Identification and Analysis of Novel Inhibitors against NS3 Helicase and NS5B RNA-Dependent RNA Polymerase from Hepatitis C Virus 1b (Con1).
Identification and Analysis of Novel Inhibitors against NS3 Helicase and NS5B RNA-Dependent RNA Polymerase from Hepatitis C Virus 1b (Con1).
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丙型肝炎病毒 1b (Con1) 中 NS3 解旋酶和 NS5B RNA 依赖性 RNA 聚合酶新型抑制剂的鉴定和分析
DOI：
10.3389/fmicb.2017.02153
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发表时间：
2017
影响因子：
5.2
通讯作者：
Song F
中科院分区：
文献类型：
--
作者：
Yang N;Sun C;Zhang L;Liu J;Song F
丙型肝炎病毒（HCV）可导致严重的肝脏疾病，包括肝纤维化、肝硬化和肝细胞癌。非结构蛋白3解旋酶（NS 3 h）和非结构蛋白5 B RNA依赖性RNA聚合酶（NS 5 B）参与HCV RNA基因组的复制，已被证明是发现直接作用抗病毒药物的良好靶点。本研究构建了两种高通量筛选系统，即基于荧光偏振（FP）的ssDNA结合试验和基于荧光强度（FI）的dsRNA形成试验，分别用于筛选候选NS 3 h和NS 5 B抑制剂。筛选了来自海洋微生物或从中国国家化合物资源中心购买的约800个小分子和粗提物的文库，选择了三个命中物用于进一步研究。从海洋真菌中分离得到的天然化合物3A 5对NS 3 h活性有抑制作用，IC_（50）值为2.8 μM。我们进一步证明了化合物No.3A5抑制NS 3 h在电泳迁移率变动分析中结合ssDNA和水解ATP的能力。在我们的稀释试验中，化合物No. 3A 5的NS 3 h抑制活性是可逆的，这表明没有形成稳定的NS 3 h-No. 3A 5复合物。另外，化合物3A 5对大肠杆菌的NS 3 h或单链结合蛋白没有结合选择性。在NS 5 B试验中，发现先前报道为激酶抑制剂的商业化合物No.39和No.94破坏dsRNA形成，其IC 50值分别为62.9和18.8 μM。这些结果强调了如何识别现有药物的新用途是发现新型HCV抑制剂的有效方法。据我们所知，本研究中报道的所有抑制剂最初都是在体外发现的抗HCV非结构蛋白活性。
Hepatitis C virus (HCV) leads to severe liver diseases, including liver fibrosis, cirrhosis and hepatocellular carcinoma. Non-structural protein 3 helicase (NS3h) and non-structural protein 5B RNA-dependent RNA polymerase (NS5B) are involved in the replication of HCV RNA genome, and have been proved to be excellent targets for discovery of direct-acting antivirals. In this study, two high-throughput screening systems, fluorescence polarization (FP)-based ssDNA binding assay and fluorescence intensity (FI)-based dsRNA formation assay, were constructed to identify candidate NS3h and NS5B inhibitors, respectively. A library of approximately 800 small molecules and crude extracts, derived from marine microorganisms or purchased from the National Compound Resource Center, China, were screened, with three hits selected for further study. Natural compound No.3A5, isolated from marine fungi, inhibited NS3h activity with an IC50 value of 2.8 μM. We further demonstrated that compound No.3A5 inhibited the abilities of NS3h to bind ssDNA in electrophoretic mobility shift assay and to hydrolyze ATP. The NS3h-inhibitory activity of compound No.3A5 was reversible in our dilution assay, which indicated there was no stable NS3h-No.3A5 complex formed. Additionally, compound No.3A5 exhibited no binding selectivity on NS3h or single strand binding protein of Escherichia coli. In NS5B assays, commercial compounds No.39 and No.94 previously reported as kinase inhibitors were found to disrupt dsRNA formation, and their IC50 values were 62.9 and 18.8 μM, respectively. These results highlight how identifying new uses for existing drugs is an effective method for discovering novel HCV inhibitors. To our knowledge, all inhibitors reported in this study were originally discovered with HCV anti-non-structural protein activities in vitro.
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DOI：
10.1038/nrd1853
发表时间：
2005-10
期刊：
Nature reviews. Drug discovery
影响因子：
--
作者：
Kwong AD;Rao BG;Jeang KT
通讯作者：
Jeang KT
DOI：
10.1080/07391102.2015.1050696
发表时间：
2015-11-02
影响因子：
4.4
作者：
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通讯作者：
Shafique, Shagufta
影响因子：
7.3
作者：
Li, Kelin;Frankowski, Kevin J.;Belon, Craig A.;Neuenswander, Ben;Ndjomou, Jean;Hanson, Alicia M.;Shanahan, Matthew A.;Schoenen, Frank J.;Blagg, Brian S. J.;Aube, Jeffrey;Frick, David N.
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Frick, David N.
影响因子：
--
作者：
Eltahla, Auda A.;Lackovic, Kurt;White, Peter A.
通讯作者：
White, Peter A.
影响因子：
3.5
作者：
Shi, Fangyuan;Zhang, Yingjie;Xu, Wenfang
通讯作者：
Xu, Wenfang