Pharmacologic Characterization of LTGO-33, a Selective Small Molecule Inhibitor of the Voltage-Gated Sodium Channel NaV1.8 with a Unique Mechanism of Action
John M. Gilchrist, Nien-Du Yang, Victoria Jiang, Bryan D. Moyer
Abstract
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<sub>V</sub>s) are responsible for action potential initiation and transmission of pain signals. Na<sub>V</sub>1.8 is specifically expressed in peripheral nociceptors and has been genetically and pharmacologically validated as a human pain target. Selective inhibition of Na<sub>V</sub>1.8 can ameliorate pain while minimizing effects on other Na<sub>V</sub> 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<sub>V</sub>1.8 small molecule inhibitor. LTGO-33 inhibited Na<sub>V</sub>1.8 in the nM potency range and exhibited over 600-fold selectivity against human Na<sub>V</sub>1.1−Na<sub>V</sub>1.7 and Na<sub>V</sub>1.9. Unlike prior reported Na<sub>V</sub>1.8 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<sub>V</sub>1.8 over dog and rodent Na<sub>V</sub>1.8 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<sub>V</sub>1.8 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<sub>V</sub>1.8 small molecule pharmacologic space. <h3>SIGNIFICANCE STATEMENT</h3> Na<sub>V</sub>1.8 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<sub>V</sub>1.8 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.