一种新颖钠通道阻断剂的分子药理学机制研究

Voltage-gated sodium channels are the important drug targets for the treatment of neuropathic pain. The traditional sodium channel blockers have been demonstrated as improved symptoms of hyperalgesia. However, these drugs non-selectively inhibited multiple sodium channels, which caused unexpected side effects when used in clinic. Therefore it would be very important to develop subtype-selective sodium channel blockers, which will consequently increase the target selectivity. The applicant has identified a novel Nav1.7 sodium channel blocker WB-001 from a high-throughput screen. The compound preferentially inhibited Nav1.7 channels in the inactivation state without use-dependent inhibition. Surprisingly WB-001 inhibited TTX-resistant sodium currents in a use-dependent manner in the DRG neurons. All these results suggested that sodium channel subtype Nav1.8 may account for the use-dependent inhibition effect. To determine the subtype selectivity, DRG neurons culture, recombinant sodium channels and whole-cell patch clamp technique will be used. And site-directed mutagenesis and transient transfection will be used to determine the molecular binding site for the class of compound. Finally WB-001 will be tested for pain relief in the inflammatory pain model.

电压门控钠通道是治疗神经病理性疼痛的有效药物靶点。传统的钠通道阻断剂虽可有效的改善痛觉症状，由于对多个亚型钠通道的非特异性作用导致副作用较多。研发亚型选择性的钠通道阻断剂，将增加药物的靶向选择性。申请人实验室从高通量筛选项目中鉴定了一个新颖化合物WB-001，可阻断失活状态Nav1.7通道，却无使用依赖性阻断作用，但在DRG神经元的TTX不敏感钠通道却表现出使用依赖性阻断特性。分析各钠通道亚型的生物物理特点，Nav1.8可能是该使用依赖性阻断的直接靶标。本研究将采用分离培养的DRG神经元、重组的钠通道和全细胞膜片钳技术确认WB-001的亚型选择性作用，进一步通过定点基因突变、细胞转染和膜片钳技术发现该类化合物在钠通道的分子结合位点，最终证实该药物对疼痛的缓解作用。

长久以来，WB4101对Alpha-1肾上腺素受体的阻断作用一直被认为是其缓解疼痛作用的主要机制。一些报道提示WB4101对离子通道尤其是钙通道和钠通道的作用，也可能参与其疼痛缓解作用中。本项目详细阐述了WB4101对钠通道的作用特征。应用手动膜片钳方法，我们发现WB4101是强效的Nav1.7和Nav1.8钠通道状态依赖性阻断剂，相比静息态，WB4101对失活态和开放态钠通道有更强的阻断作用。WB4101使Nav1.7和Nav1.8的电压依赖性失活曲线向超极化方向位移约20mV。对失活缺陷型WCW突变的Nav1.7和Nav1.8通道的持续性晚钠电流仍具有强效的抑制作用。同时，WB4101还表现了对Nav1.7和Nav1.8的频率依赖性阻断作用，将Nav1.7和Nav1.8通道蛋白DIV结构域 S6跨膜段的局麻药结合位点的苯丙氨酸进行定点突变后（Nav1.7-F1737A，Nav1.8-F1710A），WB4101的频率依赖性阻断作用减弱。最后，在原代培养的大鼠DRG神经元上，WB4101对内源表达的钠离子通道同样具有状态依赖性阻断作用。最后，我们证实了WB4101对弗氏完全佐剂（CFA）诱发的炎症性疼痛具有显著的缓解作用。通过本项目，我们证实了WB4101是一个强效且状态依赖性的钠通道阻断剂，除了对α1肾上腺素受体的阻断，对神经元钠离子通道的阻断也是WB4101疼痛缓解的重要机制。