Validation and invalidation of SARS-CoV-2 main protease inhibitors using the Flip-GFP and Protease-Glo luciferase assays

  1. Chunlong Ma
  2. Haozhou Tan
  3. Juliana Choza
  4. Yuyin Wang
  5. Jun Wang

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Abstract

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  1. Version published to 10.1016/j.apsb.2021.10.026
  2. ScreenIT

    SciScore for 10.1101/2021.08.28.458041: (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

    Experimental Models: Cell Lines
    SentencesResources
    Protease-Glo luciferase assay was carried out as follows: 293T cells in 10 cm culture dish were transfected with pGlosensor-30F Mpro plasmid in the presence of transfection reagent TransIT-293 (Mirus catalog no. MIR 2700) according to the manufacturer’s protocol.
    293T
    suggested: None
    Calu-3 cells (ATCC, HTB-55) were plated in 384 well plates and grown in Minimal Eagles Medium supplemented with 1% non-essential amino acids, 1% penicillin/streptomycin, and 10% FBS.
    Calu-3
    suggested: ATCC Cat# HTB-55, RRID:CVCL_0609)
    Recombinant DNA
    SentencesResources
    Protein Expression and Purification: The tag-free SARS CoV-2 Mpro protein with native N- and C-termini was expressed in pSUMO construct as described previously3.
    pSUMO
    suggested: RRID:Addgene_170732)
    50 ng of FlipGFP-Mpro plasmid and 50 ng SARS CoV-2 Mpro expression plasmid pcDNA3.1 SARSCoV-2 Mpro were transfected into each well with transfection reagent TransIT-293 (Mirus catalog no. MIR 2700) according to the manufacturer’s protocol.
    FlipGFP-Mpro
    suggested: None
    pcDNA3.1 SARSCoV-2
    suggested: None
    Protease-Glo luciferase assay: pGlosensor-30F DEVD vector was obtained from Promega (Catlog no. CS182101).
    pGlosensor-30F DEVD
    suggested: None
    Protease-Glo luciferase assay was carried out as follows: 293T cells in 10 cm culture dish were transfected with pGlosensor-30F Mpro plasmid in the presence of transfection reagent TransIT-293 (Mirus catalog no. MIR 2700) according to the manufacturer’s protocol.
    pGlosensor-30F Mpro
    suggested: None
    Software and Algorithms
    SentencesResources
    Two days after transfection, images were taken with Cytation 5 imaging reader (Biotek) using GFP and mCherry channels via 10× objective lens and were analyzed with Gen5 3.10 software (
    Gen5
    suggested: (Gen5, RRID:SCR_017317)
    A non-linear regression curve fit analysis (GraphPad Prism 8) of POC Infection and cell viability versus the log10 transformed concentration values to calculate EC50 values for Infection and CC50 values for cell viability.
    GraphPad
    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.

    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 10.1101/2021.08.28.458041v1 on bioRxiv