-EM structure of Danio rerio OTOP1. Functional testing of the resulting hits with electrophysiology identified six small molecules that block OTOP1 currents to varying degrees. Based on the chemical scaffold of the best identified blocker, we then screened a filtered subset of the Chembridge library and successfully identified several more blockers, improving upon the results of the initial screening. These small molecules, which are predicted to bind in a potential proton conductance pathway, can be used as chemical probes in structural and functional characterization of OTOP1 to provide insight on the mechanisms of proton conductance and gating. Furthermore, the small molecules we identified provide us with a chemical scaffold for developing a pharmacophore to find better ligands for use in future in vitro and in vivo experiments.