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.

需要发现和开发针对已验证疼痛靶点的新分子，以推进疼痛疾病的治疗。电压门控钠通道 (Na _V s) 负责动作电位的启动和疼痛信号的传输。 Na _V 1.8 在外周伤害感受器中特异性表达，并已被基因和药理学验证为人类疼痛靶标。选择性抑制 Na _V 1.8 可以减轻疼痛，同时最大限度地减少对心脏、呼吸和中枢神经系统生理学必需的其他 Na _{V亚型的影响。在这里，我们介绍了新型 Na V} 1.8 小分子抑制剂LTGO-33 的药理学、相互作用位点和作用机制。 LTGO-33 在 nM 效力范围内抑制 Na _{V 1.8，并对人 Na V} 1.1–Na _V 1.7 和 Na _V 1.9表现出超过 600 倍的选择性。与之前报道的 Na _V 1.8 抑制剂优先通过孔区域与失活状态相互作用不同，LTGO-33 是状态独立的，具有类似的针对关闭和失活通道的效力。 LTGO-33 对灵长类动物 Na _V 1.8 的物种特异性优于对狗和啮齿动物 Na _V 1.8 的物种特异性，并抑制人背根神经节神经元的动作电位放电。利用嵌合体与诱变相结合，第二个电压传感结构域的细胞外裂隙被确定为通道抑制所需的关键位点。生物物理作用机制研究表明，膜去极化可缓解 LTGO-33 抑制，表明该分子稳定失活状态以防止通道打开。 LTGO-33 同样抑制与人类疼痛疾病相关的野生型和多种 Na _{V 1.8 变体。这些集体结果说明了通过 Na V} 1.8 小分子药理学空间中先前未描述的新相互作用位点和作用机制来抑制 LTGO-33 。