Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp12/7/8 RNA-dependent RNA polymerase
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Abstract
The coronavirus disease 2019 (COVID-19) global pandemic has turned into the largest public health and economic crisis in recent history impacting virtually all sectors of society. There is a need for effective therapeutics to battle the ongoing pandemic. Repurposing existing drugs with known pharmacological safety profiles is a fast and cost-effective approach to identify novel treatments. The COVID-19 etiologic agent is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a single-stranded positive-sense RNA virus. Coronaviruses rely on the enzymatic activity of the replication–transcription complex (RTC) to multiply inside host cells. The RTC core catalytic component is the RNA-dependent RNA polymerase (RdRp) holoenzyme. The RdRp is one of the key druggable targets for CoVs due to its essential role in viral replication, high degree of sequence and structural conservation and the lack of homologues in human cells. Here, we have expressed, purified and biochemically characterised active SARS-CoV-2 RdRp complexes. We developed a novel fluorescence resonance energy transfer-based strand displacement assay for monitoring SARS-CoV-2 RdRp activity suitable for a high-throughput format. As part of a larger research project to identify inhibitors for all the enzymatic activities encoded by SARS-CoV-2, we used this assay to screen a custom chemical library of over 5000 approved and investigational compounds for novel SARS-CoV-2 RdRp inhibitors. We identified three novel compounds (GSK-650394, C646 and BH3I-1) and confirmed suramin and suramin-like compounds as in vitro SARS-CoV-2 RdRp activity inhibitors. We also characterised the antiviral efficacy of these drugs in cell-based assays that we developed to monitor SARS-CoV-2 growth.
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SciScore for 10.1101/2021.04.07.438807: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
NIH rigor criteria are not applicable to paper type.Table 2: Resources
Antibodies Sentences Resources Finally, the cells were infected by adding 20 µl of SARS-CoV-2 with a final MOI of 0.5 PFU/cell. 22 h post infection, cells were fixed, permeabilised, and stained for SARS-CoV-2 N protein using Alexa488-labelled-CR3009 antibody and cellular DNA using DRAQ7 (Abcam). DRAQ7suggested: NoneExperimental Models: Cell Lines Sentences Resources For protein expression, Sf9 cells were infected with baculovirus and collected 48 hours after infection, flash-frozen and stored at −70°C. Sf9suggested: NoneThe conditions for the viral inhibition assay were the following: 15.000 Vero E6 cells (NIBC, UK) resuspended in DMEM … SciScore for 10.1101/2021.04.07.438807: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
NIH rigor criteria are not applicable to paper type.Table 2: Resources
Antibodies Sentences Resources Finally, the cells were infected by adding 20 µl of SARS-CoV-2 with a final MOI of 0.5 PFU/cell. 22 h post infection, cells were fixed, permeabilised, and stained for SARS-CoV-2 N protein using Alexa488-labelled-CR3009 antibody and cellular DNA using DRAQ7 (Abcam). DRAQ7suggested: NoneExperimental Models: Cell Lines Sentences Resources For protein expression, Sf9 cells were infected with baculovirus and collected 48 hours after infection, flash-frozen and stored at −70°C. Sf9suggested: NoneThe conditions for the viral inhibition assay were the following: 15.000 Vero E6 cells (NIBC, UK) resuspended in DMEM containing 10% FBS were seeded into each well of 96-well imaging plates (Greiner 655090) and cultured overnight at 37°C and 5% CO2. Vero E6suggested: NoneSoftware and Algorithms Sentences Resources Data analysis: MATLAB was used to process data. MATLABsuggested: (MATLAB, RRID:SCR_001622)Results from OddPub: Thank you for sharing your data.
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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