TMPRSS2 and furin are both essential for proteolytic activation and spread of SARS-CoV-2 in human airway epithelial cells and provide promising drug targets

  1. Dorothea Bestle
  2. Miriam Ruth Heindl
  3. Hannah Limburg
  4. Thuy Van Lam van
  5. Oliver Pilgram
  6. Hong Moulton
  7. David A. Stein
  8. Kornelia Hardes
  9. Markus Eickmann
  10. Olga Dolnik
  11. Cornelius Rohde
  12. Stephan Becker
  13. Hans-Dieter Klenk
  14. Wolfgang Garten
  15. Torsten Steinmetzer
  16. Eva Böttcher-Friebertshäuser

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Abstract

In December 2019, a novel coronavirus named SARS-CoV-2 first reported in Wuhan, China, emerged and rapidly spread to numerous other countries globally, causing the current pandemic. SARS-CoV-2 causes acute infection of the respiratory tract (COVID-19) that can result in severe disease and lethality. Currently, there is no approved antiviral drug for treating COVID-19 patients and there is an urgent need for specific antiviral therapies and vaccines.

In order for SARS-CoV-2 to enter cells, its surface glycoprotein spike (S) must be cleaved at two different sites by host cell proteases, which therefore represent potential drug targets. In the present study we investigated which host cell proteases activate the SARS-CoV-2 S protein in Calu-3 human airway epithelial cells. We show that S can be cleaved by both the proprotein convertase furin at the S1/S2 site and the transmembrane serine protease 2 (TMPRSS2) at the S2’ site. We demonstrate that TMPRSS2 is essential for activation of SARS-CoV-2 S in Calu-3 cells through antisense-mediated knockdown of TMPRSS2 expression. Further, we show that SARS-CoV-2 replication can be efficiently inhibited by two synthetic inhibitors of TMPRSS2 and also by the broad range serine protease inhibitor aprotinin. Additionally, SARS-CoV-2 replication was also strongly inhibited by the synthetic furin inhibitor MI-1851. Combining various TMPRSS2 inhibitors with MI-1851 produced more potent antiviral activity against SARS-CoV-2 than an equimolar amount of any single serine protease inhibitor. In contrast, inhibition of endosomal cathepsins by E64d did not affect virus replication.

Our data demonstrate that both TMPRSS2 and furin are essential for SARS-CoV-2 activation in human airway cells and are promising drug targets for the treatment of COVID-19 either by targeting one of these proteases alone or by a combination of furin and TMPRSS2 inhibitors. Therefore, this approach has a high therapeutic potential for treatment of COVID-19.

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  1. ScreenIT

    SciScore for 10.1101/2020.04.15.042085: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    A monoclonal mouse antibody against the C-terminal Myc-tag was purchased from CellSignaling Technology (2276S)
    Myc-tag
    suggested: None
    A monoclonal mouse anti-beta actin antibody was purchased from Abcam (ab6276)
    A monoclonal mouse anti-beta actin antibody
    suggested: None
    anti-beta actin
    suggested: (Abcam Cat# ab6276, RRID:AB_2223210)
    Experimental Models: Cell Lines
    SentencesResources
    Vero E6 (ATCC® CRL-1586) and HEK293 (ATCC® CRL-1573) cells were maintained in DMEM supplemented with 10 % FCS, antibiotics and glutamine.
    Vero E6
    suggested: None
    For analysis of multicycle replication kinetics Calu-3 cells were seeded in 12-well plates and grown to 90 % confluence.
    Calu-3
    suggested: None
    Transient expression of SARS CoV-2 S protein in HEK293 cells: For transient expression of SARS-CoV-2 S protein 60 % confluent HEK293 cells were co-transfected with 1.6 µg of pCAGGS-S-Myc-6xHis and either 15 ng of empty pCAGGS vector or pCAGGS-TMPRSS2 using Liopfectamine® 2000 (Invitrogen) according to the manufacturers protocol for 48 h.
    HEK293
    suggested: None

    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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    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.

  2. Version published to 10.1101/2020.04.15.042085v1 on bioRxiv