An All-Optical Electrophysiology Screening Platform to Identify Nav Channel Modulators as Pain Therapeutics

Abstract

More than 100M people in the US suffer from chronic pain. Current treatments, including opioids and non-steroidal inflammatory agents, have severe limitations for chronic treatment due to tolerance and dose-limiting toxicities. To develop novel pain therapeutics, we have created an all-optical electrophysiology (Optopatch) screening platform using engineered optogenetic proteins. Blue and red light can be used to stimulate and record bioelectrical activity, respectively. For target-based screening application, we demonstrate Optopatch measurements in engineered excitable HEK cells (spiking HEK cells) with heterologous expression of Nav channels targets implicated in pain transmission, including Nav1.7, Nav1.8 and Nav1.9. The Nav channel Optopatch assays can distinguish compounds with different working mechanisms (state-dependent versus state independent block). To achieve a throughput of 10,000 compounds/day, we developed a next generation kinetic plate reader (SWARM) capable of recording 24 wells simultaneously. We have executed a 14,000-compound screen on Nav1.7 spiking HEK cells and identified novel Nav1.7 inhibitors with sub-micromolar potency. To further qualify candidate compounds, we have developed proprietary secondary assays for hit prioritization, which combined the Optopatch platform with an in vitro model of chronic pain, in which dorsal root ganglion (DRG) sensory neurons are exposed to a mixture of inflammatory mediators. This assay leverages our custom Firefly microscope to make highly-parallelized measurements of DRG excitability, where ∼100 neurons can be measured in parallel with single-cell precision and ms-temporal resolution. The newly identified hits are benchmarked against Nav1.7 inhibitors in Phase II trial (PF-05089771 and vixotrigine) and selected hits demonstrate more favorable features in our secondary assay. Interestingly, we found that highly selective Nav1.7 cannot fully reverse the inflammatory mediator-induced DRG hyperexcitability; we dissect Nav-subtype contributions to hyperexcitability using control pharmacology and protein knockdown with antisense oligonucleotides (ASOs).