An orally bioavailable SARS-CoV-2 main protease inhibitor exhibits improved affinity and reduced sensitivity to mutations

  1. Michael Westberg
  2. Yichi Su
  3. Xinzhi Zou
  4. Pinghan Huang
  5. Arjun Rustagi
  6. Jaishree Garhyan
  7. Puja Bhavesh Patel
  8. Daniel Fernandez
  9. Yan Wu
  10. Chenzhou Hao
  11. Chieh-Wen Lo
  12. Marwah Karim
  13. Lin Ning
  14. Aimee Beck
  15. Panatda Saenkham-Huntsinger
  16. Vivian Tat
  17. Aleksandra Drelich
  18. Bi-Hung Peng
  19. Shirit Einav
  20. Chien-Te K. Tseng
  21. Catherine Blish
  22. Michael Z. Lin

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Abstract

Inhibitors of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (M pro ) such as nirmatrelvir (NTV) and ensitrelvir (ETV) have proven effective in reducing the severity of COVID-19, but the presence of resistance-conferring mutations in sequenced viral genomes raises concerns about future drug resistance. Second-generation oral drugs that retain function against these mutants are thus urgently needed. We hypothesized that the covalent hepatitis C virus protease inhibitor boceprevir (BPV) could serve as the basis for orally bioavailable drugs that inhibit SARS-CoV-2 M pro more efficiently than existing drugs. Performing structure-guided modifications of BPV, we developed a picomolar-affinity inhibitor, ML2006a4, with antiviral activity, oral pharmacokinetics, and therapeutic efficacy similar or superior to those of NTV. A crucial feature of ML2006a4 is a derivatization of the ketoamide reactive group that improves cell permeability and oral bioavailability. Last, ML2006a4 was found to be less sensitive to several mutations that cause resistance to NTV or ETV and occur in the natural SARS-CoV-2 population. Thus, anticipatory design can preemptively address potential resistance mechanisms to expand future treatment options against coronavirus variants.

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  1. Version published to 10.1126/scitranslmed.adi0979
  2. ScreenIT

    SciScore for 10.1101/2020.09.15.275891: (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

    Experimental Models: Cell Lines
    SentencesResources
    Cell-based Mpro Activity Assay in HEK293A and Huh7: Mpro Expression and Purification: Modified versions of previous protocols based on HRV protease (40) or SUMO protease (51) processing of Mpro fusion proteins were used to obtain purified Mpro with either native or extended termini after expression in E. coli.
    HEK293A
    suggested: RRID:CVCL_6910)
    Cytotoxicity assay in Huh7 cells: 24 h prior to the drug treatment, 10000 Huh7 cells were seeded in 100 μl culture medium (DMEM with 10% FBS, 2mM L-glutamine, penicillin-streptomycin) in a 96-well white microplate (Greiner Bio-One, Austria) pre-coated with poly-D-lysine (10μg/ml, Sigma).
    Huh7
    suggested: None
    Antiviral and cytotoxicity assays in Caco-2 cells: The tested Mpro inhibitors were serially diluted from 10 mM DMSO stocks using eight log10 dilutions in test medium (MEM supplemented with 2% FBS and 50 μg/mL gentamicin) yielding a concentration range of 10 pM – 100 μM.
    Caco-2
    suggested: None
    SARSCoV2 (USA_WA1/2020 strain passaged twice in Vero 76 cells in MEM supplemented with 2% fetal bovine serum and 50 μg/ml gentamicin to prepare a working stock) was prepared at a multiplicity of infection (MOI) that would yield measurable virus titers within 72 hours.
    Vero 76
    suggested: None
    Software and Algorithms
    SentencesResources
    The IC50 curves were evaluated using the following equation in Prism (GraphPad):

    Here vrel is the experimentally measured rate of substrate cleavage normalized to the rate of substrate cleavage in the absence of inhibitor, I.

    Prism
    suggested: (PRISM, RRID:SCR_005375)

    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

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  3. Version published to 10.1101/2020.09.15.275891v2 on bioRxiv
  4. Version published to 10.1101/2020.09.15.275891v1 on bioRxiv