Atazanavir is a competitive inhibitor of SARS-CoV-2 M pro , impairing variants replication in vitro and in vivo

  1. Otávio Augusto Chaves
  2. Carolina Q. Sacramento
  3. André C. Ferreira
  4. Mayara Mattos
  5. Natalia Fintelman-Rodrigues
  6. Jairo R. Temerozo
  7. Douglas Pereira Pinto
  8. Gabriel P. E. da Silveira
  9. Laís Bastos da Fonseca
  10. Heliana Martins Pereira
  11. Aluana Santana Carlos
  12. Joana da Costa Pinto d’Ávila
  13. João P.B. Viola
  14. Robson Q. Monteiro
  15. Leonardo Vazquez
  16. Patrícia T. Bozza
  17. Hugo Caire Castro-Faria-Neto
  18. Thiago Moreno L. Souza

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Abstract

Atazanavir (ATV) has already been considered as a potential repurposing drug to 2019 coronavirus disease (COVID-19), however, there are controversial reports on its mechanism of action and effectiveness as anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Through the pre-clinical chain of experiments: enzymatic, molecular docking, cell-based, and in vivo assays, it is demonstrated here that both SARS-CoV-2 B.1 lineage and variant of concern gamma are susceptible to this antiretroviral. Enzymatic assays and molecular docking calculations showed that SARS-CoV-2 main protease (M pro ) was inhibited by ATV, with Morrison’s inhibitory constant (Ki) 1.5-fold higher than boceprevir (GC376, a positive control). ATV was a competitive inhibition, increasing the M pro ’s Michaelis-Menten (K m ) more than 6-fold. Cell-based assays indicated that SARS-CoV-2 gamma is more susceptible to ATV than its predecessor strain B.1. Using oral administration of ATV in mice to reach plasmatic exposure similar to humans, transgenic mice expression in human angiotensin converting enzyme 2 (K18-hACE2) were partially protected against lethal challenge with SARS-CoV-2 gamma. Moreover, less cell death and inflammation were observed in the lung from infected and treated mice. Our studies may contribute to a better comprehension of the M pro /ATV interaction, which could pave the way to the development of specific inhibitors of this viral protease.

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    SciScore for 10.1101/2021.11.24.469775: (What is this?)

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

    Table 1: Rigor

    EthicsEuthanasia Agents: Infection was performed in 96-well plates (Nalge Nunc Int, Rochester, New York, USA) for 1 h at 37°C in 5 % of CO2.
    IACUC: The experiments performed in this section were approved by the Committee on the Use of Laboratory Animals of the Oswaldo Cruz Foundation (CEUA-FIOCRUZ, license L003/21). 2.8.
    IRB: In vivo assays – Mice treatment and infections: Experiments with transgenic mice expressing human ACE-2 receptor (K18-hACE2-mice), were performed in Animal Biosafety Level 3 (ABSL-3) multiuser facility, according to the animal welfare guidelines of the Ethics Committee of Animal Experimentation (CEUA-INCa, License 005/2021) and WHO guidelines [14].
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Cells and virus: African green monkey kidney (Vero, subtype E6) and human lung epithelial (Calu-3) cells were cultured in high-glucose Dulbecco’s modified Eagle medium (DMEM - HyClone, Logan, Utah) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin (P/S - Thermo Fisher Scientific®, Massachusetts, USA), and 10% fetal bovine serum (FBS - HyClone, Logan, Utah).
    Calu-3
    suggested: BCRJ Cat# 0264, RRID:CVCL_0609)
    The SARS-CoV-2 B.1 lineage (GenBank #MT710714) and gamma variant (also known as P1 or B.1.1.28 lineage; #EPI_ISL_1060902) were isolated on Vero E6 cells from nasopharyngeal swabs of confirmed cases.
    Vero E6
    suggested: RRID:CVCL_XD71)
    Cytotoxic assays: Vero cells (2.0 × 104 cell/well) were treated for 3 days with different concentrations of ATV or RDV (ranging from 1 to 600 μM) as previously described by us [7,23].
    Vero
    suggested: RRID:CVCL_ZW93)
    Experimental Models: Organisms/Strains
    SentencesResources
    Pharmacokinetic assays: ATV’s concentration in the plasma and lungs of adult Swiss-Webster mice (8-15 weeks) was evaluated over time.
    Swiss-Webster
    suggested: None
    Software and Algorithms
    SentencesResources
    Cells and virus: African green monkey kidney (Vero, subtype E6) and human lung epithelial (Calu-3) cells were cultured in high-glucose Dulbecco’s modified Eagle medium (DMEM - HyClone, Logan, Utah) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin (P/S - Thermo Fisher Scientific®, Massachusetts, USA), and 10% fetal bovine serum (FBS - HyClone, Logan, Utah).
    Thermo Fisher Scientific®
    suggested: (Thermo Fisher Scientific, RRID:SCR_008452)
    After fluorescence quantification, the Michaelis-Menten constant (Km) and maximum velocity (Vmax) were calculated by non-linear regression using GraphPad Prism 9.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    The molecular docking calculations were performed with GOLD 2020.2 software (Cambridge Crystallographic Data Center Software Ltd., CCDC, CB2 1EZ, UK) [20].
    GOLD
    suggested: (GOLD, RRID:SCR_000188)
    It was defined 8 □ radius around the active binding site and the figures were generated with PyMOL Delano Scientific LLC software (DeLano Scientific LLC: San Carlos, CA, USA) [21]. 2.5.
    PyMOL
    suggested: (PyMOL, RRID:SCR_000305)
    All experiments were carried out at least three independent times, including a minimum of two technical replicates in each assay, and each data was analyzed from Prism GraphPad software 8.0 (Windows GraphPad Software, San Diego, California USA).
    Prism GraphPad
    suggested: None
    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.
    • 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

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