Structural Basis of Nav1.7 Inhibition by an Isoform-Selective Small Molecule Antagonist

Abstract

Voltage-gated sodium (Nav) channels are therapeutic targets for many cardiovascular and neurological disorders. Unfortunately, selective inhibitors have been challenging to design because the nine mammalian Nav channel isoforms share high sequence identity and remain recalcitrant to high-resolution structural studies. Recently, a series of highly selective small-molecule Nav channel antagonists (the aryl-sulfonamide series) has been identified that is able to achieve isoform selectivity by binding to the relatively poorly-conserved surface of voltage-sensor domain IV (VSD4). Understanding how these antagonists work and the details of their binding site has been a recent focus of the pharmaceutical industry which has hopes of identifying selective inhibitors of Nav1.7, an important target for novel pain drugs. Here, we describe the structural basis of Nav1.7 inhibition by this novel class of small molecule antagonist using X-ray crystallography, and thus present important experimental structures of a gating modifier in complex with a voltage-gated ion channel. To enable these unique mammalian Nav channel crystal structures, we exploited the established portability of VSDs and the presumed structural relatedness between human and bacterial Nav channels to develop a robust protein production and crystallization strategy. GX-936 and related aryl-sulfonamide inhibitors bind to the activated state of VSD4, where their anionic aryl sulfonamide warhead directly engages the R4 gating charge on the S4 helix. By opposing VSD4 deactivation, which stabilizes inactivated states of the channel, these compounds inhibit Nav1.7 through a voltage-sensor trapping mechanism. Residues from the S2 and S3 helices are key determinants of isoform selectivity and bound phospholipids implicate the membrane as a modulator of channel function and pharmacology. Our results shed light on the molecular basis of voltage sensing and establish the first structural blueprints to design selective Nav channel antagonists.