Mitoquinone mesylate targets SARS-CoV-2 infection in preclinical models

  1. Anton Petcherski
  2. Madhav Sharma
  3. Sandro Satta
  4. Maria Daskou
  5. Hariclea Vasilopoulos
  6. Cristelle Hugo
  7. Eleni Ritou
  8. Barbara Jane Dillon
  9. Eileen Fung
  10. Gustavo Garcia
  11. Claudio Scafoglio
  12. Arunima Purkayastha
  13. Brigitte N Gomperts
  14. Gregory A Fishbein
  15. Vaithilingaraja Arumugaswami
  16. Marc Liesa
  17. Orian S Shirihai
  18. Theodoros Kelesidis

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Abstract

To date, there is no effective oral antiviral against SARS-CoV-2 that is also anti-inflammatory. Herein, we show that the mitochondrial antioxidant mitoquinone/mitoquinol mesylate (Mito-MES), a dietary supplement, has potent antiviral activity against SARS-CoV-2 and its variants of concern in vitro and in vivo . Mito-MES had nanomolar in vitro antiviral potency against the Beta and Delta SARS-CoV-2 variants as well as the murine hepatitis virus (MHV-A59). Mito-MES given in SARS-CoV-2 infected K18-hACE2 mice through oral gavage reduced viral titer by nearly 4 log units relative to the vehicle group. We found in vitro that the antiviral effect of Mito-MES is attributable to its hydrophobic dTPP+ moiety and its combined effects scavenging reactive oxygen species (ROS), activating Nrf2 and increasing the host defense proteins TOM70 and MX1. Mito-MES was efficacious reducing increase in cleaved caspase-3 and inflammation induced by SARS-CoV2 infection both in lung epithelial cells and a transgenic mouse model of COVID-19. Mito-MES reduced production of IL-6 by SARS-CoV-2 infected epithelial cells through its antioxidant properties (Nrf2 agonist, coenzyme Q10 moiety) and the dTPP moiety. Given established safety of Mito-MES in humans, our results suggest that Mito-MES may represent a rapidly applicable therapeutic strategy that can be added in the therapeutic arsenal against COVID-19. Its potential long-term use by humans as diet supplement could help control the SARS-CoV-2 pandemic, especially in the setting of rapidly emerging SARS-CoV-2 variants that may compromise vaccine efficacy.

One-Sentence Summary

Mitoquinone/mitoquinol mesylate has potent antiviral and anti-inflammatory activity in preclinical models of SARS-CoV-2 infection.

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    SciScore for 10.1101/2022.02.22.481100: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: Tissues were procured under Institutional Review Board-approved protocols at the David Geffen School of Medicine at UCLA.
    Field Sample Permit: All work was conducted under protocols approved by the Institutional Animal Care and Use Committee (IACUC).
    IACUC: All work was conducted under protocols approved by the Institutional Animal Care and Use Committee (IACUC).
    Sex as a biological variableIn total, we utilized 55 male and female 4 to12-week-old specific pathogen–free hemizygous for Tg(K18-ACE2)2Prlmn (Strain B6.Cg-Tg(K18-ACE2)2Prlmn/J, the Jackson laboratory strain 034860) mice.
    RandomizationLittermates of the same sex were randomly assigned to experimental groups and all animal studies included both male and female mice.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Two to three days later, SARS-CoV-2 S or NP protein staining was assessed using an anti-SARS-CoV-2 S or NP protein antibody.
    NP protein
    suggested: None
    Cells were then washed with PBS and stained with 1:5,000 (anti-Spike S antibody clone 1A9) or 1:10,000 (anti-NP antibody clone ARC2372) in permeabilization buffer at 37 °C.
    anti-Spike S
    suggested: None
    anti-NP
    suggested: None
    The following antibodies and dilutions were used: polyclonal rabbit anti-Tom70 (1:100), rabbit anti-SARS-CoV-2 Spike S1 (clone #007) (1:100), polyclonal rabbit anti-cleaved caspase-3 (1:200), polyclonal rabbit anti-SARS-CoV-2 NP (1:1000).
    anti-Tom70
    suggested: (Thermo Fisher Scientific Cat# PA1-46006, RRID:AB_923450)
    anti-SARS-CoV-2 Spike S1
    suggested: None
    anti-cleaved caspase-3
    suggested: None
    anti-SARS-CoV-2 NP
    suggested: None
    Secondary antibodies were goat anti-rabbit Alexa Fluor 488 IgG, goat anti-mouse Alexa Fluor 546, goat anti-rabbit DyLight 650.
    anti-rabbit
    suggested: None
    The following antibodies were used for staining in flow cytometry: PE/Cy7 anti-mouse CD3 (clone 17A2, 1:25), BV 605 anti-mouse CD8a (clone 53-6.7, 1:10), Alexa Fluor 647 anti-mouse/human CD11b (clone M1/70, 1:50), BV421 anti-mouse CD11c (clone N418, 1:10), PerCP/Cy5.5 anti-mouse CD19 (clone 1D3/CD19, 1:10), PE/Cy7 anti-mouse CD31 (clone 390, 1:100), BV785 anti-mouse CD45 (clone 30-F11, 1:40), PE/Cy7 anti-human CD147 (clone HIM6, 1:10), BV421 anti-human CD304 (Neuropilin-1, clone 12C2, 1:20), BV605 anti-mouse CD326 (Ep-CAM, clone G8.8, 1:8)), PE anti-mouse CD335 (NKp46, clone 29A1.4, 1:10), BV711 MHC II anti-mouse I-A/I-E (clone M5/114.15.2, 1:25), APC/Cy7 anti-mouse Podoplanin (clone 8.1.1, 1:50), BV421 anti-mouse TER-119/Erythroid Cells (clone TER-119, 1:33), APC/Cy7 anti-mouse Ly-6G/Ly-6C (Gr-1, clone RB6-8C5, 1:25), rabbit anti-SARS-CoV-2 NP (clone ARC2372. 1:50), rabbit anti-SARS-CoV-2 Spike (clone 007, 1:50), mouse anti-SARS-CoV-2 Spike (clone 1A9, 1:50), anti-ACE-2 (polyclonal, 1:10), rabbit anti-human TMPRSS2 (polyclonal, 1:10), Alexa Fluor 647 anti-human Furin (clone 222722, 1:10), rabbit anti-human MAVS (clone D5A9E, 1:10), rabbit anti-human TOM70 (polyclonal,1:5), rabbit anti-human/mouse Alexa Fluor 647 cleaved caspase-3 (clone D3E9, 1:25), mouse anti-human, HO-1 (clone HO-1-2, 1:20), anti-MX1 (polyclonal, 1:10), Alexa Fluor 647 anti-human STING (clone 723505, 1:10).
    anti-human
    suggested: (R and D Systems Cat# MAB7169, RRID:AB_10971940)
    Neuropilin-1
    suggested: (BioLegend Cat# 354533, RRID:AB_2876674)
    Ep-CAM
    suggested: (Miltenyi Biotec Cat# 130-111-007, RRID:AB_2657511)
    anti-mouse
    suggested: (Millipore Cat# AP1058-50UG, RRID:AB_1084125)
    Gr-1
    suggested: (Bio-Rad Cat# MCA2387A647T, RRID:AB_2115660)
    anti-SARS-CoV-2
    suggested: (Thermo Fisher Scientific Cat# 51-6490-82, RRID:AB_2884044)
    anti-ACE-2
    suggested: None
    anti-human/mouse
    suggested: None
    anti-MX1
    suggested: None
    Rabbit anti-NRF2 (polyclonal, 1:50) anti-FOXJ1-CF647 (polyclonal, 1:10).
    anti-NRF2
    suggested: None
    To determine SARS-CoV-2 NP expression at the single cell level among different cell subtypes in murine lung [lung EPCAM (+) epithelial cells, ciliated FOXJ-1(+) lung EPCAM epithelial cells, alveolar type 1 (AT1), alveolar type 2 (AT2), CD31(+) CD45(-) endothelial cells, CD45(+) immune cells], one aliquot of cells was stained with a mastermix of antibodies for surface (CD326, MHCII, CD45, CD31, podoplanin, red blood cell marker Ter119) and intracellular antigens (SARS-CoV-2 NP, FOXJ-1).
    AT2
    suggested: None
    CD326
    suggested: None
    CD45
    suggested: None
    CD31
    suggested: None
    Ter119
    suggested: None
    To determine lung inflammation at the single cell level and cellular infiltration of murine lung with different immune cell subtypes [neutrophils, monocytes, macrophages, myeloid dendritic cells (mDCs), lymphoid dendritic cells (lymphoid DCs), natural killer cells (NK cells), T lymphocytes and B lymphocytes), one aliquot of lung cells was stained with a mastermix of antibodies for surface markers [CD11b, CD11c, CD8, CD3, CD19, CD45, NKp46, MHCII, Ly-6G/Ly-6C (Gr-1), red blood cell marker Ter119)].
    CD11c
    suggested: (Nanostring Cat# 121300104, RRID:AB_2893077)
    CD8
    suggested: (BioLegend Cat# 391503, RRID:AB_2721611)
    CD3
    suggested: (BioLegend Cat# 133313, RRID:AB_2715571)
    CD19
    suggested: None
    NKp46
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    HEK293-ACE2 cells were maintained at 37 °C and 5% CO2 in MEM supplemented with 10% (v/v) FBS and hygromycin.
    HEK293-ACE2
    suggested: None
    The antiviral activity of Mito-MES was evaluated in Calu3, Vero-E6, HEK293T and HSAECs ALI cell cultures.
    Vero-E6
    suggested: None
    HEK293T
    suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)
    HSAECs
    suggested: None
    AT1 cells were gated as m-CD45(-)/m-CD31(-)/m-EPCAM (+)/m-podoplanin (+) (AT1 marker)/ MHCII (-) (AT2 marker) cells.
    AT1
    suggested: None
    Luminex immunoassay was used to measure human cytokines [interleukin-1β (IL-1β), IL-8, IL-10, TNF-α, Vascular endothelial growth factor (VEGF)] secreted by Calu3 cells and airway lung epithelial cells in cell culture supernatants according to the manufacturer (Bio-Techne, Minneapolis, MN).
    Calu3
    suggested: KCLB Cat# 30055, RRID:CVCL_0609)
    Experimental Models: Organisms/Strains
    SentencesResources
    In total, we utilized 55 male and female 4 to12-week-old specific pathogen–free hemizygous for Tg(K18-ACE2)2Prlmn (Strain B6.Cg-Tg(K18-ACE2)2Prlmn/J, the Jackson laboratory strain 034860) mice.
    Tg(K18-ACE2)2Prlmn (Strain B6.Cg-Tg(K18-ACE2)2Prlmn/J
    suggested: RRID:IMSR_JAX:034860)
    Cohort B included 20 female K18-hACE2 mice between 4 to 8 weeks of age (16-21 g).
    K18-hACE2
    suggested: RRID:IMSR_GPT:T037657)
    Software and Algorithms
    SentencesResources
    The half maximal inhibitory concentration (IC50) for each experiment were determined using the Prism (GraphPad Holdings, San Diego, CA) software.
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    Samples were acquired using an LSRFortessa™ flow cytometer and FACSDiva™ software (Becton Dickinson, Franklin Lakes, NJ).
    FACSDiva™
    suggested: (BD FACSDiva Software, RRID:SCR_001456)
    Data were analyzed using FlowJo™ software (Becton Dickinson, Franklin Lakes, NJ).
    FlowJo™
    suggested: (FlowJo, RRID:SCR_008520)
    Cells were then analyzed using an LSR Fortessa flow cytometer and FACSDiva software), and data were analyzed using FlowJo software (Becton Dickinson, Franklin Lakes, NJ).
    FACSDiva
    suggested: (BD FACSDiva Software, RRID:SCR_001456)
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    All qRT-PCR reactions were performed using BIO-RAD CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA) on 96-well plates.
    Bio-Rad Laboratories
    suggested: (Bio-Rad Laboratories, RRID:SCR_008426)
    Wells were imaged using an LSM880 Zeiss confocal microscope (Carl Zeiss GmbH, Jena, Germany) with a 10X air objective and cell counts and infection ratios determined using CellProfiler 2.0.
    CellProfiler
    suggested: None
    All analyses were performed with GraphPad, version 8.0 (GraphPad Holdings, San Diego, CA).
    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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  2. Version published to 10.1101/2022.02.22.481100 on bioRxiv