Characterization of aurintricarboxylic acid as a potent hepatitis C virus replicase inhibitor.
Characterization of aurintricarboxylic acid as a potent hepatitis C virus replicase inhibitor.
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DOI：
10.3851/imp1286
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发表时间：
2009-09-25
影响因子：
--
通讯作者：
Kaushik-Basu, Neerja
中科院分区：
文献类型：
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作者：
Chen, Ye;Bopda-Waffo, Alain;Kaushik-Basu, Neerja
背景技术背景：丙型肝炎病毒（HCV）NS 5 B是病毒复制机制的重要组成部分，也是抗病毒干预的重要靶点。金精三羧酸（ATA）是一种广谱的抗病毒药物，本研究以重组NS 5 B、HCV复制酶和携带1b基因型亚基因组复制子的Huh-7细胞为研究对象，对ATA的体外抗NS 5 B活性和在HCV复制子细胞中的抗NS 5 B活性进行了研究。体外ATA活性分析显示，重组NS 5 B和HCV复制酶在亚微摩尔范围内具有等效抑制作用（50%抑制浓度[IC（50）]约为150 nM）。生物化学和机理研究揭示了ATA抑制的双峰机制，具有焦磷酸盐模拟物和非核苷抑制剂的特征。分子建模和竞争置换研究与这些参数一致，表明ATA可能与NS 5 B的苯并噻二嗪变构口袋3或其催化中心结合。动力学研究表明，ATA抑制RNA和UTP底物的混合模式。在单循环测定条件下，ATA抑制HCV NS 5 B起始和从预结合RNA延伸，但与连续聚合条件相比效力降低>或= 5倍。ATA对天然复制酶复合物的IC（50）值为145 nM。在HCV复制子细胞中，ATA治疗消融HCV RNA复制（50%有效浓度=75 nM）与伴随的NS 5 B表达的减少和没有明显的细胞毒性effects.CONCLUSIONS：本研究确定ATA作为一种有效的抗NS 5 B抑制剂，并表明其独特的作用模式可能被利用的结构改进和开发新的抗NS 5 B剂。
BACKGROUND: Hepatitis C virus (HCV) NS5B is an essential component of the viral replication machinery and an important target for antiviral intervention. Aurintricarboxylic acid (ATA), a broad-spectrum antiviral agent, was evaluated and characterized for its anti-NS5B activity in vitro and in HCV replicon cells.METHODS: Recombinant NS5B, HCV replicase and Huh-7 cells harbouring the subgenomic HCV replicon of genotype 1b were employed for biochemical and mechanistic investigations.RESULTS: Analysis of ATA activity in vitro yielded equipotent inhibition of recombinant NS5B and HCV replicase in the submicromolar range (50% inhibition concentration [IC(50)] approximately 150 nM). Biochemical and mechanistic studies revealed a bimodal mechanism of ATA inhibition with characteristics of pyrophosphate mimics and non-nucleoside inhibitors. Molecular modelling and competition displacement studies were consistent with these parameters, suggesting that ATA might bind to the benzothiadiazine allosteric pocket 3 of NS5B or at its catalytic centre. Kinetic studies revealed a mixed mode of ATA inhibition with respect to both RNA and UTP substrates. Under single-cycle assay conditions, ATA inhibited HCV NS5B initiation and elongation from pre-bound RNA, but with > or =fivefold decreased potency compared with continuous polymerization conditions. The IC(50) value of ATA for the native replicase complex was 145 nM. In HCV replicon cells, ATA treatment ablated HCV RNA replication (50% effective concentration =75 nM) with concomitant decrease in NS5B expression and no apparent cytotoxic effects.CONCLUSIONS: This study identified ATA as a potent anti-NS5B inhibitor and suggests that its unique mode of action might be exploited for structural refinement and development of novel anti-NS5B agents.