Uppal Timsy, Tuffo Kai, Khaiboullina Svetlana, Reganti Sivani, Pandori Mark, Verma Subhash C
Department of Microbiology and Immunology, University of Nevada, Reno, NV, 89557, USA.
Nevada State Public Health Laboratory, Reno, NV, 89557, USA.
Cell Insight. 2022 Jun 29;1(4):100046. doi: 10.1016/j.cellin.2022.100046. eCollection 2022 Aug.
COVID-19 (Coronavirus Disease 2019) caused by SARS-CoV-2 (Severe Acute Respiratory Syndrome CoronaVirus-2) continues to pose an international public health threat and thus far, has resulted in greater than 6.4 million deaths worldwide. Vaccines are critical tools to limit COVID-19 spread, but antiviral drug development is an ongoing global priority due to fast-spreading COVID-19 variants that may elude vaccine efficacies. The RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 is an essential enzyme of viral replication and transcription machinery complex. Therefore, the RdRp is an attractive target for the development of effective anti-COVID-19 therapeutics. In this study, we developed a cell-based assay to determine the enzymatic activity of SARS-CoV-2 RdRp through a luciferase reporter system. The SARS-CoV-2 RdRp reporter assay was validated using known inhibitors of RdRp polymerase, remdesivir along with other anti-virals including ribavirin, penciclovir, rhoifolin, 5'CT, and dasabuvir. Dasabuvir (an FDA-approved drug) exhibited promising RdRp inhibitory activity among these inhibitors. Anti-viral activity of dasabuvir was also tested on the replication of SARS-CoV-2 through infection of Vero E6 cells. Dasabuvir inhibited the replication of SARS-CoV-2, USA-WA1/2020 as well as B.1.617.2 (delta variant) in Vero E6 cells in a dose-dependent manner with EC values 9.47 μM and 10.48 μM, for USA-WA1/2020 and B.1.617.2 variants, respectively. Our results suggest that dasabuvir can be further evaluated as a therapeutic drug for COVID-19. Importantly, this system provides a robust, target-specific, and high-throughput screening compatible (z- and z'-factors of >0.5) platforms that will be a valuable tool for screening SARS-CoV-2 RdRp inhibitors.
由严重急性呼吸综合征冠状病毒2（SARS-CoV-2）引起的2019冠状病毒病（COVID-19）继续构成国际公共卫生威胁，迄今为止，已在全球导致超过640万人死亡。疫苗是限制COVID-19传播的关键工具，但由于COVID-19变体传播迅速，可能逃避疫苗效力，抗病毒药物研发仍是全球当前的重点。SARS-CoV-2的RNA依赖性RNA聚合酶（RdRp）是病毒复制和转录机制复合体的一种必需酶。因此，RdRp是开发有效抗COVID-19疗法的一个有吸引力的靶点。在本研究中，我们开发了一种基于细胞的检测方法，通过荧光素酶报告系统来测定SARS-CoV-2 RdRp的酶活性。使用已知的RdRp聚合酶抑制剂瑞德西韦以及其他抗病毒药物（包括利巴韦林、喷昔洛韦、根皮苷、5'CT和达沙布韦）对SARS-CoV-2 RdRp报告基因检测进行了验证。达沙布韦（一种美国食品药品监督管理局批准的药物）在这些抑制剂中表现出有前景的RdRp抑制活性。还通过感染Vero E6细胞测试了达沙布韦对SARS-CoV-2复制的抗病毒活性。达沙布韦在Vero E6细胞中以剂量依赖性方式抑制SARS-CoV-2、美国-WA1/2020以及B.1.617.2（德尔塔变体）的复制，对于美国-WA1/2020和B.1.617.2变体，其半数有效浓度（EC）值分别为9.47 μM和10.48 μM。我们的结果表明，达沙布韦可作为COVID-19治疗药物作进一步评估。重要的是，该系统提供了一个强大、靶点特异性且与高通量筛选兼容（z和z'因子>0.5）的平台，这将是筛选SARS-CoV-2 RdRp抑制剂的一个有价值的工具。
