A comparative analysis of SARS-CoV-2 antivirals in human airway models characterizes 3CL pro inhibitor PF-00835231 as a potential new treatment for COVID-19

  1. Maren de Vries
  2. Adil S Mohamed
  3. Rachel A Prescott
  4. Ana M Valero-Jimenez
  5. Ludovic Desvignes
  6. Rebecca O’Connor
  7. Claire Steppan
  8. Joseph C Devlin
  9. Ellie Ivanova
  10. Alberto Herrera
  11. Austin Schinlever
  12. Paige Loose
  13. Kelly Ruggles
  14. Sergei B Koralov
  15. Annaliesa S. Anderson
  16. Joseph Binder
  17. Meike Dittmann

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Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of Coronavirus Disease 2019 (COVID-19). There is a dire need for novel effective antivirals to treat COVID-19, as the only approved direct-acting antiviral to date is remdesivir, targeting the viral polymerase complex. A potential alternate target in the viral life cycle is the main SARS-CoV-2 protease 3CL pro (M pro ). The drug candidate PF-00835231 is the active compound of the first anti-3CL pro regimen in clinical trials. Here, we perform a comparative analysis of PF-00835231, the pre-clinical 3CL pro inhibitor GC-376, and the polymerase inhibitor remdesivir, in alveolar basal epithelial cells modified to express ACE2 (A549 +ACE2 cells). We find PF-00835231 with at least similar or higher potency than remdesivir or GC-376. A time-of-drug-addition approach delineates the timing of early SARS-CoV-2 life cycle steps in A549 +ACE2 cells and validates PF-00835231’s early time of action. In a model of the human polarized airway epithelium, both PF-00835231 and remdesivir potently inhibit SARS-CoV-2 at low micromolar concentrations. Finally, we show that the efflux transporter P-glycoprotein, which was previously suggested to diminish PF-00835231’s efficacy based on experiments in monkey kidney Vero E6 cells, does not negatively impact PF-00835231 efficacy in either A549 +ACE2 cells or human polarized airway epithelial cultures. Thus, our study provides in vitro evidence for the potential of PF-00835231 as an effective SARS-CoV-2 antiviral and addresses concerns that emerged based on prior studies in non-human in vitro models.

Importance

The arsenal of SARS-CoV-2 specific antiviral drugs is extremely limited. Only one direct-acting antiviral drug is currently approved, the viral polymerase inhibitor remdesivir, and it has limited efficacy. Thus, there is a substantial need to develop additional antiviral compounds with minimal side effects and alternate viral targets. One such alternate target is its main protease, 3CL pro (M pro ), an essential component of the SARS-CoV-2 life cycle processing the viral polyprotein into the components of the viral polymerase complex. In this study, we characterize a novel antiviral drug, PF-00835231, which is the active component of the first-in-class 3CL pro -targeting regimen in clinical trials. Using 3D in vitro models of the human airway epithelium, we demonstrate the antiviral potential of PF-00835231 for inhibition of SARS-CoV-2.

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  1. Version published to 10.1101/2020.08.28.272880v2 on bioRxiv
  2. ScreenIT

    SciScore for 10.1101/2020.08.28.272880: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    BlindingCompound characterization at NYU was done in a blinded manner.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Surface ACE2 was visualized by staining A549 and A549+ACE2 cells at 4°C with anti-ACE2 (1:500, R&D Biosystems AF933) and AlexaFluor 647 secondary antibody and DAPI.
    anti-ACE2
    suggested: None
    Fixed cells were permeabilized with Triton-X and stained with mouse monoclonal SARS-CoV anti-N antibody 1C7, which cross-reacts with SARS-CoV-2 N (kind gift of Thomas Moran),
    anti-N
    suggested: None
    In addition, SARS-CoV-2 (2x) was incubated with SARS-CoV-2 (2019-nCov) rabbit polyclonal spike neutralizing antibody (nAB, 2x)
    SARS-CoV-2
    suggested: None
    Cultures were stained with i) rabbit polyclonal anti-SARS Nucleocapsid Protein antibody, which cross reacts with SARS-CoV-2 N (1:1000
    anti-SARS
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Confluent 6-well A549 and A549+ACE2 cells were collected in RLT lysis buffer supplemented with beta-mercaptoethanol and total RNA was extracted using Qiagen RNeasy mini kit.
    A549
    suggested: None
    Cells were then infected at 0.425 multiplicity of infection (MOI), based on Vero E6 titer, at 37°C. 1 hour post virus addition, virus was removed, and media containing compound/carrier was added.
    Vero E6
    suggested: None
    l analysis: Antiviral activities of PF-00835231 and remdesivir in A549+ACE2 cells were determined by the following method.
    A549+ACE2
    suggested: None
    Software and Algorithms
    SentencesResources
    Images were collected on the BZ-X810 (RRID:SCR_016979, Keyence, Osaka, Japan) fluorescence microscope.
    detected: BZ-X700 microscope ( RRID:SCR_016979)
    The Cellranger software suite (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger) from 10X was used to demultiplex cellular barcodes, align reads to the human genome (GRCh38 ensemble, http://useast.ensembl.org/Homo_sapiens/Info/Index) and perform UMI counting.
    https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger
    suggested: (Cell Ranger , RRID:SCR_017344)
    Other statistical data analyses were performed in GraphPad Prism 7.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: Thank you for sharing your data.

    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.

    About SciScore

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  3. Version published to 10.1101/2020.08.28.272880v1 on bioRxiv