A cell-based assay to discover inhibitors of SARS-CoV-2 RNA dependent RNA polymerase.
A cell-based assay to discover inhibitors of SARS-CoV-2 RNA dependent RNA polymerase.
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基于细胞的检测发现SARS-CoV-2 RNA依赖性RNA聚合酶的抑制剂
DOI：
10.1016/j.antiviral.2021.105078
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发表时间：
2021-06
影响因子：
7.6
通讯作者：
Cen S
中科院分区：
文献类型：
--
作者：
Zhao J;Guo S;Yi D;Li Q;Ma L;Zhang Y;Wang J;Li X;Guo F;Lin R;Liang C;Liu Z;Cen S
抗病毒治疗是控制和结束这场毁灭性的新冠肺炎大流行的有效途径之一。SARS-CoV-2的病毒RNA依赖RNA聚合酶(RdRp)被认为是抗病毒药物的重要靶点。然而，无细胞的SARS-CoV-2 RdRp生化检测需要将核苷酸前体药物转化为活性三磷酸形式，这在细胞中经常发生，但在体外是一个复杂的多步骤化学过程，因此阻碍了这种无细胞检测在快速发现RdRp抑制剂方面的应用。此外，SARS-CoV-2外切核糖核酸酶提供了对病毒RdRp的校对能力，从而产生了病毒RdRp对核苷酸类似物较高的耐药性阈值，这一点必须在开发这类抗病毒药物时进行检测和评估。在这里，我们报告了一种基于细胞的测试，以评估核苷酸类似物对SARS-CoV-2RdRp的有效性，并评估它们对病毒外切核酸酶介导的校对的耐受性。通过对7种常用的核苷酸类似病毒聚合酶抑制剂雷米昔韦、莫鲁匹拉韦、利巴韦林、法比拉韦、喷昔洛韦、恩替卡韦和替诺福韦的检测，发现莫鲁匹拉韦和雷米昔韦对SARS-CoV-2RdRp均有较强的抑制作用，EC50值分别为0.22gμM和0.67gμM。此外，我们的结果表明，外切核糖核酸酶nsp14增加了SARS-CoV-2 RdRp对核苷酸类似物的抵抗力。我们还确定Remdevir对细胞中的病毒外切核糖核酸酶活性具有最高的抗性。因此，我们建立了一种基于细胞的SARS-CoV-2RdRp实验方法，该方法可以用于发现治疗新冠肺炎患者迫切需要的RdRp抑制剂。
Antiviral therapeutics is one effective avenue to control and end this devastating COVID-19 pandemic. The viral RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 has been recognized as a valuable target of antivirals. However, the cell-free SARS-CoV-2 RdRp biochemical assay requires the conversion of nucleotide prodrugs into the active triphosphate forms, which regularly occurs in cells yet is a complicated multiple-step chemical process in vitro, and thus hinders the utility of this cell-free assay in the rapid discovery of RdRp inhibitors. In addition, SARS-CoV-2 exoribonuclease provides the proof-reading capacity to viral RdRp, thus creates relatively high resistance threshold of viral RdRp to nucleotide analog inhibitors, which must be examined and evaluated in the development of this class of antivirals. Here, we report a cell-based assay to evaluate the efficacy of nucleotide analog compounds against SARS-CoV-2 RdRp and assess their tolerance to viral exoribonuclease-mediated proof-reading. By testing seven commonly used nucleotide analog viral polymerase inhibitors, Remdesivir, Molnupiravir, Ribavirin, Favipiravir, Penciclovir, Entecavir and Tenofovir, we found that both Molnupiravir and Remdesivir showed the strong inhibition of SARS-CoV-2 RdRp, with EC50 value of 0.22 μM and 0.67 μM, respectively. Moreover, our results suggested that exoribonuclease nsp14 increases resistance of SARS-CoV-2 RdRp to nucleotide analog inhibitors. We also determined that Remdesivir presented the highest resistance to viral exoribonuclease activity in cells. Therefore, we have developed a cell-based SARS-CoV-2 RdRp assay which can be deployed to discover SARS-CoV-2 RdRp inhibitors that are urgently needed to treat COVID-19 patients.
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影响因子：
4.4
作者：
Chien M;Anderson TK;Jockusch S;Tao C;Li X;Kumar S;Russo JJ;Kirchdoerfer RN;Ju J
通讯作者：
Ju J
影响因子：
3.9
作者：
Min, Jung Sun;Kim, Geon-Woo;Jin, Young-Hee
通讯作者：
Jin, Young-Hee
影响因子：
158.5
作者：
Grein, J.;Ohmagari, N.;Flanigan, T.
通讯作者：
Flanigan, T.
影响因子：
16.6
作者：
Shannon, Ashleigh;Selisko, Barbara;Canard, Bruno
通讯作者：
Canard, Bruno
DOI：
10.1073/pnas.1323705111
发表时间：
2014-09-16
影响因子：
11.1
作者：
Subissi, Lorenzo;Posthuma, Clara C.;Imbert, Isabelle
通讯作者：
Imbert, Isabelle