Identification of Novel Pore Interactions for Selective Nav1.8 Inhibitors

Abstract

Nav1.8 voltage gated sodium channels are expressed predominantly in peripheral nociceptors, playing a key role in action potential propagation in these neurons. Nav1.8 knockout and antisense studies indicate that the channel has a major function in nociceptive processing in preclinical models of pain and histological samples suggest that Nav1.8 in expressed on primary afferent terminals innervating ‘painful’ human tissues. Small molecule modulators selective for Nav1.8 from several chemical series have been disclosed including PF-1247324 (IC50: 199 nM against human Nav1.8) and A-803467 (IC50: 9 nM). Here we describe the in vitro biophysical properties of these compounds and identify amino acid residues within the pore domain that are important for compound-channel interaction. While the compounds are broadly similar in terms of selectivity, they can be differentiated by their unique use- and state-dependence of inhibition. PF-1247324 exhibits significant positive use- and state-dependence of block similar to that observed for local anesthetics (LA). In contrast, A-803467 shows negative use and state dependence (partial loss of block) that depends on the magnitude and duration of membrane depolarization. These different biophysical profiles of the two chemotypes appear to result from distinct interactions with the channel. Alanine mutations were constructed at two sites important for LA binding (F1710A and Y1717A). In addition, unique residues present in the S6 segments of Domains II and III of Nav1.8 but not TTX-sensitive Nav channels were mutated. We found that the affinity of PF-1247324 was significantly reduced by mutations at the LA binding site as well as Nav1.8 specific residues, whereas A-803467 affinity was reduced most dramatically by mutation of one unique site. The data suggest PF-1247324 and A-803467 interact with distinct yet partially overlapping regions of the pore domain of Nav1.8.