针对肠道病毒71（EV71）关键蛋白3C 蛋白酶的新型抑制剂的研究

Entrovirus 71 (EV71) is the primary pathogen of Hand-foot-mouth disease (HFMD) in China. The EV71 encoded 3C protein, a cystein protease, is responsible for the cleavage and modification of EV71 polyprotein. The 3C protease is crucial for the replication of the virus, which makes it an attract target for anti-EV71 drug discovery. This project is focused on the design, synthesis and biological evaluation of six member ring lactam derivatives as the inhibitors against 3C protease. Based on the crystal structure of 3C protease, the inhibitor will be designed with structure based drug design approach. Through the investigation of cyanohydrin, the metobolism stability of inhibitor would be opimized and novel noncovalent “ warhead” of 3C protease inhibitor would be developed. The interaction between inhibitor and 3C protease as well as the binding mode of inhibitor are expected to be enlaborated through the thorough study of structure-activity relationship. A pharmacophore is expected to be fully established and optimized eventually. Moreover, lead compound will be selected based on overall properties of the inhibitors including in vivo efficacy, in vivo PK and preliminary safety evaluation. The project will be of importance for the discovery and development of novel, proprietary lead compound for the treatment of hand-foot-mouth disease by targeting 3C protease of EV71.

人肠道病毒71型（Enterovirus 71, EV71）是近年来在我国爆发的人手足口病的主要病原体。EV71基因组编码的3C蛋白是一类半胱氨酸蛋白酶，主要行使特异性蛋白酶的功能，在病毒复制过程中发挥重要作用，一旦3C蛋白酶的功能丧失，则病毒进一步的转录和复制将无法正常继续进行。本项目针对EV71病毒关键蛋白3C蛋白酶，基于3C蛋白酶的晶体结构，以六元内酰胺为结构母核，设计与合成新型的3C蛋白酶抑制剂。针对本团队发现的羟基氰这一高活性、非共价结合的功能性基团，改造和优化分子结构，改善化合物的代谢稳定性，发掘高活性、高选择性和代谢稳定性强的3C蛋白酶抑制剂；系统深入地研究六元内酰胺类小分子抑制剂结构与活性的关系，明确抑制剂与3C蛋白酶活性位点的作用模式，建立3C蛋白酶抑制剂药效团模型；开展药效学、药物动力学和初步的安全性评价，发展具有自主知识产权的抗EV71病毒的先导化合物。

本项目主要针对肠病毒71的3C蛋白酶，以六元内酰胺为结构母核，深入开展3C蛋白酶抑制剂的研究，发展高活性小分抑制剂，取得阶段性的研究成果。（1）基于前期发现的氰醇类抑制剂的结构，开展结构改造和优化，发展了一类亚胺基噁唑烷-2-酮类3C蛋白酶抑制剂，保持了抑制剂的高活性，显著提高了抑制剂的稳定性，计算机模拟对接的结果显示与氰醇化合物相似的结合模式，可作为氰醇结构单元的非经典的生物电子等排体用于药物设计。（2）在传统迈克尔受体的双键上引入第二个活化吸电子基团，通过酶动力学和质谱对构效关系和抑制机制进行系统的讨论，发展了一类具有高活性、高选择性且可逆的共价类3C蛋白酶抑制剂。本项目在执行期间，针对冠状病毒的3CL蛋白酶，在获得冠状病毒主蛋白酶的晶体结构基础上，构建酶活分析体系和确定蛋白酶的催化活性中心，对3CL蛋白酶的催化机制进行了系统性研究，取得重要的研究进展，为发展抗冠状病毒药物打下了良好的基础。. 本项目在执行期间，已经发表10篇研究论文，申报专利5项，授权专利1项，独立培养6名硕士研究生，联合培养4名博士研究生和3名硕士研究生。