Pharmacologic Characterization of LTGO-33, a Selective Small Molecule Inhibitor of the Voltage-Gated Sodium Channel Na1.8 with a Unique Mechanism of Action.

Discovery and development of new molecules directed against validated pain targets is required to advance the treatment of pain disorders. Voltage-gated sodium channels (Nas) are responsible for action potential initiation and transmission of pain signals. Na1.8 is specifically expressed in peripheral nociceptors and has been genetically and pharmacologically validated as a human pain target. Selective inhibition of Na1.8 can ameliorate pain while minimizing effects on other Na 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 Na1.8 small molecule inhibitor. LTGO-33 inhibited Na1.8 in the nM potency range and exhibited over 600-fold selectivity against human Na1.1-Na1.7 and Na1.9. Unlike prior reported Na1.8 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 Na1.8 over dog and rodent Na1.8 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 Na1.8 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 Na1.8 small molecule pharmacologic space. SIGNIFICANCE STATEMENT: Na1.8 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 Na1.8 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.