新型五环三萜-环糊精多价物的合成、抗流感进入构效关系及分子机制研究

Due to the high mutation rate of gene replication of influenza virus and the recombination of cross-species influenza virus, currently anti-influenza virus drugs targeting replication and release process are easy to produce drug-resistance variants, which is a major threat to millions of people worldwide. The virus entry into the cell provides a new opportunity for the research and development of new antiviral drugs. Based on our previously established “visual” model of fluorescence labeled A/WSN/33 (H1N1) infecting MCDK cell line, this project take the anti-influenza virus entry activity of the heptavalent pentacyclic triterpene-β-cyclodextrin conjugate, the aglycon of which was found in Radix Isatidis, as lead compound (IC50 =1.60 µM) to investigate the anti-influenza entry activity, broad spectrum and resistance of several series of novel cyclodextrin-triterpene multivalent conjugates, which using three kinds of nature cyclodextrin as scaffold and five kinds of pentacyclic triterpene as pharmacophore connected by different flexible or rigid linker. The molecular mechanism manipulated by those novel multivalent conjugates targeting influenza virus entry during the virus-host recognition will be investigated by multiple techniques including time-of-addition, hemagglutination inhibition, immunofluorescence and surface plasmon resonance experiments, and the binding affinity between them and HA protein will be also determined. Further, the pharmacology and acute toxicity of the candidate entry inhibitors with high activity and low toxicity effects will be carried out in vivo. This project will be helpful for developing novel broad spectrum anti-influenza virus drugs derived from traditional Chinese medicine with treatment and prevention functionality.

由于流感病毒基因复制的高突变率及跨物种流感病毒之间的重组，使得针对病毒复制和释放的抗流感药物较易产生耐药性，严重危害人类健康。以“病毒进入细胞”为靶的抗流感药物的研究是抗病毒药物研发的热点。本课题基于荧光标记A/WSN/33病毒感染MDCK细胞的“可视化”模型，以川续断中发现的活性五环三萜与β-环糊精形成的七价物为先导物（IC50=1.60µM），设计并合成以三种天然环糊精为骨架、以五种五环三萜为药效团、以柔性或刚性臂连接的新型多价物，运用细胞病变及噬斑实验研究其抗病毒进入活性、广谱性及耐药性；进而通过加药时间点、血凝抑制、免疫荧光及表面等离子共振等实验确认其作用于病毒感染宿主细胞的进入阶段并测定其与病毒进入密切相关的HA蛋白的结合常数，阐明其抗流感作用的分子机制；进一步对活性强而毒性低的候选抑制剂进行体内活性及急毒评价，为研发兼具治疗和预防功能、源于中药的广谱抗流感药物提供新的思路。

流感病毒具有极强的传染性和危害性，其周期性爆发严重危害人类健康。由于流感病毒基因复制的高突变率及跨物种流感病毒之间的基因重组，使得现有针对病毒复制和释放的抗流感药物较易产生耐药性。“以病毒进入细胞”为靶的抗流感药物的研究是抗病毒药物研究的热点之一。本课题基于基于荧光标记A/WSN/33病毒感染MDCK细胞的“可视化”模型及流感病毒进入细胞过程中的“多价效应”，以五种常见的五环三萜（齐墩果酸、次囊酸、熊果酸、白桦脂酸及甘草次酸）为药效团，以三种天然的α-, β-及γ-环糊精为核心骨架，以烷基链、哌嗪链、芳香链、寡聚乙二醇链等为连接臂，通过点击化学及酰胺化、酯化等化学反应，设计并合成了多个系列基于环糊精骨架的五环三萜多价物，为了便于比较，同时平行合成了部分环糊精-五环三萜单价化合物。对上述合成的环糊精-五环三萜多价化合物及单价化合物的抗流感活性进行了构效关系的研究，发现部分化合物显示出了较好的抗流感活性，进而综合运用加药时间点实验、血凝抑制实验、免疫荧光、表面等离子共振等技术对其抗流感作用机制及耐药性进行了研究，揭示了五环三萜类天然产物广谱抗病毒活性的生源功能，为新型具有抗流感病毒进入活性的多价抑制剂的研究提供了实验依据。本项目相关研究共发表SCI论文十四篇，其中在本领域权威刊物Biomaterials发表论文一篇，Eur. J. Med. Chem发表论文六篇，Tetrahedron封面论文一篇，鉴于本研究在相关领域中的影响，应邀在国际刊物Med. Res. Rev.发表综述一篇；此外，相关研究获得中国专利授权三项，参加会议报告三人次，获快闪报告奖一人次，培养博士生3名（含1名在读），硕士生6名（含3名在读）。