A Novel Membrane Potential Assay to Identify Nav1.7-Selective Blockers

Abstract

The voltage-gated sodium channel Nav1.7 is one of the most validated pain targets, as gain-of-function mutations cause excessive pain, whereas loss-of-function mutations produce insensitivity to pain. Targeting Nav1.7 holds great promise in treating pain, yet presents a tantalizing challenge, as few Nav1.7 selective chemical scaffolds exist. So far millions of compounds have been screened by using fluorescence membrane potential (MP) assays, but very few selective hits have been identified. Here we show that the conventional MP approach, which uses Nav1.7 WT channel and the activator veratridine, is intrinsically flawed: it is biased toward non-selective pore blockers which compete with veratridine, and fails to detect highly potent, selective compounds such as the aryl sulfonamide PF-771. By using a mutant channel and an alternative activator, we developed a novel method that not only robustly detects known Nav1.7 blockers, but also decreases the number of non-selective pore blocker hits. We conducted a high-throughput screen using this methodology and identified novel Nav1.7-selective blockers.