Remdesivir Is Effective in Combating COVID-19 because It Is a Better Substrate than ATP for the Viral RNA-Dependent RNA Polymerase.
Remdesivir Is Effective in Combating COVID-19 because It Is a Better Substrate than ATP for the Viral RNA-Dependent RNA Polymerase.
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DOI：
10.1016/j.isci.2020.101849
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发表时间：
2020-12-18
期刊：
影响因子：
5.8
通讯作者：
Johnson KA
中科院分区：
文献类型：
--
作者：
Dangerfield TL;Huang NZ;Johnson KA
COVID-19由严重急性呼吸道综合征冠状病毒2（SARS-CoV-2）引起，目前正在使用Remdesivir进行治疗，Remdesivir是一种抑制RNA依赖性RNA聚合酶（RdRp）的核苷类似物。然而，Remdesivir的酶促机制和效率尚未确定，迫切需要可靠的新抑制剂筛选。在这里，我们提出了我们的工作，以优化在大肠杆菌中的表达，然后通过纯化和动力学分析的未标记的NSP 12/7/8 RdRp复合物。预稳态动力学分析表明，我们的重组RdRp催化快速（kcat = 240-680 s-1）和进行性（koff = 0.013 s-1）RNA聚合。Remdesivir三磷酸（RTP）掺入的特异性常数（kcat/Km）（1.29 μM− 1 s −1）高于竞争ATP（0.74 μM−1 s−1）。这项工作提供了第一个强大的分析RNA聚合和RTP掺入的SARS-CoV-2 RdRp和设置标准的发展信息酶检测筛选新的抑制剂。NSP 12/7/8与伴侣蛋白在大肠杆菌中的共表达产生可溶性SARS CoV 2 RdRp无标签RdRp复合物催化快速和进行性RNA聚合速率足以在2分钟内复制30 kb基因组Remdesivir以ATP生物科学;生物化学;分子生物学;分子生物学观察到的特异性常数的两倍掺入
COVID-19 is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and is currently being treated using Remdesivir, a nucleoside analog that inhibits the RNA-dependent-RNA polymerase (RdRp). However, the enzymatic mechanism and efficiency of Remdesivir have not been determined, and reliable screens for new inhibitors are urgently needed. Here we present our work to optimize expression in E. coli, followed by purification and kinetic analysis of an untagged NSP12/7/8 RdRp complex. Pre-steady-state kinetic analysis shows that our reconstituted RdRp catalyzes fast (kcat = 240–680 s−1) and processive (koff = 0.013 s−1) RNA polymerization. The specificity constant (kcat/Km) for Remdesivir triphosphate (RTP) incorporation (1.29 μM−1s−1) is higher than that for the competing ATP (0.74 μM−1 s−1). This work provides the first robust analysis of RNA polymerization and RTP incorporation by the SARS-CoV-2 RdRp and sets the standard for development of informative enzyme assays to screen for new inhibitors. Co-expression of NSP12/7/8 with chaperones in E. coli gives soluble SARS CoV2 RdRp Tag-free RdRp complex catalyzes fast and processive RNA polymerization Polymerization rates are sufficient to replicate the 30 kb genome in 2 min Remdesivir is incorporated with a specificity constant twice that observed for ATP Biological Sciences; Biochemistry; Molecular Biology; Molecular Biology Experimental Approach
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影响因子：
14.9
作者：
ASLANIDIS, C;DEJONG, PJ
通讯作者：
DEJONG, PJ
影响因子：
4.8
作者：
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158.5
作者：
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DOI：
10.1016/j.ddtec.2012.09.003
发表时间：
2012
期刊：
Drug discovery today. Technologies
影响因子：
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作者：
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通讯作者：
Schinazi RF
影响因子：
2.9
作者：
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通讯作者：
Blom, Thomas