An Enzymatic TMPRSS2 Assay for Assessment of Clinical Candidates and Discovery of Inhibitors as Potential Treatment of COVID-19

  1. Jonathan H. Shrimp
  2. Stephen C. Kales
  3. Philip E. Sanderson
  4. Anton Simeonov
  5. Min Shen
  6. Matthew D. Hall

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Abstract

SARS-CoV-2 is the viral pathogen causing the COVID19 global pandemic. Consequently, much research has gone into the development of pre-clinical assays for the discovery of new or repurposing of FDA-approved therapies. Preventing viral entry into a host cell would be an effective antiviral strategy. One mechanism for SARS-CoV-2 entry occurs when the spike protein on the surface of SARS-CoV-2 binds to an ACE2 receptor followed by cleavage at two cut sites (“priming”) that causes a conformational change allowing for viral and host membrane fusion. TMPRSS2 has an extracellular protease domain capable of cleaving the spike protein to initiate membrane fusion. A validated inhibitor of TMPRSS2 protease activity would be a valuable tool for studying the impact TMPRSS2 has in viral entry and potentially be an effective antiviral therapeutic. To enable inhibitor discovery and profiling of FDA-approved therapeutics, we describe an assay for the biochemical screening of recombinant TMPRSS2 suitable for high throughput application. We demonstrate effectiveness to quantify inhibition down to subnanomolar concentrations by assessing the inhibition of camostat, nafamostat and gabexate, clinically approved agents in Japan. Also, we profiled a camostat metabolite, FOY-251, and bromhexine hydrochloride, an FDA-approved mucolytic cough suppressant. The rank order potency for the compounds tested are: nafamostat (IC 50 = 0.27 nM), camostat (IC 50 = 6.2 nM), FOY-251 (IC 50 = 33.3 nM) and gabexate (IC 50 = 130 nM). Bromhexine hydrochloride showed no inhibition of TMPRSS2. Further profiling of camostat, nafamostat and gabexate against a panel of recombinant proteases provides insight into selectivity and potency.

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  1. ScreenIT

    SciScore for 10.1101/2020.06.23.167544: (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.

    Table 2: Resources

    Experimental Models: Organisms/Strains
    SentencesResources
    Peptides custom ordered from LifeTein (Somerset, NJ) include: Cbz-D-Arg-Gly-Arg-AMC, Cbz-D-Arg-Pro-Arg-AMC.
    Cbz-D-Arg-Gly-Arg-AMC
    suggested: None
    Software and Algorithms
    SentencesResources
    Data normalization, visualization and curve fitting were performed using Prism (GraphPad, San Diego, CA)
    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: 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.
    • No funding statement was detected.
    • 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 10.1101/2020.06.23.167544 on bioRxiv