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bioRxiv · 10.1101/2022.04.04.486994

Screening of SARS-CoV-2 Antivirals Through a Cell-Based RNA-Dependent RNA Polymerase (RdRp) Reporter Assay

Uppal, T.·Tuffo, K.·Khaiboullina, S.·Reganti, S.·PANDORI, M. C.·Verma, S. C.
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Abstract

COVID-19 (Coronavirus Disease 2019) caused by SARS-CoV-2 (Severe Acute Respiratory Syndrome CoronaVirus-2) continues to pose international public health threat and thus far, has resulted in greater than 5.6 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 vaccines 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 luciferase reporter system. The SARS-CoV-2 RdRp reporter assay was validated using a known inhibitors of RdRp polymerase, remdesivir along with other anti-virals including ribavirin, penciclovir, rhoifolin, 5CT, and dasabuvir. Among these inhibitors, dasabuvir (FDA-approved drug) exhibited promising RdRp inhibitory activity. 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 IC50 values 9.47 M and 10.48 M, for USA-WA1/2020 and B.1.617.2 variants, respectively). Our results suggests that dasabuvir can be further evaluated as a therapeutic drug for COVID-19. In addition, our assays provide robust, target-specific, and high-throughput screening compatible (z- and z-factors of > 0.5) platforms that will be a valuable tool for the screening SARS-CoV-2 RdRp inhibitors. SignificanceSARS-CoV-2 has caused a major public crisis world has seen in recent history. Development of vaccines and emergency use authorization of anti-virals are helping in reducing the burden of SARS-CoV-2 caused hospitalization and deaths. However, there is still need for optimal anti-viral(s) that can efficiently block viral propagation, and targeting viral polymerase (RdRp) is an among the most suitable targets for clamping viral replication. In this study, we developed a cell-based assay to screen potential compounds capable of blocking RdRp activity. The efficacy of our assay was validated by using already approved anti-virals, which reduced RdRp activity and slowed the replication of two SARS-CoV-2 variants (WA1 USA-WA1/2020 and B.1.617.2) in a cell culture model. This confirmed that our system can be used for identifying potential anti-SARS-CoV-2 anti-virals.

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Uppal, T., Tuffo, K., Khaiboullina, S., Reganti, S., PANDORI, M. C., Verma, S. C.. 2022-04-05. Screening of SARS-CoV-2 Antivirals Through a Cell-Based RNA-Dependent RNA Polymerase (RdRp) Reporter Assay. https://doi.org/10.1101/2022.04.04.486994

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