三种具有镇痛活性的Nav1.8抑制剂的作用机制研究

Voltage-gated sodium channel subtype Nav1.8 is an idea target for the development of analgesic drugs. Recently, scientists from basic study field and the pharmaceutical industry mainly focus on specific inhibitor discovery and action mechanism investigation. It should note that it is still hard for the heterologous expression of Nav1.8 in mammalian cells, which limits the progress of the discovery of Nav1.8 inhibitors. MrVIB and A-803467 are two Nav1.8 inhibitors with antinociceptive potential, but their action mechanisms still remain at large. In our previous study, we developed an effective Nav1.8 expression system through construction of chimeric channels. By using this system, we identified a high specific inhibitor of Nav1.8, named μ-EPTX-Na1a, through a large screening of peptide toxins. The specificity of μ-EPTX-Na1a on Nav1.8 is over other Nav1.8 inhibitors identified so far. Furthermore, it has analgesic effect better than morphine and shows low side effects on animal models. Basing on previous studies, the three Nav1.8 inhibitors (μ-EPTX-Na1a, MrVIB and A-803467) are rather different in molecular structure, inhibitory activity on Nav1.8 and antinociceptive effects, indicating their distinct action mechanisms. Subsequently, in the present study, we will try to elucidate the action mechanisms through investigating their effects on kinetics of Nav1.8 current, construction of inhibitor-channel complex and determination of their analgesic behaviors, and therefore clarify the relationship between Nav1.8 inhibition and analgesic. This study will provide basis for the analgesic drug development by using the three Nav1.8 inhibitors as drug leads.

靶向钠通道Nav1.8成为镇痛药研发的新策略，近年来主要聚焦于专一性抑制剂及作用机制研究。因缺少适于高通量筛选的Nav1.8哺乳动物细胞表达系统，目前进展缓慢，两种抑制剂MrVIB和A-803467备受关注，但因作用机制尚未深入阐明，应用受到限制，故国际上该研究领域需求迫切。为此，利用钠通道亚型嵌合策略构建了一种高效Nav1.8表达系统，并在本室储备的多肽毒素库中开展较大规模筛选，鉴定了一个目前专一性最高的Nav1.8抑制剂μ-EPTX-Na1a，其具有优于吗啡的镇痛效果和低毒副作用，是一个优质镇痛药先导分子；且相比于上述两种抑制剂，具有完全不同的分子结构和作用模式。因此，本项目以三种抑制剂为研究对象，在电生理活性、抑制剂-通道复合物、镇痛活性三个层面阐明其作用机制，揭示Nav1.8抑制与镇痛活性的内在联系，为基于三种抑制剂的镇痛新药研发提供理论依据，为靶向Nav1.8研发镇痛药提供思路。

临床遗传学、动物模型和药理学研究表明电压门控钠通道亚型Nav1.8是炎性和神经性等疼痛治疗的新型药物靶点。作用于疼痛相关钠通道专一性抑制剂的筛选和结构与功能的研究，有助于新型镇痛药物先导分子的研发和利用专一性多肽探针探究钠离子通道参与疼痛的调节。本项目对已发现的Nav1.8专一性抑制剂μ-EPTX-Na1a（Na1a），从镇痛动物模型、药物安全性方面进行了进一步评价；其次，成功地从海南捕鸟蛛毒液中筛选得到Nav1.8的专一性延缓失活剂δ-TRTX-Hhn1a（Hhn1a），其具双重作用，低浓度抑制峰电流，高浓度抑制通道的失活，可用作研究毒素通道相互作用的分子探针；最后成功构建了基于HEK293T细胞系的Nav1.8嵌合体通道异源表达系统，便于体外进行大规模的通道筛选。通过本项目研究发表了高水平SCI研究论文8篇，申请国家发明专利4项，培养博士研究生3名，硕士研究生4名，国内学术报告3次。