A high throughput screening assay for inhibitors of SARS-CoV-2 pseudotyped particle entry

  1. Miao Xu
  2. Manisha Pradhan
  3. Kirill Gorshkov
  4. Jennifer D. Petersen
  5. Min Shen
  6. Hui Guo
  7. Wei Zhu
  8. Carleen Klumpp-Thomas
  9. Sam Michael
  10. Misha Itkin
  11. Zina Itkin
  12. Marco R. Straus
  13. Joshua Zimmerberg
  14. Wei Zheng
  15. Gary R. Whittaker
  16. Catherine Z. Chen

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Abstract

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  1. Version published to 10.1016/j.slasd.2021.12.005
  2. ScreenIT

    SciScore for 10.1101/2021.10.04.463106: (What is this?)

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

    Table 1: Rigor

    EthicsField Sample Permit: SARS-CoV-2 cytopathic effect (CPE) assay: SARS-CoV-2 CPE assay was conducted at a contract research organization Southern Research Institute (Birmingham, AL) 2.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Mouse-anti-firefly luciferase antibody was purchased from Santa Cruz (sc-74548).
    Mouse-anti-firefly luciferase antibody
    suggested: None
    Mouse-anti-firefly luciferase
    suggested: None
    Permeabilization/blocking solution was removed, 1:1000 primary mouse-anti-luciferase antibody (Santa Cruz) was added, and incubated overnight at 4 °C.
    mouse-anti-luciferase
    suggested: None
    Then, grids were transferred across two drops of blocking solution for 10 minutes before incubation of with 10 nm gold-conjugated goat-α-rabbit secondary antibody diluted 1:20 in blocking solution for 30 min.
    secondary
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    7E6 HEK293T cells were seeded in a 10 cm2 dish in 16 ml culture medium (DMEM, 10% FBS) without antibiotics that were incubated for 24 hr at 37 °C with 5% CO2.
    HEK293T
    suggested: None
    The SARS-CoV-2-S PP entry assay and ATP content cytotoxicity assay in HEK293-ACE2 cells, were used to screen the NPC and MIPE libraries in parallel.
    HEK293-ACE2
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Materials: Dulbecco’s Modified Eagle’s Medium (DMEM) (11965092), OPTI-MEM reduced serum medium (11058021), Pen/Strep (15140), TrypLE (12604013), PBS -/-(w/o Ca2+ or Mg2+) (10010049), Lipofectamine 3000 Transfection Reagent (L3000015), HCS Cell Mask Green (H32714), goat anti-mouse AlexaFluor 647 (A28175), and Hoechst 33342 (H3570) were purchased from ThermoFisher.
    PBS -/-
    suggested: None
    Recombinant DNA
    SentencesResources
    The DNA solution was prepared by diluting 8 μg of pTG-Luc, 6 μg of pCMV-MLVgag-pol, and 6 μg of pcDNA-SARS-CoV-2-S(ΔC19) in 1 ml of Opti-MEM medium (ThermoFisher).
    pTG-Luc
    suggested: None
    pCMV-MLVgag-pol
    suggested: None
    pcDNA-SARS-CoV-2-S(ΔC19
    suggested: None
    Software and Algorithms
    SentencesResources
    Average values, standard deviations, and data counts were generated using pivot tables in Microsoft Excel and data was plotted in Graphpad Prism.
    Microsoft Excel
    suggested: (Microsoft Excel, RRID:SCR_016137)
    Graphpad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Half-maximal efficacious concentration (EC50) and half-maximal cytotoxicity concentration (CC50) of compounds were calculated using Prism software (GraphPad Software, San Diego, CA).
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    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 10.1101/2021.10.04.463106v1 on bioRxiv