Yang Na, Sun Chaomin, Zhang Lixin, Liu Jianguo, Song Fuhang
Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China.
Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, China.
Front Microbiol. 2017 Nov 2;8:2153. doi: 10.3389/fmicb.2017.02153. eCollection 2017.
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 IC 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 . In NS5B assays, commercial compounds No.39 and No.94 previously reported as kinase inhibitors were found to disrupt dsRNA formation, and their IC 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 .
丙型肝炎病毒（HCV）会引发严重的肝脏疾病，包括肝纤维化、肝硬化和肝细胞癌。非结构蛋白3解旋酶（NS3h）和非结构蛋白5B RNA依赖性RNA聚合酶（NS5B）参与HCV RNA基因组的复制，并且已被证明是发现直接作用抗病毒药物的理想靶点。在本研究中，构建了两种高通量筛选系统，即基于荧光偏振（FP）的单链DNA结合测定和基于荧光强度（FI）的双链RNA形成测定，分别用于鉴定NS3h和NS5B抑制剂的候选物。对一个包含约800种小分子和粗提物的文库进行了筛选，这些小分子和粗提物源自海洋微生物或购自中国国家化合物资源中心，从中选出了三个有活性的化合物进行进一步研究。从海洋真菌中分离出的天然化合物No.3A5抑制NS3h活性，其IC值为2.8 μM。我们进一步证明，化合物No.3A5在电泳迁移率变动分析中抑制NS3h结合单链DNA的能力以及水解ATP的能力。在我们的稀释试验中，化合物No.3A5对NS3h的抑制活性是可逆的,这表明没有形成稳定的NS3h-No.3A5复合物。此外，化合物No.3A5对NS3h或 的单链结合蛋白没有结合选择性。在NS5B测定中，先前报道为激酶抑制剂的市售化合物No.39和No.94被发现可破坏双链RNA的形成，其IC值分别为62.9和18.8 μM。这些结果凸显了确定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通过HCV抗非结构蛋白活性发现的。
