Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, 4301 West Markham Street, Little Rock, AR 72205, USA.
Department of Pharmaceutical Sciences, University of Arkansas for Medical Sciences, 4301 West Markham Street, Little Rock, AR 72205, USA.
Bioorg Med Chem Lett. 2019 Feb 1;29(3):430-434. doi: 10.1016/j.bmcl.2018.12.026. Epub 2018 Dec 13.
The hepatitis C virus (HCV) represents a substantial threat to human health worldwide. The virus expresses a dual-function protein, NS3 having both protease and RNA helicase activities that are essential for productive viral replication and sustained infections. While viral protease and polymerase inhibitors have shown great successes in treating chronic HCV infections, drugs that specifically target the helicase activity have not advanced. A robust and quantitative 96-well plate-based fluorescent DNA unwinding assay was used to screen a class of indole thio-barbituric acid (ITBA) analogs using the full-length, recombinant HCV NS3, and identified three naphthoyl-containing analogs that efficiently inhibited NS3 helicase activity in a dose-dependent manner, with observed IC values of 21-24 µM. Standard gel electrophoresis helicase assays using radiolabeled duplex DNA and RNA NS3 substrates confirmed the inhibition of NS3 unwinding activity. Subsequent anisotropy measurements demonstrated that the candidate compounds did not disrupt NS3 binding to nucleic acids. Additionally, the rate of ATP hydrolysis and the protease activity were also not affected by the inhibitors. Thus, these results indicate that the three ITBA analogs containing N-naphthoyl moieties are the foundation of a potential series of small molecules capable of inhibiting NS3 activity via a novel interaction with the helicase domain that prevents the productive unwinding of nucleic acid substrates, and may represent the basis for a new class of therapeutic agents with the potential to aid in the treatment and eradication of hepatitis C virus.
丙型肝炎病毒 (HCV) 是全球人类健康的重大威胁。该病毒表达一种具有双重功能的蛋白，NS3 既有蛋白酶活性，也有 RNA 解旋酶活性，这对病毒的复制和持续感染至关重要。虽然病毒蛋白酶和聚合酶抑制剂在治疗慢性 HCV 感染方面取得了巨大成功，但专门针对解旋酶活性的药物尚未取得进展。本研究使用全长重组 HCV NS3 ，采用一种基于 96 孔板的荧光 DNA 解链检测法，对一类吲哚硫代巴比妥酸 (ITBA) 类似物进行了筛选，发现了三个含萘酰基的类似物，它们能够以剂量依赖的方式有效地抑制 NS3 解旋酶活性，观察到的 IC 值为 21-24 μM。使用放射性标记的双链 DNA 和 RNA NS3 底物的标准凝胶电泳解旋酶分析证实了 NS3 解旋活性的抑制。随后的各向异性测量表明，候选化合物不会破坏 NS3 与核酸的结合。此外，ATP 水解的速率和蛋白酶活性也不受抑制剂的影响。因此，这些结果表明，三种含有 N-萘酰基部分的 ITBA 类似物是一类具有潜在应用前景的小分子的基础，它们能够通过与解旋酶结构域的新型相互作用抑制 NS3 活性，从而阻止核酸底物的有效解链，可能代表一类具有治疗潜力的新型治疗剂的基础，有助于治疗和根除丙型肝炎病毒。
