Mechanism-specific assay design facilitates the discovery of Nav1.7-selective inhibitors.

Many ion channels, including Nav1.7, Cav1.3, and Kv1.3, are linked to human pathologies and are important therapeutic targets. To develop efficacious and safe drugs, subtype-selective modulation is essential, but has been extremely difficult to achieve. We postulate that this challenge is caused by the poor assay design, and investigate the Nav1.7 membrane potential assay, one of the most extensively employed screening assays in modern drug discovery. The assay uses veratridine to activate channels, and compounds are identified based on the inhibition of veratridine-evoked activities. We show that this assay is biased toward nonselective pore blockers and fails to detect the most potent, selective voltage-sensing domain 4 (VSD4) blockers, including PF-05089771 (PF-771) and GX-936. By eliminating a key binding site for pore blockers and replacing veratridine with a VSD-4 binding activator, we directed the assay toward non-pore-blocking mechanisms and discovered Nav1.7-selective chemical scaffolds. Hence, we address a major hurdle in Nav1.7 drug discovery, and this mechanistic approach to assay design is applicable to Cav3.1, Kv1.3, and many other ion channels to facilitate drug discovery.

Discovery of Acyl-sulfonamide Nav1.7 Inhibitors GDC-0276 and GDC-0310.

Nav1.7 is an extensively investigated target for pain with a strong genetic link in humans, yet in spite of this effort, it remains challenging to identify efficacious, selective, and safe inhibitors. Here, we disclose the discovery and preclinical profile of GDC-0276 (1) and GDC-0310 (2), selective Nav1.7 inhibitors that have completed Phase 1 trials. Our initial search focused on close-in analogues to early compound 3. This resulted in the discovery of GDC-0276 (1), which possessed improved metabolic stability and an acceptable overall pharmacokinetics profile. To further derisk the predicted human pharmacokinetics and enable QD dosing, additional optimization of the scaffold was conducted, resulting in the discovery of a novel series of N-benzyl piperidine Nav1.7 inhibitors. Improvement of the metabolic stability by blocking the labile benzylic position led to the discovery of GDC-0310 (2), which possesses improved Nav selectivity and pharmacokinetic profile over 1.

Structural basis of Nav1.7 inhibition by an isoform-selective small-molecule antagonist.

Voltage-gated sodium (Nav) channels propagate action potentials in excitable cells. Accordingly, Nav channels are therapeutic targets for many cardiovascular and neurological disorders. 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. Targeting the human Nav1.7 channel involved in pain perception, we present a protein-engineering strategy that has allowed us to determine crystal structures of a novel receptor site in complex with isoform-selective antagonists. GX-936 and related inhibitors bind to the activated state of voltage-sensor domain IV (VSD4), where their anionic aryl sulfonamide warhead engages the fourth arginine gating charge on the S4 helix. By opposing VSD4 deactivation, these compounds inhibit Nav1.7 through a voltage-sensor trapping mechanism, likely by stabilizing inactivated states of the channel. 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 help to elucidate the molecular basis of voltage sensing and establish structural blueprints to design selective Nav channel antagonists.

Treatment of Na(v)1.7-mediated pain in inherited erythromelalgia using a novel sodium channel blocker.

Mutations in the SCN9A gene leading to deficiency of its protein product, Na(v)1.7, cause congenital indifference to pain (CIP). CIP is characterized by the absence of the ability to sense pain associated with noxious stimuli. In contrast, the opposite phenotype to CIP, inherited erythromelalgia (IEM), is a disorder of spontaneous pain caused by missense mutations resulting in gain-of-function in Na(v)1.7 that promote neuronal hyperexcitability. The primary aim of this study was to demonstrate that Na(v)1.7 antagonism could alleviate the pain of IEM, thereby demonstrating the utility of this opposite phenotype model as a tool for rapid proof-of-concept for novel analgesics. An exploratory, randomized, double-blind, 2-period crossover study was conducted in 4 SCN9A mutation-proven IEM patients. In each treatment period (2days), separated by a 2-day washout period, patients were orally administered XEN402 (400mg twice daily) or matching placebo. In 3 patients, pain was induced by heat or exercise during each treatment arm. A fourth patient, in constant severe pain, required no induction. Patient-reported outcomes of pain intensity and/or relief were recorded, and the time taken to induce pain was measured. The ability to induce pain in IEM patients was significantly attenuated by XEN402 compared with placebo. XEN402 increased the time to maximal pain induction and significantly reduced the amount of pain (42% less) after induction (P=.014). This pilot study showed that XEN402 blocks Na(v)1.7-mediated pain associated with IEM, thereby demonstrating target engagement in humans and underscoring the use of rare genetic disorders with mutant target channels as a novel approach to rapid proof-of-concept.