Discovery and development of new molecules directed against validated pain targets is required to advance the treatment of pain disorders. Voltage-gated sodium channels (Na_Vs) are responsible for action potential initiation and transmission of pain signals. Na_V1.8 is specifically expressed in peripheral nociceptors and has been genetically and pharmacologically validated as a human pain target. Selective inhibition of Na_V1.8 can ameliorate pain while minimizing effects on other Na_V isoforms essential for cardiac, respiratory, and central nervous system physiology. Here we present the pharmacology, interaction site, and mechanism of action of LTGO-33, a novel Na_V1.8 small molecule inhibitor. LTGO-33 inhibited Na_V1.8 in the nM potency range and exhibited over 600-fold selectivity against human Na_V1.1-Na_V1.7 and Na_V1.9. Unlike prior reported Na_V1.8 inhibitors that preferentially interacted with an inactivated state via the pore region, LTGO-33 was state-independent with similar potencies against closed and inactivated channels. LTGO-33 displayed species specificity for primate Na_V1.8 over dog and rodent Na_V1.8 and inhibited action potential firing in human dorsal root ganglia neurons. Using chimeras combined with mutagenesis, the extracellular cleft of the second voltage-sensing domain was identified as the key site required for channel inhibition. Biophysical mechanism of action studies demonstrated that LTGO-33 inhibition was relieved by membrane depolarization, suggesting the molecule stabilized the deactivated state to prevent channel opening. LTGO-33 equally inhibited wild-type and multiple Na_V1.8 variants associated with human pain disorders. These collective results illustrate LTGO-33 inhibition via both a novel interaction site and mechanism of action previously undescribed in Na_V1.8 small molecule pharmacologic space. SIGNIFICANCE STATEMENT: Na_V1.8 sodium channels primarily expressed in peripheral pain-sensing neurons represent a validated target for the development of novel analgesics. Here we present the selective small molecule Na_V1.8 inhibitor LTGO-33 that interdicts a distinct site in a voltage-sensor domain to inhibit channel opening. These results inform the development of new analgesics for pain disorders.

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