鄂木斯克出血热病毒NS2B-NS3蛋白酶药物筛选体系的建立及抑制剂筛选  

Establishment of A Drug-Screening System for the Omsk Hemorrhagic Fever Virus NS2B-NS3 Protease and Inhibitor Selection

作　　者：陈雅丽 封守琴 杨海涛 王泽方 肖云杰 CHEN Yali;FENG Shouqin;YANG Haitao;WANG Zefang;XIAO Yunjie(Tianjin University,Tianjin 300072,China;ShanghaiTech University,Shanghai 201210,China)

机构地区：[1]天津大学生命科学学院,天津300072 [2]上海科技大学生命科学与技术学院,上海201210

出　　处：《病毒学报》2024年第5期986-995,共10页Chinese Journal of Virology

基　　金：国家自然科学基金面上项目(项目号:8217033959),题目:功能未知小囊泡在膀胱血-尿屏障形成中的作用和机制研究;国家自然科学基金青年项目(项目号:8220082920),题目:Wolbachia诱导胞质不相容性表型多样性分子机制研究。

摘　　要：鄂木斯克出血热病毒(Omsk hemorrhagic fever virus,OHFV)是一种蜱传黄病毒,主要分布在俄罗斯西伯利亚地区,通过蜱虫叮咬、接触感染物等途径感染人类。感染后症状包括头痛、咳嗽、发烧并伴有出血。目前尚无针对该病毒的特效药物。鄂木斯克出血热病毒基因组经翻译后形成多聚蛋白,其中NS3蛋白酶可将多聚蛋白切割从而发挥其各自在病毒复制周期中的作用。NS2B的亲水区可以作为NS3蛋白酶的辅因子维持其水解活性,在该病毒的生命周期中发挥着不可或缺的作用。该文旨在以NS2B-NS3蛋白酶为靶点开发有效抑制鄂木斯克出血热的小分子抑制剂。选用大肠杆菌表达系统表达鄂木斯克出血热病毒NS2B-NS3蛋白酶,以获得纯度高且性质均一的蛋白。同时建立药物筛选体系,开展抑制剂筛选。最终建立的药物筛选体系为20 mmol/L Tris,20%甘油,pH 8.5,蛋白终浓度为2μmol/L,底物终浓度为100μmol/L。利用该体系进行抑制剂筛选,筛选出对OHFV NS2B-NS3蛋白酶抑制率高达99%的化合物吡啶硫酮锌,并对该抑制剂进行了IC50的测定和抑制剂类型的判定,其为可逆抑制剂。吡啶硫酮锌有望成为抗鄂木斯克出血热病毒的先导化合物,为针对该病毒今后的药物开发提供研究基础。Omsk hemorrhagic fever virus(OHFV)is a tick-borne flavivirus found primarily in Siberian(Russia).It infects humans through tick bites or contact with infectious materials.Symptoms of infection include headache,cough,fever,and bleeding.Specific drugs targeting this virus are not available.The translated genome of OHFV forms polyproteins,among which the NS3 protease cleaves polyproteins to fulfill their respective roles in the viral replication cycle.The hydrophilic region of NS2B acts as a cofactor to maintain the hydrolytic activity of NS3 protease,having an indispensable role in the viral lifecycle.We aimed to develop small-molecule inhibitors of Omsk hemorrhagic fever by targeting the NS2B-NS3 protease.The NS2B-NS3 of OHFV protease was expressed using the Escherichia coli expression system to obtain highly pure and homogeneous protein.Simultaneously,a drug screening system was established to conduct inhibitor screening.The final drug-screening system consisted of Tris(20 mmol/L)and 20%glycerol at pH 8.5,with a final protein concentration of 2μmol/L and a substrate concentration of 100μmol/L.Utilizing this system for inhibitor screening,the compound zinc pyrithione,was identified with percent inhibition up to 99%against NS2B-NS3 protease of OHFV.The half-maximal inhibitory concentration of this inhibitor was determined,and its inhibitory type was identified as reversible.Zinc pyrithione shows promise to be a lead compound against Omsk hemorrhagic fever virus,providing a research basis for drug development targeting this virus.

关 键 词：鄂木斯克出血热 黄病毒 NS2B-NS3蛋白酶 蛋白酶抑制剂 

分 类 号：R373.3[医药卫生—病原生物学]