Probing remdesivir nucleotide analogue insertion to SARS-CoV-2 RNA dependent RNA polymerase in viral replication
Probing remdesivir nucleotide analogue insertion to SARS-CoV-2 RNA dependent RNA polymerase in viral replication
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DOI：
10.1039/d1me00088h
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发表时间：
2021-09-13
影响因子：
3.6
通讯作者：
Yu, Jin
中科院分区：
文献类型：
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作者：
Romero, Moises Ernesto;Long, Chunhong;Yu, Jin
雷姆德西韦（RDV）前药可代谢成三磷酸形式的核苷酸类似物（RDV- tp），结合并插入病毒RNA依赖RNA聚合酶（RdRp）的活性位点，进一步干扰病毒基因组复制。在这项工作中，我们通过计算研究了RDV-TP如何与天然核苷酸底物三磷酸腺苷（ATP）结合并插入SARS-CoV-2 RdRp活性位点。为此，我们首先构建了初始结合复合物（活性位点开放）和底物插入复合物（活性位点关闭）的原子结构模型，基于最近确定的SARS-CoV-2 RdRp或非结构蛋白（nsp） 12的高分辨率低温电镜结构，以及辅助蛋白因子nsp7和nsp8的复合物。通过伞式采样策略对开放状态和封闭状态RdRp复合物之间的核苷酸插入进行全原子分子动力学模拟，我们的研究表明，RDV-TP最初可以以相对稳定的状态与病毒RdRp活性位点结合，因为它主要与模板尿嘧啶核苷酸（nt +1）形成碱基叠加，在自由波动下支持RDV-TP插入的低自由能垒（类似于1.5 kcal mol(-1)）。相比之下，相应的天然底物ATP在与模板nt的沃森-克里克碱基配对中首先结合到RdRp活性位点，并在模板nt的波动得到很好的抑制时以中低的自由能垒（类似于2.6 kcal mol(-1)）插入到活性位点。模拟结果还表明，基序C-S759、基序S682（基序B附近）和基序G-K500与模板主干可以特异性地稳定RDV-TP与模板的初始碱基叠加。尽管motif F-R555/R553与三磷酸的相互作用会阻碍RDV-TP的插入，但这种相互作用似乎会促进ATP的插入。插入的RDV-TP和ATP可以通过与motif B-T687和motif A-D623的特异性糖相互作用进一步区分。
Remdesivir (RDV) prodrug can be metabolized into a triphosphate form nucleotide analogue (RDV-TP) to bind and insert into the active site of viral RNA dependent RNA polymerase (RdRp) to further interfere with viral genome replication. In this work, we computationally studied how RDV-TP binds and inserts to the SARS-CoV-2 RdRp active site, in comparison with natural nucleotide substrate adenosine triphosphate (ATP). To do that, we first constructed atomic structural models of an initial binding complex (active site open) and a substrate insertion complex (active site closed), based on high-resolution cryo-EM structures determined recently for SARS-CoV-2 RdRp or non-structural protein (nsp) 12, in complex with accessory protein factors nsp7 and nsp8. By conducting all-atom molecular dynamics simulation with umbrella sampling strategies on the nucleotide insertion between the open and closed state RdRp complexes, our studies show that RDV-TP can initially bind in a comparatively stabilized state to the viral RdRp active site, as it primarily forms base stacking with the template uracil nucleotide (nt +1), which under freely fluctuations supports a low free energy barrier of the RDV-TP insertion (similar to 1.5 kcal mol(-1)). In comparison, the corresponding natural substrate ATP binds initially to the RdRp active site in Watson-Crick base pairing with the template nt, and inserts into the active site with a medium low free energy barrier (similar to 2.6 kcal mol(-1)), when the fluctuations of the template nt are well quenched. The simulations also show that the initial base stacking of RDV-TP with the template can be specifically stabilized by motif C-S759, S682 (near motif B) with the base, and motif G-K500 with the template backbone. Although the RDV-TP insertion can be hindered by motif F-R555/R553 interaction with the triphosphate, the ATP insertion seems to be facilitated by such interactions. The inserted RDV-TP and ATP can be further distinguished by specific sugar interaction with motif B-T687 and motif A-D623, respectively.