Mechanism of Inhibition of Ebola Virus RNA-Dependent RNA Polymerase by Remdesivir
Mechanism of Inhibition of Ebola Virus RNA-Dependent RNA Polymerase by Remdesivir
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DOI：
10.3390/v11040326
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发表时间：
2019-04-01
期刊：
影响因子：
4.7
通讯作者：
Gotte, Matthias
中科院分区：
文献类型：
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作者：
Tchesnokov, Egor P.;Feng, Joy Y.;Gotte, Matthias
雷米西韦(GS-5734)是一种1-氰基取代的腺苷核苷酸类似物前药，对多种RNA病毒具有广谱的抗病毒活性。这种化合物目前正在临床开发中，用于治疗埃博拉病毒病(EVD)。虽然在细胞培养和非人类灵长类动物中已经证明了抗病毒作用，但埃博拉病毒(EBOV)抑制雷德韦尔的作用机制仍未完全阐明。最近表达和纯化了EBOV RNA依赖的RNA聚合酶(RdRp)复合体，从而能够对雷米德韦的相关三磷酸(TP)形式及其假定的靶点进行生化研究。在这项研究中，我们证实了雷米昔韦-TP能够与三磷酸腺苷(ATP)竞争掺入。酶动力学分析表明，EBOV RdRp与呼吸道合胞病毒(RSV)RdRp结合ATP和Redesivir-TP的效率相近。ATP对redesivir-Tp的选择性与EBOV RdRp类似于4，对RSV RdRp类似于3。相反，纯化的人线粒体RNA聚合酶(h-mtRNAP)能有效地识别雷米昔韦-TP，选择性值接近500倍。对于EBOV RdRp，位置I的掺入抑制剂不影响随后在I+1位置的核苷酸掺入事件。对于RSV RdRp，我们在I+1位置测量到类似于6倍的抑制，尽管RNA合成没有终止。在这两种情况下，链终止都是延迟的，主要出现在I+5位。这种模式是redesivir-TP及其1-氰基修饰所特有的。2位修饰的化合物表现出不同的抑制模式。当2-C-甲基-ATP不被掺入时，Ara-ATP起到非排他性链终止子的作用，阻止I+1位的核苷酸掺入。综上所述，我们的生化数据表明，瑞希韦对EBOV RNA合成的抑制主要归因于延迟的链终止。被掺入的核苷酸类似物与其抑制作用之间的5个残基之间的距离值得进一步研究。
Remdesivir (GS-5734) is a 1-cyano-substituted adenosine nucleotide analogue prodrug that shows broad-spectrum antiviral activity against several RNA viruses. This compound is currently under clinical development for the treatment of Ebola virus disease (EVD). While antiviral effects have been demonstrated in cell culture and in non-human primates, the mechanism of action of Ebola virus (EBOV) inhibition for remdesivir remains to be fully elucidated. The EBOV RNA-dependent RNA polymerase (RdRp) complex was recently expressed and purified, enabling biochemical studies with the relevant triphosphate (TP) form of remdesivir and its presumptive target. In this study, we confirmed that remdesivir-TP is able to compete for incorporation with adenosine triphosphate (ATP). Enzyme kinetics revealed that EBOV RdRp and respiratory syncytial virus (RSV) RdRp incorporate ATP and remdesivir-TP with similar efficiencies. The selectivity of ATP against remdesivir-TP is similar to 4 for EBOV RdRp and similar to 3 for RSV RdRp. In contrast, purified human mitochondrial RNA polymerase (h-mtRNAP) effectively discriminates against remdesivir-TP with a selectivity value of similar to 500-fold. For EBOV RdRp, the incorporated inhibitor at position i does not affect the ensuing nucleotide incorporation event at position i+1. For RSV RdRp, we measured a similar to 6-fold inhibition at position i+1 although RNA synthesis was not terminated. Chain termination was in both cases delayed and was seen predominantly at position i+5. This pattern is specific to remdesivir-TP and its 1-cyano modification. Compounds with modifications at the 2-position show different patterns of inhibition. While 2-C-methyl-ATP is not incorporated, ara-ATP acts as a non-obligate chain terminator and prevents nucleotide incorporation at position i+1. Taken together, our biochemical data indicate that the major contribution to EBOV RNA synthesis inhibition by remdesivir can be ascribed to delayed chain termination. The long distance of five residues between the incorporated nucleotide analogue and its inhibitory effect warrant further investigation.