PF-06526290 can both enhance and inhibit conduction through voltage-gated sodium channels.

BACKGROUND AND PURPOSE: Pharmacological agents that either inhibit or enhance flux of ions through voltage-gated sodium (Nav ) channels may provide opportunities for treatment of human health disorders. During studies to characterize agents that modulate Nav 1.3 function, we identified a compound that appears to exhibit both enhancement and inhibition of sodium ion conduction that appeared to be dependent on the gating state that the channel was in. The objective of the current study was to determine if these different modulatory effects are mediated by the same or distinct interactions with the channel. EXPERIMENTAL APPROACH: Electrophysiology and site-directed mutation were used to investigate the effects of PF-06526290 on Nav channel function. KEY RESULTS: PF-06526290 greatly slows inactivation of Nav channels in a subtype-independent manner. However, upon prolonged depolarization to induce inactivation, PF-06526290 becomes a Nav subtype-selective inhibitor. Mutation of the domain 4 voltage sensor modulates inhibition of Nav 1.3 or Nav 1.7 channels by PF-06526290 but has no effect on PF-06526290 mediated slowing of inactivation. CONCLUSIONS AND IMPLICATIONS: These findings suggest that distinct interactions may underlie the two modes of Nav channel modulation by PF-06526290 and that a single compound can affect sodium channel function in several ways.

Discovery and in Vitro Optimization of 3-Sulfamoylbenzamides as ROMK Inhibitors.

Inhibitors of the renal outer medullary potassium channel (ROMK) show promise as novel mechanism diuretics, with potentially lower risk of diuretic-induced hypokalemia relative to current thiazide and loop diuretics. Here, we report the identification of a novel series of 3-sulfamoylbenzamide ROMK inhibitors. Starting from HTS hit 4, this series was optimized to provide ROMK inhibitors with good in vitro potencies and well-balanced ADME profiles. In contrast to previously reported small-molecule ROMK inhibitors, members of this series were demonstrated to be highly selective for inhibition of human over rat ROMK and to be insensitive to the N171D pore mutation that abolishes inhibitory activity of previously reported ROMK inhibitors.

GI-530159, a novel, selective, mechanosensitive two-pore-domain potassium (K2P ) channel opener, reduces rat dorsal root ganglion neuron excitability.

BACKGROUND AND PURPOSE: TREK two-pore-domain potassium (K2P ) channels play a critical role in regulating the excitability of somatosensory nociceptive neurons and are important mediators of pain perception. An understanding of the roles of TREK channels in pain perception and, indeed, in other pathophysiological conditions, has been severely hampered by the lack of potent and/or selective activators and inhibitors. In this study, we describe a new, selective opener of TREK channels, GI-530159. EXPERIMENTAL APPROACH: The effect of GI-530159 on TREK channels was demonstrated using 86 Rb efflux assays, whole-cell and single-channel patch-clamp recordings from recombinant TREK channels. The expression of K2P 2.1 (TREK1), K2P 10.1 (TREK2) and K2P 4.1 (TRAAK) channels was determined using transcriptome analysis from single dorsal root ganglion (DRG) cells. Current-clamp recordings from cultured rat DRG neurons were used to measure the effect of GI-530159 on neuronal excitability. KEY RESULTS: For recombinant human TREK1 channels, GI-530159 had similar low EC50 values in Rb efflux experiments and electrophysiological recordings. It activated TREK2 channels, but it had no detectable action on TRAAK channels nor any significant effect on other K channels tested. Current-clamp recordings from cultured rat DRG neurones showed that application of GI-530159 at 1 µM resulted in a significant reduction in firing frequency and a small hyperpolarization of resting membrane potential. CONCLUSIONS AND IMPLICATIONS: This study provides pharmacological evidence for the presence of mechanosensitive TREK K2P channels in sensory neurones and suggests that development of selective K2P channel openers like GI-530159 could aid in the development of novel analgesic agents. LINKED ARTICLES: This article is part of a themed section on Recent Advances in Targeting Ion Channels to Treat Chronic Pain. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.12/issuetoc.

Addition of a single methyl group to a small molecule sodium channel inhibitor introduces a new mode of gating modulation.

BACKGROUND AND PURPOSE: Aryl sulfonamide Nav 1.3 or Nav 1.7 voltage-gated sodium (Nav ) channel inhibitors interact with the Domain 4 voltage sensor domain (D4 VSD). During studies to better understand the structure-activity relationship of this interaction, an additional mode of channel modulation, specifically slowing of inactivation, was revealed by addition of a single methyl moiety. The objective of the current study was to determine if these different modulatory effects are mediated by the same or distinct interactions with the channel. EXPERIMENTAL APPROACH: Electrophysiology and site-directed mutation were used to compare the effects of PF-06526290 and its desmethyl analogue PF-05661014 on Nav channel function. KEY RESULTS: PF-05661014 selectively inhibits Nav 1.3 versus Nav 1.7 currents by stabilizing inactivated channels via interaction with D4 VSD. In contrast, PF-06526290, which differs from PF-05661014 by a single methyl group, exhibits a dual effect. It greatly slows inactivation of Nav channels in a subtype-independent manner. However, upon prolonged depolarization to induce inactivation, PF-06526290 becomes a Nav subtype selective inhibitor similar to PF-05661014. Mutation of the D4 VSD modulates inhibition of Nav 1.3 or Nav 1.7 by both PF-05661014 and PF-06526290, but has no effect on the inactivation slowing produced by PF-06526290. This finding, along with the absence of functional inhibition of PF-06526290-induced inactivation slowing by PF-05661014, suggests that distinct interactions underlie the two modes of Nav channel modulation. CONCLUSIONS AND IMPLICATIONS: Addition of a methyl group to a Nav channel inhibitor introduces an additional mode of gating modulation, implying that a single compound can affect sodium channel function in multiple ways.

Voltage sensor interaction site for selective small molecule inhibitors of voltage-gated sodium channels.

Voltage-gated sodium (Nav) channels play a fundamental role in the generation and propagation of electrical impulses in excitable cells. Here we describe two unique structurally related nanomolar potent small molecule Nav channel inhibitors that exhibit up to 1,000-fold selectivity for human Nav1.3/Nav1.1 (ICA-121431, IC50, 19 nM) or Nav1.7 (PF-04856264, IC50, 28 nM) vs. other TTX-sensitive or resistant (i.e., Nav1.5) sodium channels. Using both chimeras and single point mutations, we demonstrate that this unique class of sodium channel inhibitor interacts with the S1-S4 voltage sensor segment of homologous Domain 4. Amino acid residues in the "extracellular" facing regions of the S2 and S3 transmembrane segments of Nav1.3 and Nav1.7 seem to be major determinants of Nav subtype selectivity and to confer differences in species sensitivity to these inhibitors. The unique interaction region on the Domain 4 voltage sensor segment is distinct from the structural domains forming the channel pore, as well as previously characterized interaction sites for other small molecule inhibitors, including local anesthetics and TTX. However, this interaction region does include at least one amino acid residue [E1559 (Nav1.3)/D1586 (Nav1.7)] that is important for Site 3 α-scorpion and anemone polypeptide toxin modulators of Nav channel inactivation. The present study provides a potential framework for identifying subtype selective small molecule sodium channel inhibitors targeting interaction sites away from the pore region.