Biophysical Mode-of-Action and Selectivity Analysis of Allosteric Inhibitors of Hepatitis C Virus (HCV) Polymerase
Biophysical Mode-of-Action and Selectivity Analysis of Allosteric Inhibitors of Hepatitis C Virus (HCV) Polymerase
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DOI：
10.3390/v9060151
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发表时间：
2017-06-01
期刊：
影响因子：
4.7
通讯作者：
Danielson, U. Helena
中科院分区：
文献类型：
--
作者：
Abdurakhmanov, Eldar;Solbak, Sara Oie;Danielson, U. Helena
丙型肝炎病毒（HCV）非结构蛋白5B（NS5B）聚合酶的变构抑制剂可有效治疗基因型1，尽管其作用模式和抑制其他分离株和基因型的潜力尚未充分确定。我们已经使用生物物理技术和一种新的基于生物传感器的实时聚合酶测定法来研究四种抑制剂对基因型1b（BK和Con1）和3a的酶的作用模式和选择性。两个拇指抑制剂（洛美布韦和非利布韦）与所有三个NS5B变体相互作用，尽管对3a酶的亲和力很低。在两种测试的棕榈抑制剂（dasabuvir和nesbuvir）中，只有dasabuvir与1b变体相互作用，而nesbuvir与NS5B 3a相互作用。洛米布韦、非利布韦和达沙布韦稳定了两种1b变体的结构，但对3a酶没有影响。拇指化合物干扰酶和RNA之间的相互作用，并阻止从起始到延伸的过渡。两种别构抑制剂类型具有不同的抑制机制。序列和结构分析揭示了1b和3a变体的结合位点的差异，解释了对基因型3a NS5B的不良影响。在设计变构化合物时需要考虑间接作用模式。目前的方法提供了一种有效的策略，用于识别和优化变构抑制剂靶向HCV基因型3a。
Allosteric inhibitors of hepatitis C virus (HCV) non-structural protein 5B (NS5B) polymerase are effective for treatment of genotype 1, although their mode of action and potential to inhibit other isolates and genotypes are not well established. We have used biophysical techniques and a novel biosensor-based real-time polymerase assay to investigate the mode-of-action and selectivity of four inhibitors against enzyme from genotypes 1b (BK and Con1) and 3a. Two thumb inhibitors (lomibuvir and filibuvir) interacted with all three NS5B variants, although the affinities for the 3a enzyme were low. Of the two tested palm inhibitors (dasabuvir and nesbuvir), only dasabuvir interacted with the 1b variant, and nesbuvir interacted with NS5B 3a. Lomibuvir, filibuvir and dasabuvir stabilized the structure of the two 1b variants, but not the 3a enzyme. The thumb compounds interfered with the interaction between the enzyme and RNA and blocked the transition from initiation to elongation. The two allosteric inhibitor types have different inhibition mechanisms. Sequence and structure analysis revealed differences in the binding sites for 1b and 3a variants, explaining the poor effect against genotype 3a NS5B. The indirect mode-of-action needs to be considered when designing allosteric compounds. The current approach provides an efficient strategy for identifying and optimizing allosteric inhibitors targeting HCV genotype 3a.