Pharmacological Characterization of the Mechanism of Action of R523062, a Promising Antiviral for Enterovirus D68.
Pharmacological Characterization of the Mechanism of Action of R523062, a Promising Antiviral for Enterovirus D68.
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DOI：
10.1021/acsinfecdis.0c00383
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发表时间：
2020-08-14
影响因子：
5.3
通讯作者：
Wang J
中科院分区：
文献类型：
--
作者：
Ma C;Hu Y;Zhang J;Wang J
肠道病毒D 68（EV-D 68）是一种重新出现的病毒，可导致儿童中度至重度呼吸道疾病。在严重的情况下，EV-D 68感染可导致神经系统并发症，称为急性弛缓性肌病（AFM）。目前还没有针对EV-D 68的抗病毒药物或疫苗。本研究的目的是描绘一个有前途的抗病毒候选药物R523062的作用机制，该候选药物是通过基于表型细胞病变效应（CPE）的高通量筛选确定的。R523062抑制多种当代EV-D 68毒株，EC 50值为个位数微摩尔，对肠道病毒A71毒株的有效性较低。通过连续病毒传代实验鉴定的抗性突变体定位于四种病毒蛋白，包括VP 1-G178 S、2A-V112 I、2C-I227 L/Q322 R和3A-V54 A。通过加药时间试验、蛋白酶酶法和重组病毒空斑试验分别排除了VP 1-G178 S、2A-V112 I和3A-V54 A突变体参与耐药的可能性。相反，编码2C-I227 L/Q322 R双突变体的重组病毒赋予显著的耐药性，这与连续传代实验的结果一致。热位移结合分析显示R523062结合野生型EV-D 68 2C、2C-Q322 R，但不结合2C-I227 L或2C-I227 L/Q322 R，证实2C为R523062的直接药物靶标，且2C-I227 L单独赋予耐药性。2C抑制剂R523062还显示出与病毒2A蛋白酶抑制剂特拉匹韦以及病毒衣壳VP 1抑制剂R856932相加的抗病毒活性。总的来说，这项研究确定了一种有希望的EV-D 68抗病毒候选药物R523062，其作用机制通过靶向病毒2C蛋白得到证实。
Enterovirus D68 (EV-D68) is a re-emerging virus that causes moderate to severe respiratory diseases in children. In severe cases, EV-D68 infection can lead to neurological complications called acute flaccid myelitis (AFM). There is currently no antiviral or vaccine available for EV-D68. The goal of this study is to delineate the mechanism of action of a promising antiviral drug candidate R523062 that was identified through a phenotypic cytopathic effect (CPE)-based high-throughput screening. R523062 inhibits multiple contemporary EV-D68 strains with single-digit micromolar EC50 values and is less effective against the enterovirus A71 strains. Resistant mutants identified through serial viral passage experiments were mapped to four viral proteins including VP1-G178S, 2A-V112I, 2C-I227L/Q322R, and 3A-V54A. The involvements of VP1-G178S, 2A-V112I, and 3A-V54A mutants in drug resistance were ruled out by the drug time-of-addition experiment, protease enzymatic assay, and the plaque assay with recombinant virus, respectively. In contrast, recombinant virus encoding the 2C-I227L/Q322R double mutants confers significant drug resistance, which is consistent with the result from serial passage experiments. Thermal shift binding assay showed R523062 binds to the wild-type EV-D68 2C, 2C-Q322R, but not 2C-I227L or 2C-I227L/Q322R, confirming 2C as the direct drug target of R523062 and 2C-I227L alone confers drug resistance. The 2C inhibitor R523062 also showed additive antiviral activity with the viral 2A protease inhibitor telaprevir as well as the viral capsid VP1 inhibitor R856932. Collectively, this study identified a promising EV-D68 antiviral drug candidate R523062 with a confirmed mechanism of action by targeting the viral 2C protein.
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