Development of a simple in vitro assay to identify and evaluate nucleotide analogs against SARS-CoV-2 RNA-dependent RNA polymerase

  1. Gaofei Lu
  2. Xi Zhang
  3. Weinan Zheng
  4. Jialei Sun
  5. Lan Hua
  6. Lan Xu
  7. Xin-jie Chu
  8. Sheng Ding
  9. Wen Xiong

Reviewed by

Read the full article See related articles

Discuss this preprint

Start a discussion What are Sciety discussions?

Listed in

Log in to save this article

Abstract

Nucleotide analogs targeting viral RNA polymerase have been approved to be an effective strategy for antiviral treatment and are attracting antiviral drugs to combat the current SARS-CoV-2 pandemic. In this report, we develop a robust in vitro nonradioactive primer extension assay to evaluate the incorporation efficiency of nucleotide analog by SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) quantitively. Our results show that many nucleotide analogs can be incorporated into RNA by SARS-CoV-2 RdRp, and that the incorporation of some of them leads to chain termination. The discrimination values of nucleotide analog over those of natural nucleotide were measured to evaluate the incorporation efficiency of nucleotide analog by RdRp. We found that the incorporation efficiency of Remdesivir-TP is higher than ATP, and we did not observe chain termination or delayed chain termination caused by single Remdesivir-TP incorporation, while multiple incorporations of Remdesivir-TP caused chain termination in our assay condition. The incorporation efficiency of Ribavirin-TP and Favipiravir-TP is very low either as ATP or GTP analogs, which suggested that mutagenesis may not be the mechanism of action of those two drugs against SARS-CoV-2. Incorporation of Sofosbuvir-TP is also very low suggesting that sofosbuvir may not be very effective in treating SARS-CoV-2 infection. As a comparison, 2’-C-Methyl-GTP can be incorporated into RNA efficiently, and the derivative of 2’-C-Methyl-GTP may have therapeutic application in treating SARS-CoV-2 infection. This report provides a simple screening method that should be useful in evaluating nucleotide-based drugs targeting SARS-CoV-2 RdRp, and for studying the mechanism of action of selected nucleotide analog.

Article activity feed

  1. ScreenIT

    SciScore for 10.1101/2020.07.16.205799: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    Data were analyzed using GraphPad Prism 7.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    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.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2020.07.16.205799 on bioRxiv