Challenges for targeting SARS-CoV-2 proteases as a therapeutic strategy for COVID-19

  1. Kas Steuten
  2. Heeyoung Kim
  3. John C. Widen
  4. Brett M. Babin
  5. Ouma Onguka
  6. Scott Lovell
  7. Oguz Bolgi
  8. Berati Cerikan
  9. Mirko Cortese
  10. Ryan K. Muir
  11. John M. Bennett
  12. Ruth Geiss-Friedlander
  13. Christoph Peters
  14. Ralf Bartenschlager
  15. Matthew Bogyo

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Abstract

Two proteases produced by the SARS-CoV-2 virus, M pro and PL pro , are essential for viral replication and have become the focus of drug development programs for treatment of COVID-19. We screened a highly focused library of compounds containing covalent warheads designed to target cysteine proteases to identify new lead scaffolds for both M pro and PL pro proteases. These efforts identified a small number of hits for the M pro protease and no viable hits for the PL pro protease. Of the M pro hits identified as inhibitors of the purified recombinant protease, only two compounds inhibited viral infectivity in cellular infection assays. However, we observed a substantial drop in antiviral potency upon expression of TMPRSS2, a transmembrane serine protease that acts in an alternative viral entry pathway to the lysosomal cathepsins. This loss of potency is explained by the fact that our lead M pro inhibitors are also potent inhibitors of host cell cysteine cathepsins. To determine if this is a general property of M pro inhibitors, we evaluated several recently reported compounds and found that they are also effective inhibitors of purified human cathepsin L and B and showed similar loss in activity in cells expressing TMPRSS2. Our results highlight the challenges of targeting M pro and PL pro proteases and demonstrate the need to carefully assess selectivity of SARS-CoV-2 protease inhibitors to prevent clinical advancement of compounds that function through inhibition of a redundant viral entry pathway.

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  1. ScreenIT

    SciScore for 10.1101/2020.11.21.392753: (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 AuthenticationContamination: Cell lines: VeroE6 and A549 cells were obtained from American Type Culture Collection (ATCC) and tested at regular intervals for mycoplasma contamination.

    Table 2: Resources

    Antibodies
    SentencesResources
    After three times washing with PBS, bound primary antibody was detected with a secondary antibody (anti-mouse IgG), conjugated to horseradish peroxidase.
    anti-mouse IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    This Heidelberg strain was passaged in VeroE6 cells, aliquoted and stored at −80° C.
    VeroE6
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Cell lines: VeroE6 and A549 cells were obtained from American Type Culture Collection (ATCC) and tested at regular intervals for mycoplasma contamination.
    A549
    suggested: NCI-DTP Cat# A549, RRID:CVCL_0023)
    Lentivirus stocks were produced by transfection of HEK293T cells with a pWPI plasmid encoding for TMPRSS2 and the pCMV-Gag-Pol and pMD2-VSV-G packaging plasmids (kind gifts from D. Trono, Geneva).
    HEK293T
    suggested: None
    For all other assays, A549+ACE2 cells were cultured in Roswell Park Memorial Institute (
    A549+ACE2
    suggested: None
    Software and Algorithms
    SentencesResources
    PLpro expression and purification: For cloning of PLpro with an N-terminal 6xHis-SUMO1 tag, synthetic fragments of the Nsp3 coding sequence derived from the original Wuhan strain were purchased from BioCat.
    BioCat
    suggested: (BioCAT, RRID:SCR_001440)
    All data were analyzed using GraphPad Prism (v8.4)
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Densitometric analysis of protein bands on gels was performed using ImageJ (v1.52p)
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    The LC-MS systems used was either a Thermo Fisher Finnigan Surveyor Plus equipped with an Agilent Zorbax 300SB-C18 column (3.5 μm, 3.0 × 150 mm) coupled to a Finnigan LTQ mass spectrometer or an Agilent 1100 Series HPLC equipped with a Luna 4251-E0 C18 column (3 μm, 4.6 x 150 mm) coupled to a PE SCIEX API 150EX mass spectrometer (wavelengths monitored = 220, 254 and 646 nm).
    Thermo Fisher Finnigan Surveyor Plus
    suggested: None

    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: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT04535167RecruitingFirst-In-Human Study To Evaluate Safety, Tolerability, And P…

    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

    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 10.1101/2020.11.21.392753 on bioRxiv