FASN inhibitor TVB-3166 prevents S-acylation of the spike protein of human coronaviruses

  1. Katrina Mekhail
  2. Minhyoung Lee
  3. Michael Sugiyama
  4. Audrey Astori
  5. Jonathan St-Germain
  6. Elyse Latreille
  7. Negar Khosraviani
  8. Kuiru Wei
  9. Zhijie Li
  10. James Rini
  11. Warren L. Lee
  12. Costin Antonescu
  13. Brian Raught
  14. Gregory D. Fairn

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Abstract

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  1. Version published to 10.1016/j.jlr.2022.100256
  2. Version published to 10.1101/2020.12.20.423603v2 on bioRxiv
  3. ScreenIT

    SciScore for 10.1101/2020.12.20.423603: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    RandomizationFollowing recovery, mice were randomly separated into weight matched groups and immediately received solvent control or TVB-3166 (30 mg/kg) by oral gavage.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableMHV-S infection of mice: Four-week-old female A/J mice were purchased from Jackson Laboratories.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Antibodies and antibody-conjugated beads: C9 antibody (Clone 1D4, Santa Cruz, sc-57432), anti-C9 agarose bead (Cube Biotech), HA antibody (Abcam, ab9110), myc antibody (Bio X Cell, Lebanon, NH), SARS-CoV-2 Spike antibody, (Abcam, ab272504), ZDHHC5 antibody (Sigma, HPA014670), fluorescent secondary antibodies (Jackson Laboratories, Invitrogen, LI-COR). siRNA Transfection: MRC-5 cells seeded on 6-well tissue culture plates were transfected 2X, 24 hours apart, followed by a 24 to 48-hour recovery before infection with 229E.
    antibody-conjugated beads: C9
    suggested: None
    anti-C9
    suggested: None
    ZDHHC5
    suggested: (Sigma-Aldrich Cat# HPA014670, RRID:AB_2257442)
    Pre-cleared samples were incubated with anti-C9 or anti-HA primary antibody for overnight at 4°C.
    anti-HA
    suggested: None
    To form primary antibody-bead complex, Protein G beads were added and incubated for 30min.
    30min
    suggested: None
    Cells were then incubated in a 1:50 dilution of mouse anti-229E Spike protein antibody by inverted drop for 1 hour at room temperature.
    anti-229E Spike protein
    suggested: None
    After washing, cells were incubated in 1:1000 anti-mouse Alexa Fluor 488 secondary antibody (Jackson ImmunoResearch) and DAPI (1 mg/mL) for 1h at room temperature, followed by mounting on slides with DAKO mounting media.
    anti-mouse
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Metabolic labelling/ on-bead click chemistry assay: HEK293T cells seeded in T25 Flask (25cm2) were transfected with pcDNA3.1-SARS CoV-2-Spike-C9 and incubated overnight in DMEM supplemented with 10% (v/v) charcoal-stripped FBS with or without 100 μM 15-HPYA (Click Chemistry Tools, Scottsdale, Arizona).
    HEK293T
    suggested: None
    Propagation of the Human 229E Coronavirus: Human coronavirus 229E (ATCC® VR-TM 740) was purchased from American Type Culture Collection (ATCC), Manassas, VA. Stocks of the 229E virus were produced by propagation in MRC-5 cells.
    MRC-5
    suggested: ICLC Cat# HL95001, RRID:CVCL_0440)
    Experimental Models: Organisms/Strains
    SentencesResources
    For each well 2.5 mg of either: 229E Spike-C9 + HA-ZDHHC5, 229E Spike-C9+ EGFP-C1, SARS-CoV-2 Spike-C9 and HA-ZDHHC5 or SARS-CoV2 Spike-C9 + EGFP-C1 DNA mixtures were diluted in Opti-mem and mixed with Lipofectamine 3000 according to the manufacturer’s instructions.
    Spike-C9 + HA-ZDHHC5, 229E Spike-C9+ EGFP-C1
    suggested: None
    MHV-S infection of mice: Four-week-old female A/J mice were purchased from Jackson Laboratories.
    A/J
    suggested: RRID:IMSR_JAX:000646)
    Software and Algorithms
    SentencesResources
    Images were acquired using Metamorph software on a Hamamatsu ORCA-Flash 4.0 V2 sCMOS camera.
    Metamorph
    suggested: None
    Total fluorescent signal was quantified with ImageJ.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)

    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: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT03Trial number did not resolve on clinicaltrials.gov. Is the number correct?NA

    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.

  4. Version published to 10.1101/2020.12.20.423603v1 on bioRxiv