S-217622, a SARS-CoV-2 main protease inhibitor, decreases viral load and ameliorates COVID-19 severity in hamsters

  1. Michihito Sasaki
  2. Koshiro Tabata
  3. Mai Kishimoto
  4. Yukari Itakura
  5. Hiroko Kobayashi
  6. Takuma Ariizumi
  7. Kentaro Uemura
  8. Shinsuke Toba
  9. Shinji Kusakabe
  10. Yuki Maruyama
  11. Shun Iida
  12. Noriko Nakajima
  13. Tadaki Suzuki
  14. Shinpei Yoshida
  15. Haruaki Nobori
  16. Takao Sanaki
  17. Teruhisa Kato
  18. Takao Shishido
  19. William W. Hall
  20. Yasuko Orba
  21. Akihiko Sato
  22. Hirofumi Sawa

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Abstract

In parallel with vaccination, oral antiviral agents are highly anticipated to act as countermeasures for the treatment of the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Oral antiviral medication demands not only high antiviral activity but also target specificity, favorable oral bioavailability, and high metabolic stability. Although a large number of compounds have been identified as potential inhibitors of SARS-CoV-2 infection in vitro, few have proven to be effective in vivo. Here, we show that oral administration of S-217622 (ensitrelvir), an inhibitor of SARS-CoV-2 main protease (M pro ; also known as 3C-like protease), decreases viral load and ameliorates disease severity in SARS-CoV-2–infected hamsters. S-217622 inhibited viral proliferation at low nanomolar to submicromolar concentrations in cells. Oral administration of S-217622 demonstrated favorable pharmacokinetic properties and accelerated recovery from acute SARS-CoV-2 infection in hamster recipients. Moreover, S-217622 exerted antiviral activity against SARS-CoV-2 variants of concern, including the highly pathogenic Delta variant and the recently emerged Omicron BA.5 and BA.2.75 variants. Overall, our study provides evidence that S-217622, an antiviral agent that is under evaluation in a phase 3 clinical trial (clinical trial registration no. jRCT2031210350), has remarkable antiviral potency and efficacy against SARS-CoV-2 and is a prospective oral therapeutic option for COVID-19.

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  1. Version published to 10.1126/scitranslmed.abq4064
  2. ScreenIT

    SciScore for 10.1101/2022.02.14.480338: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: The protocol was reviewed and approved by the Institutional Animal Care and Use Committee of Hokkaido University (approval no. 20-0060).
    Euthanasia Agents: Virus infection and treatment of hamsters: Five-week old male Syrian hamsters (Japan SLC) were intranasally inoculated with 5,000 pfu of SARS-CoV-2 in 200 μl of PBS under anesthesia with isoflurane inhalation.
    Sex as a biological variablePharmacokinetics studies: Five-week old male Syrian hamsters (Japan SLC) were orally administered at 10, 30, and 100 mg/kg of S-217622 under the non-fasting conditions (n = 3, each).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Vero-TMPRSS2 cells at 24 hpi and Calu-3 cells at 72 hpi were fixed with 3.7% formaldehyde in PBS, followed by permeabilization with 0.5% Triton X-100 in PBS for 5 min and staining with anti-SARS-CoV-2 nucleocapsid rabbit monoclonal antibody (GTX635679, GeneTex) in 25% Block Ace (KAC) in PBS for 1 h.
    anti-SARS-CoV-2 nucleocapsid
    suggested: None
    GTX635679
    suggested: (GeneTex Cat# GTX635679, RRID:AB_2888553)
    Alexa Fluor 488-conjugated anti-rabbit IgG antibody (Invitrogen; Thermo Fisher Scientific) was used as the secondary antibody.
    Alexa Fluor 488-conjugated anti-rabbit IgG antibody
    suggested: None
    anti-rabbit IgG
    suggested: None
    Tissues were further incubated for 4 days with secondary antibody staining solution; Alexa Fluor Plus 647-conjugated anti-rabbit IgG (A32795, Invitrogen) diluted in PBSBT with 0.1% saponin and filtrated though a 0.45 μm syringe filter.
    A32795
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Vero-TMPRSS2 cells [Vero E6 cells (ATCC, CRL-1586
    Vero E6
    suggested: None
    293T (RIKEN BRC, RCB2202) cells were maintained in high glucose DMEM containing 10% FBS.
    293T
    suggested: None
    RCB2202
    suggested: None
    293T-ACE2-TMPRSS2 cells stably expressing human TMPRSS2 and ACE2 were established by a lentiviral vector transduction system as previously described35 and maintained in high glucose DMEM containing 10%
    293T-ACE2-TMPRSS2
    suggested: None
    Vero-TMPRSS2 cells at 24 hpi and Calu-3 cells at 72 hpi were fixed with 3.7% formaldehyde in PBS, followed by permeabilization with 0.5% Triton X-100 in PBS for 5 min and staining with anti-SARS-CoV-2 nucleocapsid rabbit monoclonal antibody (GTX635679, GeneTex) in 25% Block Ace (KAC) in PBS for 1 h.
    Vero-TMPRSS2
    suggested: JCRB Cat# JCRB1818, RRID:CVCL_YQ48)
    Fluorescent images were captured using IX73 fluorescence microscope (Olympus). qRT-PCR: Vero-TMPRSS2 and Calu-3 cells were inoculated with SARS-CoV-2 at MOIs of 0.01 or 0.1, respectively.
    Calu-3
    suggested: BCRJ Cat# 0264, RRID:CVCL_0609)
    Recombinant DNA
    SentencesResources
    Biosensor assay for viral protease activity: The DNA fragments encoding SARS-CoV-2 NSP3 or NSP4, NSP5, N-terminal NSP6 (NSP4/5/6N) were cloned into pCMV-derivative pCXSN vector to generate pSARS-CoV-2 PLpro and pSARS-CoV-2 Mpro, respectively.
    pCMV-derivative pCXSN
    suggested: None
    pSARS-CoV-2
    suggested: None
    The amino acid sequence AVLQS (for the cleavage by Mpro) or RLKGG (for the cleavage by PLpro) were inserted into pGloSensor-30F vector backbone (CS182101, Promega) to generate firefly luciferase-based biosensor expressing plasmid pGS-AVLQS or pGS-RLKGG, respectively.
    pGloSensor-30F
    suggested: None
    293T cells on 96-well plate were co-transfected with a set of pGS-AVLQS and pSARS-CoV-2 Mpro or a set of pGS-RLKGG and pSARS-CoV-2 PLpro using TranIT-LT1
    pGS-AVLQS
    suggested: None
    pGS-RLKGG
    suggested: None
    pSARS-CoV-2 PLpro
    suggested: None
    At 24 h post transfection, the luminescence signals of biosensors and renilla luciferase (an internal control reporter) from pGS vectors were measured by Dual-Glo Luciferase Assay System (
    pGS
    suggested: None
    Software and Algorithms
    SentencesResources
    The concentration achieving 50% inhibition of cytopathic effect (effective concentration; EC50) was defined in GraphPad Prism version 8.4.3 (GraphPad Software) with a variable slope (four parameters)
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Image data was converted and processed into 3D reconstruction by Imaris software (Oxford Instruments).
    Imaris
    suggested: (Imaris, RRID:SCR_007370)

    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: We detected the following sentences addressing limitations in the study:
    We note some limitations of our study. First, therapeutic treatment with S-217622 was insufficient to control pneumonia in SARS-CoV-2-infected hamsters, although they regained lost body weight earlier compared to vehicle controls. We assume that the initial virus proliferation stimulated host immunity and subsequent inflammation31, 32, and combination with antiviral and anti-inflammatory medication may lead to better results and outcomes33. Second, this study was conducted using hamsters as a COVID-19 model and the efficacy in human patients cannot be inferred. However, S-217622 is currently under evaluation in a phase II/III clinical trial. Third, the development of a resistant viral clone against S-217622 and its virological properties will need to be investigated in future studies. In summary, our study has demonstrated the remarkable antiviral activity of S-217622 through in vitro and in vivo experiments. This scientific evidence will be invaluable when considering the application of S-217622 as a medication for COVID-19.

    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: Please consider improving the rainbow (“jet”) colormap(s) used on page 41. 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.
    • No protocol registration statement was detected.

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

    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.

  3. Version published to 10.1101/2022.02.14.480338v1 on bioRxiv