阿兹夫定体外抗登革病毒活性研究  

The study of azvudine activity against dengue viruses in vitro

作　　者：张春涛 罗荣华[2] 陈欢[2] 刘光明 常俊标[3] 杨柳萌[2] 郑永唐[2] ZHANG Chun-tao;LUO Rong-hua;CHEN Huan;LIU Guang-ming;CHANG Jun-biao;YANG Liu-meng;ZHENG Yong-tang(College of Pharmacy and Chemistry, Dali University, Dali 671000, China;Key Laboratory of Bioactive Peptides of Yunnan Province/Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China;Henan Normal University, Xinxiang 453007, China)

机构地区：[1]大理大学药学与化学学院,云南大理671000 [2]中国科学院昆明动物研究所,云南省活性多肽研究与利用重点实验室/中国科学院动物模型与人类疾病重点实验室,云南昆明650223 [3]河南师范大学,河南新乡453007

出　　处：《药学学报》2018年第6期944-949,共6页Acta Pharmaceutica Sinica

基　　金：国家重点研发计划资助项目(2016YFC1201000); 云南省重大科技专项计划课题(2017ZF007)

摘　　要：本文通过检测化合物对登革Ⅱ型重组病毒(DENV-ⅡLuc+)表达海肾荧光素酶活性的影响,进行化合物筛选,发现阿兹夫定(azvudine,FNC)、盐酸阿兹夫定(hydrochloride salt of azvudine,FNC-HCl)和三磷酸酯阿兹夫定(triphosphate azvudine,FNC-TP)能够抑制DENV-ⅡLuc+的复制,并研究其体外抗登革病毒(dengue virus,DENV)活性。首先通过噬斑法检测FNC、FNC-HCl和FNC-TP对DENV的抑制活性;并通过蛋白印迹实验检测3种化合物对DENV-Ⅱ囊膜蛋白E(envelope protein E)表达的影响,最后通过实时定量PCR检测化合物对DENV-Ⅱ病毒RNA的抑制作用。结果显示,FNC、FNC-HCl和FNC-TP抑制DENV复制的半数有效浓度(EC50)在0.54~25.42μmol·L^(-1)之间,阳性药物利巴韦林抑制DENV-Ⅱ的EC50为40.78±1.02μmol·L^(-1)。蛋白印迹实验及定量结果显示,3种化合物均能抑制DENV-Ⅱ囊膜蛋白E的表达,并且显著地抑制DENV-Ⅱ病毒RNA的复制。通过MTT法检测发现FNC、FNC-HCl和FNC-TP的细胞毒性很小,半数细胞毒性浓度(CC50)均大于3 000.00μmol·L^(-1)。本研究表明FNC、FNC-HCl和FNC-TP在细胞水平、蛋白水平及RNA水平均能抑制DENV的复制,且抑制作用优于利巴韦林,有望成为抗登革病毒的候选药物。In this study, azvudine （FNC）, hydrochloride salt of azvudine （FNC-HCl） and triphosphate azovudine （FNC-TP） were tested against DENV-Ⅱ recombinant virus （DENV-Ⅱ Luc＋）. The inhibitory activity of FNC, FNC-HCl and FNC-TP on DENVs were detected by plaque assay. The effect on the expression of DENV-Ⅱ envelope protein E was detected by Western blot; the inhibitory of DENV-Ⅱ viral RNA by compounds was detected by real-time quantitative PCR. MTT assay was used to determine the cytotoxicity of the three compounds on Vero cells. The results showed that FNC, FNC-HCl and FNC-TP inhibited the viral replication by inhibition of renilla luciferase activity of DENV-Ⅱ Luc＋. The 50% effective concentration （EC50） of FNC, FNC-HCl and FNC-TP in the inhibition of DENVs replication were from 0.54-25.42 μmol·L-1, while that of ribavirin was 40.78 ±1.02 μmol·L-1 as the positive control. Western blot and real time quantitative PCR results showed that FNC, FNC-HCl and FNC-TP significantly inhibited the expression of DENV-Ⅱ E protein, and the replication of DENV-Ⅱ viral RNA. The 50% cytotoxic concentrations of FNC, FNC-HCl and FNC-TP were all greater than 3 000.00 μmol·L-1. The results suggest that in vitro anti-DENVs activities of FNC, FNC-HCl and FNC-TP are superior to ribavirin, which are expected to become new candidates of anti-DENV drugs.

关 键 词：阿兹夫定 登革病毒 抗病毒活性 体外 

分 类 号：R966[医药卫生—药理学]