An oral SARS-CoV-2 M pro inhibitor clinical candidate for the treatment of COVID-19

  1. Dafydd R. Owen
  2. Charlotte M. N. Allerton
  3. Annaliesa S. Anderson
  4. Lisa Aschenbrenner
  5. Melissa Avery
  6. Simon Berritt
  7. Britton Boras
  8. Rhonda D. Cardin
  9. Anthony Carlo
  10. Karen J. Coffman
  11. Alyssa Dantonio
  12. Li Di
  13. Heather Eng
  14. RoseAnn Ferre
  15. Ketan S. Gajiwala
  16. Scott A. Gibson
  17. Samantha E. Greasley
  18. Brett L. Hurst
  19. Eugene P. Kadar
  20. Amit S. Kalgutkar
  21. Jack C. Lee
  22. Jisun Lee
  23. Wei Liu
  24. Stephen W. Mason
  25. Stephen Noell
  26. Jonathan J. Novak
  27. R. Scott Obach
  28. Kevin Ogilvie
  29. Nandini C. Patel
  30. Martin Pettersson
  31. Devendra K. Rai
  32. Matthew R. Reese
  33. Matthew F. Sammons
  34. Jean G. Sathish
  35. Ravi Shankar P. Singh
  36. Claire M. Steppan
  37. Al E. Stewart
  38. Jamison B. Tuttle
  39. Lawrence Updyke
  40. Patrick R. Verhoest
  41. Liuqing Wei
  42. Qingyi Yang
  43. Yuao Zhu

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Abstract

The rapid development of vaccines has been crucial in battling the ongoing COVID-19 pandemic. However, access challenges remain, breakthrough infections occur, and emerging variants present increased risk. Developing antiviral therapeutics is therefore a high priority for the treatment of COVID-19. Some drug candidates in clinical trials act against the viral RNA-dependent RNA polymerase, but there are other viral enzymes that have been considered good targets for inhibition by drugs. Owen et al . report the discovery and characterization of a drug against the main protease involved in the cleavage of polyproteins involved in viral replication. The drug, PF-07321332, can be administered orally, has good selectivity and safety profiles, and protects against infection in a mouse model. In a phase 1 clinical trial, the drug reached concentrations expected to inhibit the virus based on in vitro studies. It also inhibited other coronaviruses, including severe acute respiratory syndrome coronavirus 1 and Middle East respiratory syndrome coronavirus, and could be in the armory against future viral threats. —VV

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  1. Version published to 10.1126/science.abl4784
  2. ScreenIT

    SciScore for 10.1101/2021.07.28.21261232: (What is this?)

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

    Table 1: Rigor

    EthicsEuthanasia Agents: The filter cake was washed with a mixture of diethyl ether and heptane (1:1, 4 x 2 ml) to afford N- [(2S)-1-(((1S)-1-cyano-2-[(3S)-2-oxopyrrolidin-3-yl]ethyl)amino)-4-methyl-1-oxopentan-2-yl]- 4-methoxy-1H-indole-2-carboxamide (2) as a solid.
    IACUC: The study procedures were conducted with approval by the Institutional Animal Care and Use Committee at Utah State University.
    IRB: Drug Metabolism Studies and Methods: Research was conducted on human tissue acquired from a third party that has been verified as compliant with Pfizer policies, including Institutional Review Board/Independent Ethics Committee approval.
    Field Sample Permit: All definitive studies were conducted in accordance with US FDA GLP regulations in an OECD MAD member state.
    Consent: The protocol was approved by an independent institutional review board and all participants provided informed consent before screening.
    Sex as a biological variableRat intestinal microsomes (pool of 200, male Sprague Dawley,), monkey intestinal microsomes (pool of 6, male Cynomolgus,), human intestinal microsomes (pool of 9, male and female,), and human liver microsomes (HLM) (custom pool of 50 donors, male and female), were purchased from Sekisui XenoTech (Kansas City, KS) or BioIVT (Baltimore, MD)
    RandomizationThe design for the single ascending dose portion was investigator- and participant- blinded, sponsor-open, randomized, 4-period cross-over (with Period 3 and 4 being optional) in 2 interleaving cohorts with placebo substitution.
    BlindingAnderson (study 1)) for processing and blinded evaluation by an experienced veterinary pathologist and yielded similar results.
    Power Analysisnot detected.
    Cell Line AuthenticationAuthentication: Plasma samples were analyzed for PF-07321332 concentrations at Pfizer (Groton, CT) using a validated sensitive and specific LC-MS/MS method in compliance with the Sponsor SOPs.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Ki values were fit to the tight binding Morrison equation with fixed parameters for enzyme concentration, substrate concentration and the Km parameter using ActivityBase software (IDBS) indicated below Cellular Antiviral Activity: The ability of compounds to inhibit viral induced cytopathic effect (CPE) against human coronaviruses (SARS-CoV-1, SARS-CoV-2, hCoV-229E, MERS) was assessed by monitoring cell viability using two different assay endpoints in VeroE6, MRC-5 or Vero81 cells.
    MRC-5
    suggested: None
    Vero81
    suggested: None
    VeroE6 cells that are enriched for hACE2 expression were batched innoculated with SARS- CoV-2 (USA_WA1/2020) at a multiplicity of infection (MOI) of 0.002 in a BSL-3 lab (Southern Research Institute)
    VeroE6
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    The virus titer was then quantified by infecting Vero76 cells in a standard endpoint dilution assay and virus dose that was able to infect 50% of the cell cultures (CCID50 per ml) was calculated (53).
    Vero76
    suggested: None
    Briefly, TK6 cells were incubated with (4 or 24 h) vehicle or PF-07321332 in the presence or absence of metabolic activation (Aroclor 1254- induced rat liver S9/NADPH), harvested, fixed, stained with acridine orange and scored for frequency of micronuclei in mononucleated cells by light microscopy.
    TK6
    suggested: None
    In brief, human embryonic kidney (HEK) cells stably expressing the human Kv11.1 (hERG) channel were cultured in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) supplemented with 10% (v.
    HEK
    suggested: None
    CHO cells stably expressing the human Nav1.5 sodium channel (Catalogue No. CT6007; Charles River Cleveland, OH, USA) were cultured in Ham’s F12 media supplemented with 10% FBS (10% (v/v) and G418 (0.25 mg/ml).
    CHO
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Reactions were monitored by thin layer chromatography (TLC) performed on Analtech, Inc. silica gel GF 250 μm plates or Merck silica gel plates (60 F254) and were visualized with UV light (254 nm) and/or KMnO4 staining or by UPLC-MS (Waters Acquity, ESCI (ESI +/-, APCI +/-)).
    ESI +/-
    suggested: None
    A total of 24 BALB/c mice (Charles River, 8 week old female, n=6 mice/group) were divided into 4 groups: group 1: untreated, infected control; group 2: 300 mg/kg PF-07321332 (6); group 3: 1000 mg/kg PF-07321332 (6), and group 4: untreated, uninfected control (for pharmacokinetic analysis and normal weight).
    BALB/c
    suggested: None
    Rat intestinal microsomes (pool of 200, male Sprague Dawley,), monkey intestinal microsomes (pool of 6, male Cynomolgus,), human intestinal microsomes (pool of 9, male and female,), and human liver microsomes (HLM) (custom pool of 50 donors, male and female), were purchased from Sekisui XenoTech (Kansas City, KS) or BioIVT (Baltimore, MD)
    Sprague Dawley
    suggested: None
    Samples were directly analyzed for depletion of 6 by LC-MS/MS. Plasma Protein Binding Determination for PF-07321332 (6): On the day of each incubation, fresh blood in K2EDTA was collected from male Wistar- Hannover rats (n=5, pooled), male Cynomolgus monkeys (n=2, pooled), and humans (n=1 male and n=1 female, pooled).
    Wistar- Hannover
    suggested: None
    Chronic Toxicity Studies with PF-07321332 (6): Briefly, groups of male and female Wistar Han rats or Mauritian cynomolgus monkeys were administered vehicle [2% (v/v
    Wistar Han
    suggested: None
    Software and Algorithms
    SentencesResources
    Upon request and subject to certain criteria, conditions and exceptions, Pfizer will provide access to de-identified participant data from Pfizer-sponsored global interventional clinical studies conducted for medicines, vaccines and medical devices for indications that have been approved in the US and/or the EU or in programs that have been terminated (i.e., development of all indications has been discontinued).
    Pfizer
    suggested: (Pfizer Inc., RRID:SCR_021375)
    All structures were refined iteratively by manual model building in Coot (47) followed by refinement in BUSTER.
    Coot
    suggested: (Coot, RRID:SCR_014222)
    BUSTER
    suggested: (BUSTER, RRID:SCR_015653)
    To determine the EC50 and EC90, the CCID50/ml values were normalized to that of no drug control as a percentage of inhibition and plotted against compound concentration in GraphPad Prism software by using four-parameter logistic regression.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Post-acquisition data processing was performed with either Topspin V3.2 or MestReNova V9.1.
    Topspin
    suggested: (TopSpin, RRID:SCR_014227)
    Jugular vein-cannulated male Wistar-Hannover rats were purchased from Charles River Laboratories, Inc. (Wilmington, MA) or Vital River (Beijing, China) and were typically 7-10 weeks of age at the time of dosing.
    Charles River Laboratories
    suggested: (Charles River Laboratories, RRID:SCR_003792)

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

    IdentifierStatusTitle
    NCT04756531RecruitingSTUDY OF PF-07321332 IN HEALTHY PARTICIPANTS

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 27. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    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.
    • Thank you for including a protocol registration statement.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

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  3. Version published to 10.1101/2021.07.28.21261232v1 on medRxiv