特异性进入抑制剂L0909的抗HCV作用机制研究

The research and development of drugs against hepatitis c virus (HCV) has been achieved many progress. However, the development of entry inhibitors specific for HCV is not satisfaction. The reasons may be that the anti-HCV activity of inhibitors is not strong, or the toxicity is too strong, or druggability characteristic is poor, which prevent the development of entry inhibitors. Furthermore, it is particularly urgent to develop new innovative drugs with China's own intellectual property. During the research in my Excellent Young Scientists Fund of the National Natural Science Foundation of China, we got a HCV specific entry inhibitor L0909, a small molecular compound which is good in druggability characteristic and strongly inhibits HCV replication in cell culture with no cytotoxicity. L0909 showed an inhibition on direct-acting antiviral agents (DAAs) resistant HCV variants with an efficacy identical to that on wild type HCV, and synergistically inhibited HCV replication with other three type DAAs. Preliminary results showed that L0909 is an early HCV entry inhibitor, but it did not directly interact with HCV structure proteins which are closely related to HCV entry into cells. So the antiviral mechanism against HCV of L0909 is needed to be deeply illustrated. In this project, using L0909 as a probe and with a variety of classic and modern experimental techniques, we will step by step analyze to clarify the molecular mechanism on HCV of L0909 and the key roles of related effector molecule with L0909 in the HCV entry cells. The results will complementally clarify HCV entry mechanism and add a new antiviral theory, and on the other hand, they will also provide a scientific basis for research and development of innovative HCV entry inhibitors.

抗丙型肝炎病毒(HCV)药物研发迅速，但HCV进入抑制剂的研发由于其活性不强或毒性大或成药性特征差等原因而严重滞后，而且我国也特别亟待研发具有我国自主知识产权的创新抗HCV药物。在国家优青项目资助期间我们获得了一个具有高效低毒、成药性好的特异性抑制HCV进入的小分子合物L0909，其对现有直接抗病毒药物(DAA)常见的耐药病毒有效，还与不同类型的DAA联用均有协同抗病毒作用；初步作用机制研究结果显示L0909为HCV早期进入抑制剂，但不直接作用于与HCV进入相关的病毒结构蛋白上，其具体的抗病毒作用机制有待深入研究。本项目在此基础上，以L0909为探针，结合多种现代和经典实验技术手段，逐步深入分析以阐明其抗HCV作用的分子机制及其效应靶分子在HCV进入过程中的关键作用。研究结果一方面将补充阐明HCV进入细胞的机理和丰富抗病毒理论，另一方面还为研发创新性强的HCV进入抑制剂提供科学依据。

抗丙型肝炎病毒(HCV)药物研发迅速，但HCV进入抑制剂的研发由于其活性不强或毒性大或成药性特征差等原因而严重滞后，而且我国也特别亟待研发具有我国自主知识产权的创新抗HCV药物，因此阐明活性强、成药性特征好的进入抑制的作用机制为指导研发抗HCV新型药物具有重要意义。本项目以具有自主知识产权的新结构类型进入抑制剂L0909为探针，利用化学合成技术、病毒细胞培养和测定技术、蛋白分析技术以及分子互作等多种现代和经典实验技术手段，发现化合物L0909在HCV进入细胞过程的病毒内化后至膜融合前阶段发挥抑制作用；其作用靶点为HCV包膜蛋白 E1，在病毒进入细胞过程中被暴露后与其特异结合，从而阻止膜融合，达到抗病毒的作用。利用本项目新建的多种病毒细胞培养模型和测定方法，探索了L0909的作用特点和优势，发现化合物L0909对多种耐药病毒株有效，与低剂量临床用三联药物的联用可协同抗HCV，还可减少耐药的发生，从而进一步提高抗HCV总体疗效。另外，应用本项目技术手段，阐明了天然产物苦豆碱作为内吞抑制剂抗HCV的作用机制，以及转化生长因子 β在HCV感染中的作用，特别是其以TGF-β/SMAD 信号通路依赖和/或非 TGF-β/SMAD 信号通路依赖途径阻断 HCV 进入的机制。资助期间发表6篇SCI文章（标注基金号），授权国家专利2个，新申请国家专利1个，培养博士生4名，作为副主编编著专业书籍1部，个人获得国家高层次人才特殊支持计划科技创新领军人才的支持。相关成果为研发具有我国自主知识产权的创新HCV进入抑制剂提供科学依据，推动了HCV进入抑制剂的研发；同时为未来抗HCV感染用药，特别是为进一步提高抗HCV总体疗效和解决耐药问题，提供了新的用药策略。