Kinetic Analysis of Membrane Potential Dye Response to NaV1.7 Channel Activation Identifies Antagonists with Pharmacological Selectivity against NaV1.5

Abstract

The Na V 1.7 voltage-gated sodium channel is a highly valued target for the treatment of neuropathic pain due to its expression in pain-sensing neurons and human genetic mutations in the gene encoding Na V 1.7, resulting in either lossof-function (e.g., congenital analgesia) or gain-of-function (e.g., paroxysmal extreme pain disorder) pain phenotypes. We exploited existing technologies in a novel manner to identify selective antagonists of Na V 1.7. A full-deck high-throughput screen was developed for both Na V 1.7 and cardiac Na V 1.5 channels using a cell-based membrane potential dye FLIPR assay. In assay development, known local anesthetic site inhibitors produced a decrease in maximal response; however, a subset of compounds exhibited a concentration-dependent delay in the onset of the response with little change in the peak of the response at any concentration. Therefore, two methods of analysis were employed for the screen: one to measure peak response and another to measure area under the curve, which would capture the delay-to-onset phenotype. Although a number of compounds were identified by a selective reduction in peak response in Na V 1.7 relative to 1.5, the AUC measurement and a subsequent refinement of this measurement were able to differentiate compounds with Na V 1.7 pharmacological selectivity over Na V 1.5 as confirmed in electrophysiology.

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

“…Membrane potential assays for sodium channels have been reported by various groups ( 20 – 22 ). Consistent with previous findings, veratridine (100 μM) elicited robust fluorescence signals in Nav1.7-expressing cells loaded with the blue fluorescence membrane potential dye ( Fig.…”

“…Membrane potential assays have been used to screen tens of millions of compounds for Nav1.7 blockers ( 20 – 24 ). Despite the tremendous efforts, few Nav1.7-selective chemical scaffolds have been identified.…”

“…To identify subtype-selective chemical scaffolds, large libraries of compounds, sometimes exceeding millions of individual compounds, are screened against the targets of interest (e.g., Nav1.7), followed by selectivity tests on other isoforms (e.g., Nav1.5). The effects of compounds are determined using in vitro assays such as electrophysiology ( 17 – 19 ) or fluorescence-based membrane potential assays ( 20 – 22 ). The major considerations for assay design have been signal robustness, throughput, and cost, whereas mechanisms of action, such as drug binding sites and molecular basis for selectivity, are often not taken into consideration.…”

“…We chose Nav1.7-membrane potential assay as a test case for several reasons. First, it is one of the most extensively utilized screening assays in modern drug discovery ( 20 – 24 ). Second, drug binding sites for both nonselective (e.g., local anesthetics and veratridine) and selective arylsulfonamide compounds (e.g., GX-936 and PF-771) have been determined, allowing rational assay design ( 14 – 16 ).…”

Benzoxazolinone aryl sulfonamides as potent, selective Na v 1.7 inhibitors with in vivo efficacy in a preclinical pain model

“…In addition, potent and selective acyl sulfonamides have been reported by Amgen (i.e. compound 2) 18 , Pfizer 19 and Merck, 20 with competitive binding studies suggesting a similar binding mode to the aryl sulfonamides.…”