Department of Bioinformatics and Biotechnology, Government College University, 38000 Faisalabad, Pakistan.
Center of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan.
Infect Genet Evol. 2020 Oct;84:104371. doi: 10.1016/j.meegid.2020.104371. Epub 2020 May 31.
HCV is a viral infection posing a severe global threat when left untreated progress to end-stage liver disease, including cirrhosis and HCC. The NS5B polymerase of HCV is the most potent target that harbors four allosteric binding sites that could interfere with the HCV infection. We present the discovery of a novel synthetic compound that harbors the potential of NS5B polymerase inhibition. All eight compounds belonging to the benzothiazine family of heterocycles displayed no cellular cytotoxicity in HepG2 cells at nontoxic dose concentration (200 μM). Subsequently, among eight compounds of the series, merely compound 5b exhibited significant inhibition of the expression of the HCV NS5B gene as compared to DMSO control in semi-quantitative PCR. Based on our western blot result, 5b at the range of 50, 100 and 200 μM induced 20, 40, and 70% inhibition of NS5B protein respectively. To estimate the binding potential, 5b was docked at respective allosteric sites followed by molecular dynamics (MD) simulations for a period of 20 ns. In addition, binding free energy calculation by MM-GB/PBSA method revealed a conserved interaction profile of residues lining the allosteric sites in agreement with the reported NS5B co-crystallized inhibitors. The presented results provide important information about a novel compound 5b which may facilitate the the discovery of novel inhibitors that tends to target multiple sites on NS5B polymerase.
丙型肝炎病毒（HCV）是一种病毒感染，如果不进行治疗，可能会导致严重的全球威胁，包括肝硬化和 HCC。HCV 的 NS5B 聚合酶是最强的靶点，它具有四个变构结合位点，可能会干扰 HCV 的感染。我们发现了一种新型的合成化合物，具有抑制 NS5B 聚合酶的潜力。属于苯并噻嗪杂环家族的 8 种化合物在 HepG2 细胞中，在非毒性剂量浓度（200μM）下，没有表现出细胞毒性。随后，在所研究的 8 种化合物中，只有化合物 5b 在半定量 PCR 中与 DMSO 对照相比，对 HCV NS5B 基因的表达表现出显著的抑制作用。根据我们的 Western blot 结果，5b 在 50、100 和 200μM 的范围内分别诱导 NS5B 蛋白的 20%、40%和 70%的抑制。为了评估结合潜力，5b 被对接在各自的变构位点上，然后进行 20ns 的分子动力学（MD）模拟。此外，通过 MM-GB/PBSA 方法进行结合自由能计算，揭示了残基在变构位点上的保守相互作用模式，与已报道的 NS5B 共结晶抑制剂一致。所呈现的结果提供了关于新型化合物 5b 的重要信息，这可能有助于发现倾向于靶向 NS5B 聚合酶多个位点的新型抑制剂。
