以“病毒进入细胞”为靶的五环三萜抗HCV病毒药物研究

Due to the high mutation rate of gene replication of RNA viruses including HCV, antiviral drugs targeting virus replication lose their activities rapidly. However, the upstream and downstream of virus replication - virus entry into the cell and virus release from the infected cell, which involving the interaction of virus and host cells (low mutation rate), provide a new opportunity for the research and development of antiviral drugs. Based on our previously established "visual" model of fluorescence labeled HCV infecting Huh7 cell line, this project take the anti-HCV entry activity of triterpene saponin found in Radix Isatidis and Dipsacus Asperoides as lead compounds to investigate the structure activity relationship (SAR) of natural triterpenes as anti-HCV entry agents and develop the pharmacophore structure conserved in pentacyclic triterpene with high activity and low hemolytic effect. Further, by multiple techniques including biotin-labeled photo-crosslinking molecular probe and bi-functional molecules such as pentacyclic triterpene-sialic acid/Vc (helpful in virus recognition) conjugates, photo-crosslinking co-immunoprecipitation and proteomic mass spectrum, the project study the target protein of pentacyclic triterpene as virus entry inhibitors and the molecular mechanism manipulated by it during virus-host recognition, develop novel antiviral drugs for HCV treatment and prevention, reduce the impact on antiviral drugs caused by the drug resistance resulting from gene mutation, and explore the feasibility of the modernization of traditional Chinese Medicine, such as Radix Isatidis and so on.

以HCV为代表的RNA病毒因其基因复制的高突变率，使以"病毒复制"为靶的抗病毒药物较易失去活性；而病毒复制的上、下游过程 -"病毒进入细胞"和"新生病毒从感染细胞中释放"，涉及与宿主细胞（突变率低）的相互作用，构成了抗病毒药物研发的新契机。本课题基于前期建立的荧光标记HCV病毒感染Huh7细胞的"可视化"模型，以板蓝根和川续断中发现的活性五环三萜为起始物质,研究三萜天然物抑制HCV病毒进入细胞的构效关系和药效团，发现活性好、溶血副作用低的先导化合物；进而通过生物素标记的光交联分子探针、唾液酸/维生素C等参与病毒识别的功能分子修饰的三萜衍生物、光交联免疫共沉淀以及蛋白质组学质谱分析技术等，寻找五环三萜以"病毒进入细胞"为靶的作用蛋白和其调控病毒与宿主细胞识别的分子机制，开发治疗和预防功能兼具的新型抗病毒药物，降低因突变带来的耐药性对抗病毒药物的冲击，并探索板蓝根等传统中药现代化的可行性。

由于HCV基因复制的高突变率，使得以病毒进入细胞为靶的新型抗HCV药物的研发受到了国内外学者的广泛关注。本项目建立了安全、快速、可靠的荧光标记HCV感染Huh7细胞的筛选平台以及从中药皂荚大量制备刺囊酸的工艺流程，合成了一系列刺囊酸等五环三萜小分子化合物库并进行了抗HCV活性评价，总结了五环三萜类天然物抗HCV进入的构效关系及药效团，即五环三萜的A环、B环及E环是其抗HCV的保守基团，而D环及17位羧基是可修饰位点。在此基础上进一步针对五环三萜的可修饰位点17位羧基开展了糖基化、维生素C及唾液酸等功能分子的修饰，并综合运用加药时间点、免疫共沉淀、表面等离子共振、蛋白质谱及Autodocking等实验，对五环三萜类化合物抗HCV的作用机制进行了深入的实验研究，发现了五环三萜抗HCV进入活性的作用靶点为HCV E2蛋白。本研究不仅发现了数个活性好而毒性低的具有抗HCV进入活性的抑制剂，为进一步研发具有新作用机制的抗HCV药物的提供了候选先导化合物，而且为“五环三萜天然物是自然界调控病毒与宿主识别的生物进化产物”的学说提供了重要的理论依据。本项目相关研究发表了SCI论文15篇，总影响因子58.97，其中在本领域权威刊物Biomaterials, J. Med. Chem及Eur. J. Med. Chem发表论文7篇，在Tetrahedron刊物发表封面论文一篇，获中国药学会优秀论文二等奖一次，培养博士生2名，硕士生6名（含3名在读）。