Kite-Shaped Molecules Block SARS-CoV-2 Cell Entry at a Post-Attachment Step

  1. Shiu-Wan Chan
  2. Talha Shafi
  3. Robert C. Ford

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Abstract

Anti-viral small molecules are currently lacking for treating coronavirus infection. The long development timescales for such drugs are a major problem, but could be shortened by repurposing existing drugs. We therefore screened a small library of FDA-approved compounds for potential severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antivirals using a pseudovirus system that allows a sensitive read-out of infectivity. A group of structurally-related compounds, showing moderate inhibitory activity with IC50 values in the 2–5 μM range, were identified. Further studies demonstrated that these “kite-shaped” molecules were surprisingly specific for SARS-CoV-1 and SARS-CoV-2 and that they acted early in the entry steps of the viral infectious cycle, but did not affect virus attachment to the cells. Moreover, the compounds were able to prevent infection in both kidney- and lung-derived human cell lines. The structural homology of the hits allowed the production of a well-defined pharmacophore that was found to be highly accurate in predicting the anti-viral activity of the compounds in the screen. We discuss the prospects of repurposing these existing drugs for treating current and future coronavirus outbreaks.

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  1. Version published to 10.3390/v13112306
  2. ScreenIT

    SciScore for 10.1101/2021.05.29.446272: (What is this?)

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

    Table 1: Rigor

    NIH rigor criteria are not applicable to paper type.

    Table 2: Resources

    Antibodies
    SentencesResources
    Anti-ACE2 antibody (Proteintech) was used at 1:2000 and anti-mouse HRP (Cell Signaling Technology) at 1:1000.
    Anti-ACE2
    suggested: None
    anti-mouse HRP (Cell Signaling Technology)
    suggested: (Cell Signaling Technology Cat# 5127, RRID:AB_10892860)
    Anti-SARS-CoV-2 spike antibody (BEI Resources NR-52947) was used at 1:1000 and anti-rabbit HRP (Cell Signaling Technology) at 1:1000.
    Anti-SARS-CoV-2
    suggested: None
    anti-rabbit HRP (Cell Signaling Technology)
    suggested: (Cell Signaling Technology Cat# 5127, RRID:AB_10892860)
    Experimental Models: Cell Lines
    SentencesResources
    Cells: 293T, 293T-ACE2, A549-ACE2, Caco2, Huh-7 and Vero cells were cultured in Dulbecco’s modified Eagle’s medium with 4mM glutamate (DMEM; Sigma) and supplemented with 10% fetal calf serum (FCS; Sigma), 100 units/ml penicillin and 100μg/ml streptomycin (Sigma) at 37°C, 5% CO2.
    Caco2
    suggested: None
    Vero
    suggested: None
    The culture medium of Caco2 and Huh-7 was supplemented with 1x non-essential amino acid.
    Huh-7
    suggested: CLS Cat# 300156/p7178_HuH7, RRID:CVCL_0336)
    Calu3 cells were cultured in Minimal Essential medium supplemented with 2mM glutamate.
    Calu3
    suggested: BCRJ Cat# 0264, RRID:CVCL_0609)
    Pseudovirus system: 293T cells seeded at 4×106 per 100mm dish were co-transfected with 6μg of a plasmid encoding MLV gag-pol, 8μg of the transfer vector encoding a luciferase reporter and 6μg of a plasmid encoding an empty vector, a viral envelope glycoprotein SARS-CoV-S, SARS-CoV-2-S, MERS-CoV-S or VSV-G using calcium phosphate (125mM CaCl2, 0.7mM Na2HPO4, 70mM sodium chloride, 25mM Hepes pH 7.05) (see Fig. 1).
    293T
    suggested: None
    293T-ACE2 cells seeded at 25,000 cells per well of 96-well plates were pre-treated with 10μM of individual drugs, in duplicate, for 1h.
    293T-ACE2
    suggested: RRID:CVCL_YZ65)
    Recombinant DNA
    SentencesResources
    Pseudovirus system: 293T cells seeded at 4×106 per 100mm dish were co-transfected with 6μg of a plasmid encoding MLV gag-pol, 8μg of the transfer vector encoding a luciferase reporter and 6μg of a plasmid encoding an empty vector, a viral envelope glycoprotein SARS-CoV-S, SARS-CoV-2-S, MERS-CoV-S or VSV-G using calcium phosphate (125mM CaCl2, 0.7mM Na2HPO4, 70mM sodium chloride, 25mM Hepes pH 7.05) (see Fig. 1).
    VSV-G
    suggested: RRID:Addgene_138479)
    Software and Algorithms
    SentencesResources
    The pharmacophoric features were calculated using AutoPH4 script (80) with holo conditions, and manually optimized for maximum performance.
    AutoPH4
    suggested: None
    Docking studies were performed using GOLD software version 2020.3.0 (81). in summary, 2D depicted structures of compounds were extracted from the PubChem database (82) and were compiled in a MOE database.
    GOLD
    suggested: (GOLD, RRID:SCR_000188)
    PubChem
    suggested: (PubChem, RRID:SCR_004284)
    Statistical analysis: Statistical analysis was performed and graphs were plotted using Prism 9.0 (GraphPad).
    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: Please consider improving the rainbow (“jet”) colormap(s) used on page 41. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • No funding statement was detected.
    • 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 10.1101/2021.05.29.446272v1 on bioRxiv