抗HBV病毒新型海洋briarane萜类分子发现与作用机制研究

Hepatitis B virus (HBV) is a significant global pathogen, and still represents a major health threat with about 350 million chronically infected individuals worldwide. HBV infection leads to a wide spectrum of liver disease ranging from acute to chronic viral hepatitis, which is often associated with the development of liver cirrhosis and hepatocellular carcinoma. The ultimate goal of chronic hepatitis B virus therapy is full eradication of the virus from the liver. However, this is rarely achieved with the clinically available first-line agents due to the inability to eliminate covalently closed circular DNA (cccDNA), which persists in the nucleus of infected hepatocyte cells. Therefore, it is urgent to search structurally novel natural products which are able to target on cccDNA and finally to eliminate HBV infection. Our previous examination of anti-HBV compounds from marine benthic organisms revealed that marine gorgonian derived briarane-type diterpenoids exerted significant inhibition against HBV-DNA, HBV-RNA, and cccDNA in HepAD38 and Hepg2.2.15 cell lines. In this proposal, the bioassays for the regulation of HBV life-cycle and inhibition of cccDNA are oriented for the discovery of bioactive and structurally novel marine terpenoids which are potent for the inhibition of HBV. Two steps are involved in the chemical investigation: (1) to extend the chemical diversity and novelty of briarane-type diterpenoids, which are followed by the anti-HBV evaluation under multiple HBV-infected cell models. The structure-activity relationships are analyzed to uncover the functional groups within the briarane scaffold and to achieve the most active compounds; (2) to extend the biogenetically related terpenoids including cembranoids and biscembranoids/isobiscembranoids which are isolated from marine soft coral and gorgonian species. Comparison of the anti-HBV activities with the diverse terpenoid scaffolds will be discussed. Briarane-type analogues with the inhibition of cccDNA will be selected in priority for further mode of action investigation. The molecular pathways and mediated factors derived from host and virus which are able to regulate cccDNA will be clarified. In addition, briarane-probes are synthesized to uncover new targets for the regulation of cccDNA formation. Present project aims to discover structurally novel anti-HBV marine natural products, and to explore marine terpenoids for pharmaceutical usage.

慢性乙肝是肝硬化和肝细胞癌的高危因素，核cccDNA难以清除导致临床药物不可治愈。课题基于前期发现珊瑚来源briarane型二萜具有显著抑制HBV病毒和cccDNA等活性，设计以调控HBV感染周期和抑制HBV关键靶标为导向，通过briarane型生源相关萜类结构多样性与新颖性研究和多模型HBV感染细胞评价，建立活性分子的构-效关系。选择具有显著抑制HBV活性新型萜类分子，针对乙肝病毒复制各环节以及化合物的抗HBV特点，评价化合物对HBV复制模板cccDNA水平、HBV基因转录和转录后修饰、core蛋白表达、HBV逆转录过程，以及宿主细胞基因表达谱变化等分子机制。同时，制备briarane分子探针，以期发现抗病毒新靶标或信号通路，并以人肝嵌合HBV动物阐明抑制HBV病毒复制功效。通过挖掘抗慢性HBV新型海洋药源分子潜力研究，为海洋萜类分子的药用功能开拓新方向。

针对乙肝病毒感染仍是我国严重危害健康疾病且临床药物有限问题，课题拟以调控HBV感染周期和抑制HBV关键靶标为导向，对briarane型萜类结构多样性与新颖性研究和多模型HBV感染细胞评价，建立活性分子的构-效关系，以期发现新型抗HBV化合物并阐明作用机制，为海洋萜类分子的药用功能开拓新方向。. 应用GNPS分子网络和NMR导向，从2种柳珊瑚中制备并鉴定195种briarane-型二萜，其中103种新化合物。结合自建海洋天然产物库，用多种细胞进行细胞毒性测定，选择134种无细胞毒或低细胞毒海洋天然产物进行抗乙型肝炎病毒活性评价。采用乙肝病毒细胞HepAD38和HepG2.2.15，发现具有抗乙型肝炎病毒活性的化合物有26个，结构类型包括二萜，倍半萜，聚酮类以及生物碱。确定2个biarane型二萜显著抑制HepAD38细胞上清中的HBV DNA水平和胞内总的HBV DNA水平，且呈时-效和量-效相关性；7个生物碱类化合物显著抑制HBsAg的分泌，且不影响HBV DNA水平以及HBeAg分泌；一个倍半萜和3个其他二萜抑制HBV复制；1个聚酮具有抗HBV作用。对2种briarane-型二萜junceellolide B（1）和C（2）进行深入作用机制研究，化合物在不同HBV 感染肝细胞模型中对HBeAg、HBV DNA 和HBV RNA 具有显著抑制作用，并可显著降低HBV cccDNA，化合物1调控宿主转录因子介导HBV cccDNA转录过程。化合物1和2不同程度地抑制HepAD38细胞内HBV复制中间体（rcDNA、ssDNA）的表达；但不影响细胞培养上清中HBsAg。化合物1-2呈量-效依赖性抑制內源性HBV core 蛋白的表达，但对外源性HBV core 蛋白无抑制活性。化合物1-2对HepAD38细胞上清中HBV pgRNA有抑制活性。但是，对调控preC和pgRNA转录的HBV BCP区无影响。在HepAD38细胞被dox持续调控下，化合物1和2（10 M）仍可降低cccDNA含量，抑制率为50.5%和56.5%，对DP-rcDNA也有一定的抑制作用，提示1和2的作用靶点可能与HBV cccDNA形成过程有关。研究工作发现一类新型海洋来源二萜通过调控宿主和病毒转录相关因子多靶标抑制HBV病毒，具有进一步研发抗HBV病毒候选分子的潜力。