Identification of allosteric inhibitors blocking the hepatitis C virus polymerase NS5B in the RNA synthesis initiation step
Identification of allosteric inhibitors blocking the hepatitis C virus polymerase NS5B in the RNA synthesis initiation step
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DOI：
10.1016/j.antiviral.2009.06.009
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发表时间：
2009-10-01
影响因子：
7.6
通讯作者：
Canard, Bruno
中科院分区：
文献类型：
--
作者：
Betzi, Stephane;Eydoux, Cecilia;Canard, Bruno
丙型肝炎病毒（HCV）RNA依赖性RNA聚合酶NS5B构成了开发抗HCV药物的首选靶标。尽管许多小分子已被鉴定为NS5B的变构抑制剂，但很少有在临床应用中有活性。我们在涉及纯化的NS5B的酶促测定中筛选了17，000种化合物，以增加治疗武器库。我们希望能阐明RNA合成的精确机制。我们成功地分离了一系列21个NS5B RNA合成的原始抑制剂。当用“B”位点突变体M423 T NS5 B测定时，通过其抑制效力的降低判断，这些非核苷抑制剂（NNI）中的4种可定位于称为“B”的已知结合位点。顺便说一句，我们的计算机模型指出Y477是抑制剂结合的关键残基。在体外，Y477F突变体失去了对新发现的抑制剂的敏感性，但在延伸阶段不能延伸引物。我们的研究结果表明，“B”位点的元素参与了不同RNA合成步骤之间的转换所需的构象变化，并且靶向该位点的化合物可以将酶锁定在其起始阶段。(C)2009 Elsevier B.V.保留所有权利。
Hepatitis C virus (HCV) RNA-dependent RNA polymerase NS5B constitutes a target of choice for the development of anti-HCV drugs. Although many small molecules have been identified as allosteric inhibitors of NS5B, very few are active in clinical applications. We have screened 17,000 compounds in an enzymatic assay involving the purified NS5B in order to increase the therapeutic arsenal. We hoped to shed some light on the precise mechanism of RNA synthesis. We succeeded in isolating a series of 21 original inhibitors of the RNA synthesis by NS5B. Four of these non-nucleoside inhibitors (NNIs) could be mapped to the known binding site called 'B' as judged by the decrease in their inhibition potency when assayed with a 'B' site mutant, M423T NS5B. Incidentally, our in silico model pointed to Y477 as a key residue for inhibitor binding. In vitro, Y477F mutant loses its sensitivity to the newly discovered inhibitors but is unable to extend primers during the elongation phase. Our results demonstrate that elements of the 'B' site are involved in the conformational changes required in the switch between the different RNA synthesis steps and that compounds targeting this site could lock the enzyme in its initiation phase. (C) 2009 Elsevier B.V. All rights reserved.