杂环肽类DENV丝氨酸蛋白酶抑制剂的设计、合成及活性研究

The mosquito-borne dengue viruses (DENV) are widespread human pathogens causing dengue fever, dengue hemorrhagic fever and dengue shock syndrome, placing 40% of the world's population at risk with no effective treatment. The N-terminal 180 amino acids of nonstructural protein 3 (NS3) is a serine protease, which binds a required cofactor NS2B. The serine protease is essential for DENV replication and thus serves as one of the promising targets for development of anti-DENV drugs. In this project, we focused on the design, synthesis and biological analysis of novel DENV serine protease inhibitors. Based on the active site characteristics from the crystal structure of DENV serine protease, a series of heterodetic cyclopeptide derivatives are presented as the inhibitors of DENV serine protease. The heterodetic cyclopeptide derivatives retain the active fragments composed of Lys and Arg, which are easily recognized by the serine protease. Compared with linear peptide inhibitor, the induction of heterodetic cyclopeptide can reduce the molecular flexibility and control the conformational diversification of the inhibitor molecule. The increased proteolytic stability and enhanced binding affinity of conformationally constrained heterodetic cyclopeptides make them attractive synthetic targets as the inhibitors of DENV serine protease. Inhibition modes and mechanisms of action were determined and the effects of the linker variety (such as the length, flexibility, hydrophobicity and electricproperty) on the activities also were revealed based on the biological analysis. These results allowed the development of a structure activity relationship (SAR) for heterodetic cyclopeptide small molecule inhibitors of DENV serine protease that identified key functional groups for effective binding and inhibition. Furthermore, the pharmacophore model of heterodetic cyclopeptide was established to optimize the small molecule structure. This research will reflect a logical progression for early antiviral drug discovery which can afford theoretical basis and material basis for successful identification of the candidate anti-DENV drugs.

登革热是发病率处于快速上升期、严重危害人类健康的传染性疾病。迄今为止，临床上尚未发现有效的治疗药物。登革热病毒（DENV）丝氨酸蛋白酶在病毒的复制转录过程中发挥着关键性作用，因而成为抗DENV药物研究的重要靶点之一。本项目基于DENV丝氨酸蛋白酶晶体结构，精确解析其活性位点的三维结构，以Lys和Arg构成的活性片段为基础，首次设计合成一系列具有高活性的杂环肽类新型DENV丝氨酸蛋白酶抑制剂，既保留易被丝氨酸蛋白酶所识别的Lys和Arg活性片段，又对分子的构象进行了限制，使得抑制剂在生物体内具有更好的抗酶解能力以及与靶标大分子更强的亲和力和选择性；在活性筛选基础上研究构效关系，重点研究成环分子片段的柔性、长短、疏水性及电性对化合物活性的影响。阐述杂环肽类活性分子与靶标大分子之间的结合模式，探索杂环肽抑制剂的药效团模型，为发展具有自主知识产权的抗DENV小分子药物奠定科学基础和物质条件。

本项目主要针对黄病毒属登革热病毒丝氨酸蛋白酶，通过结构导向的药物设计、计算机辅助的虚拟筛选和天然产物结构优化三种途径，开展了小分子抑制剂的设计、合成与活性筛选研究。1）具有自主知识产权的环肽类丝氨酸蛋白酶抑制剂-生物活性结果显示环肽类抑制剂III不仅具有良好的蛋白酶水平上的抑制活性，而且可以穿透细胞膜，在细胞水平上有效抑制登革热病毒的转录复制，从而成功地解决了线性拟肽类小分子抑制剂的细胞膜穿透性差的缺陷。项目研究结果深化了登革热病毒丝氨酸蛋白酶与小分子抑制剂相互作用机制的认识，有效解决了类肽抑制剂穿膜问题。2）本课题根据DENV丝氨酸蛋白酶的晶体结构和预测的结合模式，提取、创建并优化了药效团模型。然后以此作为提问式对数据库进行3D柔性搜索得到初筛结果。再通过基于分子对接的双轮筛选和生物活性测试，得到了一个具有良好细胞水平抑制活性的新型化学实体，并有望进入下一步优化。3）石蒜碱是一种从石蒜科植物中提取的天然生物碱，对登革热病毒具有较好的抑制作用。本研究以石蒜碱为起始原料，通过对其A-环和D-环的化学改造以及对1-位和（或）2-位羟基的结构修饰，共得到64个石蒜碱衍生物（其中52个未见报道），通过体外细胞黄病毒免疫实验来测得其抗病毒活性以及细胞毒性。结果表明，衍生物2-21和2-38具有比石蒜碱更好的抗登革病毒活性和更低的细胞毒性。.本项目执行过程中手足口病在我国多次爆发，对我国人民群众健康造成重大威胁，基于本课题组在病毒蛋白酶领域的研究基础，本项目针对引起手足口病的主要病原体肠病毒71 （EV71）的关键蛋白3C蛋白酶进行了深入的研究，利用结构生物学、化学生物学和计算机手段，以3C蛋白酶为靶点进行了具有创新性小分子抑制剂的设计与合成，取得了重要的研究进展，为发展治疗手足口病的有效治疗药物打下了良好基础。到目前为止，本研究已经发表论文9篇，申报专利2项，培养博士生2人，硕士生4人。

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