BRET-Based Self-Cleaving Biosensors for SARS-CoV-2 3CLpro Inhibitor Discovery

  1. Ningke Hou
  2. Chen Peng
  3. Lijing Zhang
  4. Yuyao Zhu
  5. Qi Hu

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Abstract

The virus proteases 3CLpro are validated drug targets for developing antivirals to treat coronavirus diseases, such as COVID-19. However, the development of 3CLpro inhibitors relies heavily on BSL-3 laboratories.

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  1. Version published to 10.1128/spectrum.02559-21
  2. ScreenIT

    SciScore for 10.1101/2021.07.28.454072: (What is this?)

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

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The PVDF membrane was blocked with 1% (for anti-FLAG antibody) or 3% (for anti-HA antibody) non-fat milk in TBST buffer (Tris-buffered saline containing 0.05% Tween-20) overnight at 4 °C and incubated with anti-FLAG antibody (Sigma, F1804, 1:1000 dilution) or anti-HA antibody (Abcam, ab9110, 1:1000 dilution) for 2 hours at room temperature.
    anti-FLAG
    suggested: (Sigma-Aldrich Cat# F1804, RRID:AB_262044)
    F1804
    suggested: (Sigma-Aldrich Cat# F1804, RRID:AB_262044)
    anti-HA
    suggested: (Abcam Cat# ab9110, RRID:AB_307019)
    ab9110
    suggested: (Abcam Cat# ab9110, RRID:AB_307019)
    Experimental Models: Cell Lines
    SentencesResources
    For western blot analysis, HEK 293T cells were lysed using RIPA buffer (Beyotime, P0013B).
    HEK 293T
    suggested: None
    Recombinant DNA
    SentencesResources
    The DNA fragment encoding the biosensor pBRET-1 was synthesized at GENEWIZ (Suzhou, China) and inserted into pcDNA3.1 vector at the site after the FLAG-tag.
    pcDNA3.1
    suggested: RRID:Addgene_79663)
    The plasmid of pBRETmut-1 was constructed by introducing the 3CLpro C145A mutation into pBRET-1 through site-directed mutagenesis using primers C145A-F and C145A-R (Table S1).
    pBRETmut-1
    suggested: None
    pBRET-1
    suggested: None
    Software and Algorithms
    SentencesResources
    The DNA fragment encoding the biosensor pBRET-1 was synthesized at GENEWIZ (Suzhou, China) and inserted into pcDNA3.1 vector at the site after the FLAG-tag.
    GENEWIZ
    suggested: (GENEWIZ, RRID:SCR_003177)

    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.

    Results from scite Reference Check: We found no unreliable references.

    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.

  3. Version published to 10.1101/2021.07.28.454072v1 on bioRxiv