基于ABPP策略的天然分子京尼平抗对虾WSSV靶标的确证及机制研究

药物靶标的确证是药物研发领域的前沿方向，也是解决对虾白斑综合症病毒（WSSV）药物研发的关键。申请人前期研究发现中草药栀子和杜仲的天然成分京尼平，不仅具有显著的抗WSSV活性，还可以通过直接作用和间接作用两种方式抑制WSSV复制，是药物开发的理想先导化合物，但作用靶标和机制并不清楚。本项目通过合成生物素标记的京尼平探针，通过活性导向的蛋白谱学（ABPP）策略分别从WSSV和对虾造血组织蛋白裂解液“钓取”并鉴定京尼平的潜在靶标蛋白；进一步利用竞争性Pull-down和RNA干扰技术进行体外和体内验证靶标蛋白，明确京尼平的作用靶标；此外，通过光谱学和分子对接技术研究京尼平与靶标的结合模式和结合位点，并利用定点突变和竞争性Pull-down技术佐证，以揭示京尼平抗对虾WSSV的作用机理。本项目不但利于阐明WSSV的致病机理，也利于推进新型药物研发的速度，具有重要科学意义和应用价值。

Drug target discovery is the frontier research on new drug creation, and it is also the key of drug development against white spot syndrome virus (WSSV). Recently, we found a natural active molecule genipin, with high anti-WSSV activity through the direct effect and indirect regulation, is an ideal lead compounds for drug development. However, the role of drug target and mechanism of genipin is not clear. In the project, biotin-labeled genipin probes are synthesized, and the potential target proteins of genipin are identified by affinity chromatography from protein lysates the WSSV and hematopoietic tissue. Furthermore, competitive pull-down technology and RNA interference technology are used to verify the target protein in vitro and in vivo, so as to clarify the drug target of genipin. In addition, The binding patterns and binding sites of genipin and target are studied by spectroscopy and molecular docking techniques, and site-directed mutation technology and competitive pull-down technology are used to prove the results, so as to reveal the mechanism of action of genipin against WSSV. This project is not only conducive to elucidating the pathogenic mechanism , but also conducive to promoting the speed of new drug research, showing important scientific significance and application value.