新型登革病毒RNA聚合酶小分子抑制剂Z1降低登革病毒复制和感染的研究  

Z1,a novel DENV2 NS5 RdRp small molecular inhibitor,inhibits DENV2 replication and infection

作　　者：郭颂欣 何士俊 黄翠红 姚新刚 刘叔文 GUO Song- xin;HE Shi- jun;HUANG Cui- iiong;YAO Xin- gang;LIU Shu-wen(School of Pharmaceutical Sciences ,Guangdong Provincial Key Lab of Drug Screening ,Guangzhou Key Lab of Drug Research for Emerging Virus Prevention and Treatment,Guangzhou 510515, China)

机构地区：[1]南方医科大学药学院广东省新药筛选重点实验室广州市新发病毒防治药物研究重点实验室,广东广州510515

出　　处：《中国药理学通报》2018年第6期790-796,共7页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No 81603118);广东省医学科学技术研究基金(No A2016119);南方医科大学科研助手项目(No C1032242);国家自然科学基金-广东联合基金重点项目(K1060014)

摘　　要：目的 筛选新型的登革病毒RNA聚合酶抑制剂。方法 大肠杆菌原核表达DENV2 RNA聚合酶蛋白NS5RdRp。SPR高通量筛选小分子化合物库,寻找与NS5 RdRp蛋白结合的小分子化合物;CPE、LDH和空斑实验在细胞水平上验证化合物的抗病毒效果;实时荧光定量PCR法检测化合物对病毒RNA合成的影响;免疫荧光法检测病毒复制中间体dsRNA以及病毒蛋白合成;蛋白免疫印迹法检测化合物对病毒蛋白合成量的影响;时间点处理实验判断抗病毒作用阶段。结果 2,5-二氢-1,2-二苯基-4-环己氨基-5-氧代-1H-吡咯-3-甲酸乙酯(Z1)与NS5 RdRp具有结合活性,在细胞水平上具有抗DENV2活性,半数有效浓度(EC50)为4.75μmol·L-1。Z1抑制DENV2 RNA合成,抑制病毒dsRNA的合成,并抑制病毒蛋白的合成,其抑制作用发生在病毒进入后阶段。结论 Z1是新型的登革病毒NS5 RdRp抑制剂,其明显抑制登革病毒RNA的合成,并进一步抑制病毒蛋白的翻译合成,从而抑制子代病毒的复制和释放,其可作为新型的抗登革病毒先导化合物用于进一步的开发。Aim To screen novel NS5 inhibitors against dengue virus（DENV） replication. Methods His-tagged DENV2 NS5 RNA-dependent polymerase（NS5 RdRp） was expressed and purified in BL21 cells. The binding ability of the small molecules to NS5 polymerase was determined by SPR assay. The activity of dengue inhibition by Z1 was determined by CPE,LDH and plaque assay. RNA synthesis was assessed by Real-time PCR. The dsRNA synthesis and viral proteins were detected by immunofluorescence assay. The level of viral proteins was examined by Western blot. The stage of DENV life cycle was evaluated by time of drug-addition assay. Results A small molecular Z1 was discovered,which could bind to NS5 RdRp. Z1 inhibited DENV2 RNA replication,synthesis of dsRNA and protein synthesis in post-entry stage of dengue life-cycle. Cell based assay confirmed that Z1 inhibited DENV-induced cell death with EC50 values of 4.75μmol·L^（-1）. Conclusions The novel NS5 inhibitor Z1,inhibits DENV2 RNA replication,protein synthesis and release of progeny virus,which may be severed as an anti-DENV2 antiviral drug for further development.

关 键 词：NS5 RdRp抑制剂 登革病毒RNA聚合酶 2型登革病毒 抗病毒药物 SPR 病毒复制 

分 类 号：R373.33[医药卫生—病原生物学] R394.2[医药卫生—基础医学]