五环三萜化合物调控多种RNA病毒感染的共性分子机制和结构基础研究

Pentacyclic triterpenes are a class of secondary plant metabolites widely found in nature with a variety of biological functions. Previous studies have indicated that certain pentacyclic triterpenoids can bind to the viral structural proteins of HIV, hepatitis C, influenza, and Ebola viruses, thereby inhibiting viral entry into the respective host cells. However, it remains to determine by what mechanism pentacyclic triterpenoids achieve such attractive activities, i.e. preventing viral envelope proteins from interacting with their host receptor despite of their significant disparate. Here, we make a proposal by design and synthesis of triterpenoid probes with dual photo-crosslinking / biotin bifunctional groups, based on the identified lead compounds and the structure-function relationships, and then incubation and photo-crossing of the probes with the envelope E2 of HCV, HA of influenza and GP of Ebola viruses. The crossing peptides are enriched via co-immunoprecipitation and characterized by mass spectrum and databases. Upon identification of the conserved primary amino acid sequence, we utilize the modern technologies such as structure mimicking, computer modeling and docking and others, to identify the potential secondary- and three-dimensional common structures. In addition, based on those structure and function information, we clarify the common structural features of the interactions of different viral envelope proteins and respective host receptors, and the common mechanism by which their interactions are regulated and manipulated by triterpene natural products. Eventually we will provide theoretical and experimental evidences to prove the hypothesis that pentacyclic triterpenoid natural products might be an evolved regulator of virus-host recognition and interactions.

五环三萜是一类自然界广泛存在、且有多种生理功能的天然物质。前期研究发现某些五环三萜可以结合HIV、丙肝、流感和埃博拉等RNA病毒的膜结构蛋白,进而抑制病毒进入各自的宿主细胞。然而这些完全不同的病毒结构蛋白是如何被五环三萜共性地识别并阻止与相应宿主受体作用的呢?本课题将基于我们发现的抑制流感、丙肝和埃博拉三种病毒的五环三萜先导物及构效关系，设计和合成带有光交联/生物素双功能基团的分子探针，通过光交联、免疫共沉淀、蛋白质谱和数据库，发现五环三萜与流感HA、丙肝E2和埃博拉GP膜蛋白结合的一级氨基酸序列；进而基于结构模拟、计算机同源模建和对接等现代技术，研究它们结合的二级平面和三级立体共性结构特征。最后基于相应信息，探索不同病毒膜蛋白与宿主受体结合的共有结构基础，阐明五环三萜调控这些病毒与相应宿主识别的共性分子机制，为“五环三萜天然物是自然界调控病毒与宿主识别的进化产物”假说提供理论和实验依据。

由艾滋病毒、丙型肝炎病毒、埃博拉出血热及流感病毒等引起的病毒性恶性传染病严重危害人类健康。基于五环三萜类天然产物的广谱病毒活性，本课题进一步建立了抗埃博拉病毒进入细胞的筛选模型，对五环三萜衍生物抗埃博拉病毒的构效关系进行了研究并发现化合物Y11和Y13具有较好的抗埃博拉病毒的活性，1μM时的抑制率分别高为99%和93%。进一步真病毒活性评价发现Y11在Hela及HFF细胞中对埃博拉（Makona）的EC50分别为0.98-0.99μM及0.46-0.89μM。在此基础上综合运用光交联、免疫共沉淀、蛋白质谱、核磁共振及计算机辅助药物设计等技术对其与作用靶蛋白进行了深入的研究，发现了该探针可与埃博拉病毒膜蛋白GP的HR2结构域能特异的结合；同时，设计并合成了具有抗HIV活性的光交联探针Y20、具有抗流感病毒活性的光交联探针Y21和具有抗HCV活性的生物素探针Y22及光交联探针Y23，采用上述实验也发现了这些探针能与病毒的膜蛋白HR2结构域结合。三萜天然产物的广泛存在，特别是其通过结合病毒膜蛋白HR2赋予植物宿主自我化学防护功能，提示三萜类天然物可能是大自然调控病毒与宿主识别的进化产物。本项目实施过程中，一共发表了SCI论文21篇，其中Science、Sci. Adv.各一篇；Biomaterials两篇；Eur. J. Med. Chem. 六篇，Tetrahedron 封面论文一篇。鉴于本研究在五环三萜抗病毒领域中的影响，项目负责人应邀在国际药化权威刊物Med. Res. Rev.发表综述一篇；此外，还获得授权相关发明专利3项，发表会议论文4篇，做大会或分会报告4人次，培养博士生4名（含2名在读），毕业硕士4名，取得了较好的研究成果。