A novel highly potent inhibitor of TMPRSS2-like proteases blocks SARS-CoV-2 variants of concern and is broadly protective against infection and mortality in mice

  1. Tirosh Shapira
  2. I. Abrrey Monreal
  3. Sébastien P. Dion
  4. Mason Jager
  5. Antoine Désilets
  6. Andrea D. Olmstead
  7. Thierry Vandal
  8. David W. Buchholz
  9. Brian Imbiakha
  10. Guang Gao
  11. Aaleigha Chin
  12. William D. Rees
  13. Theodore Steiner
  14. Ivan Robert Nabi
  15. Eric Marsault
  16. Julie Sahler
  17. Avery August
  18. Gerlinde Van de Walle
  19. Gary R. Whittaker
  20. Pierre-Luc Boudreault
  21. Hector C. Aguilar
  22. Richard Leduc
  23. François Jean

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Abstract

The COVID-19 pandemic caused by the SARS-CoV-2 virus remains a global public health crisis. Although widespread vaccination campaigns are underway, their efficacy is reduced against emerging variants of concern (VOCs) 1,2 . Development of host-directed therapeutics and prophylactics could limit such resistance and offer urgently needed protection against VOCs 3,4 . Attractive pharmacological targets to impede viral entry include type-II transmembrane serine proteases (TTSPs), such as TMPRSS2, whose essential role in the virus lifecycle is responsible for the cleavage and priming of the viral spike protein 5–7 . Here, we identify and characterize a small-molecule compound, N-0385, as the most potent inhibitor of TMPRSS2 reported to date. N-0385 exhibited low nanomolar potency and a selectivity index of >10 6 at inhibiting SARS-CoV-2 infection in human lung cells and in donor-derived colonoids 8 . Importantly, N-0385 acted as a broad-spectrum coronavirus inhibitor of two SARS-CoV-2 VOCs, B.1.1.7 and B.1.351. Strikingly, single daily intranasal administration of N-0385 early in infection significantly improved weight loss and clinical outcomes, and yielded 100% survival in the severe K18-human ACE2 transgenic mouse model of SARS-CoV-2 disease. This demonstrates that TTSP-mediated proteolytic maturation of spike is critical for SARS-CoV-2 infection in vivo and suggests that N-0385 provides a novel effective early treatment option against COVID-19 and emerging SARS-CoV-2 VOCs.

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    SciScore for 10.1101/2021.05.03.442520: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: All protocols were performed under approved BSL-3 conditions and approved by the Institutional Animal Care and Use Committee at Cornell University (IACUC mouse protocol # 2017-0108 and BSL3 IBC # MUA-16371-1).
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    BlindingTissue sections (4 μm) were analyzed after staining with H&E and scored blinded by an anatomic pathologist.
    Power Analysisnot detected.
    Cell Line AuthenticationAuthentication: A region of interest (ROI, or “circle”) is then drawn around each host cell and validated against the bright field image to correspond with host cell membranes.

    Table 2: Resources

    Antibodies
    SentencesResources
    The SARS-CoV-2 nucleocapsid antibody [HL344] (GTX635679) was kindly provided by Genetex; mouse anti-dsRNA antibody (J2-1904) was purchased from Scions English and Scientific Consulting 34; Hoechst 33258 and secondary antibodies goat anti-mouse IgG Alexa Fluor 488 (A11001) and goat anti-rabbit IgG Alexa Fluor 555 (A21428) were obtained from Invitrogen.
    GTX635679
    suggested: (GeneTex Cat# GTX635679, RRID:AB_2888553)
    anti-dsRNA
    suggested: (Millipore Cat# MABE1134, RRID:AB_2819101)
    The fixative was removed, and cells were washed with PBS, permeabilized with 0.1% Triton X-100 for 5 min and blocked with 1% Bovine serum albumin (BSA) for 1 hr, followed by immunostaining with the mouse primary antibody J2 (dsRNA) and rabbit primary antibody HL344 (SARS-CoV-2 nucleocapsid) at working dilutions of 1:1000 for 1 hr at room temperature.
    rabbit
    suggested: (Thermo Fisher Scientific Cat# MA5-36251, RRID:AB_2890565)
    Secondary antibodies were used at a 1:2000 dilution and included the goat anti-mouse IgG Alexa Fluor 488 and goat anti-rabbit IgG Alexa Fluor 555 with the nuclear stain Hoechst 33342 at 1 µg/mL and F-actin staining with Alexa Fluor 647 phalloidin at a 1:300 dilution for 1 hr at room temperature in the dark.
    anti-mouse IgG
    suggested: None
    anti-rabbit IgG
    suggested: None
    To detect viral antigen, sections were labeled with anti-SARS-CoV-2 nucleocapsid protein rabbit IgG monoclonal antibody (GeneTex; GTX635679) at 1:5000 dilution and processed using a Leica Bond Max automated IHC stainer.
    anti-SARS-CoV-2 nucleocapsid protein rabbit IgG
    suggested: (Proteintech Cat# 80026-1-RR, RRID:AB_2882944)
    Experimental Models: Cell Lines
    SentencesResources
    Cell lines, antibodies, and inhibitors: Calu-3 cells (ATCC® HTB-55™) were cultivated according to ATCC recommendations.
    Calu-3
    suggested: ATCC Cat# HTB-55, RRID:CVCL_0609)
    Vero E6 cells (ATCC® CRL-I1586™; used for SARS-CoV-2 plaque assay) were cultivated in MEM supplemented with 10% FBS, 1 mM sodium pyruvate, and 0.1 nM non-essential amino acids and used at passage 19-25.
    Vero E6
    suggested: None
    Recombinant DNA
    SentencesResources
    TMPRSS2 pericellular activity screening assay and IC50 determination: Vero E6 cells were transfected with mock (pcDNA3.1), TMPRSS2 (pcDNA3.1/TMPRSS2 Uniprot: O15393-1), or TMPRSS2-S441A (pcDNA3.1/TMPRSS2-S441A) using Lipofectamine 3000 in 12-well plates.
    pcDNA3.1
    suggested: RRID:Addgene_79663)
    pcDNA3.1/TMPRSS2
    suggested: None
    pcDNA3.1/TMPRSS2-S441A
    suggested: None
    Software and Algorithms
    SentencesResources
    IC50 values were determined after generating a nonlinear regression analysis from a log([Compound]) versus a proteolytic activity plot using GraphPad Prism software (version 9.0.1).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    GraphPad Prism was used to identify and eliminate outliers (Q = 1) and assess the goodness of the fits.
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Digital image analysis was performed using QuPath software version 0.2.3 62,63.
    QuPath
    suggested: (QuPath, RRID:SCR_018257)

    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.

  2. Version published to 10.1101/2021.05.03.442520v1 on bioRxiv