key: cord-1004936-fyal3qpw
authors: Petcherski, Anton; Sharma, Madhav; Daskou, Maria; Satta, Sandro; Vasilopoulos, Hariclea; Hugo, Cristelle; Ritou, Eleni; Dillon, Barbara Jane; Fung, Eileen; Garcia, Gustavo; Scafoglio, Claudio; Purkayastha, Arunima; Gomperts, Brigitte N; Fishbein, Gregory A; Arumugaswami, Vaithilingaraja; Liesa, Marc; Shirihai, Orian S; Kelesidis, Theodoros
title: Mitoquinone mesylate targets SARS-CoV-2 and associated lung inflammation through host pathways
date: 2022-03-09
journal: bioRxiv
DOI: 10.1101/2022.02.22.481100
sha: 66e652a13f45279e9156e2e5f6a71e9020c6c356
doc_id: 1004936
cord_uid: fyal3qpw

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.

) airway respiratory epithelium ALI cultures. Our data is consistent with prior 12 evidence that Mito-MES attenuates ROS, activation of inflammasome, NFκB signaling that 13 collectively drive a cytokine storm, release of IL-1β, IL-6 (Zhang et al., 2020) and ultimately 14 lung damage in viral infections (Hu et al., 2019) . In conclusion, our results show that Mito-MES 15 not only has antiviral activity against SARS-CoV-2 but also attenuates inflammatory responses 16 of the infected epithelial cells that drive severe lung injury in COVID-19. 17 immunoassays we showed that there was a reduction of at least 2 orders of magnitude in IL-1β 1 ( Figure 6B ) and IL-6 ( Figure 6C ) in the lungs of the Mito-MES treated SARS-CoV-2 infected 2 relative to the vehicle treated group as early as 3 dpi. The Mito-MES-induced reduction in IL-1β 3 and IL-6 in the lung after 5-7 dpi was confirmed in mice from cohort B infected with the SARS-4

CoV-2 Beta variant ( Figures 6D and 6E ). Mito-MES did not change protein levels of IL-18 5 (Figures S13A and S13C) and did not consistently reduce levels of TNF-α in SARS-CoV-2 6 infected K18-hACE2 mice ( Figure S13B and S13D). To fully characterize the anti-inflammatory 7 activity of Mito-MES at the cell level, we determined the infiltration of different immune cell 8 subtypes into murine lungs by flow cytometry (see gating strategy in Figure S14 ). Mito-MES 9 reduced frequency of CD45+ immune as early as 3 dpi in mice infected with WT SARS-CoV-2 10 ( Figures 6F and 6G ) but not at 5 dpi in mice infected with the Beta variant SARS-CoV-2 (Figure 11 S13E). Mito-MES also reduced frequency of NK cells in murine lungs of mice infected with 12 both the WT and the Beta variant SARS-CoV-2 (Figures S15M-S15O). Mito-MES did not alter 13 frequency of neutrophils (Figure S15A-S15C), macrophages (Figure S15D-S15F), myeloid 14 dendritic cells (DCs) (Figure S15G-S15I), lymphoid DCs (Figure S15J-S15L), T cells ( Figure  15 S15P-S15R) and B cells (Figures S15S and S15T). Immunofluorescence analysis showed that 16

Mito-MES inhibited SARS-CoV-2-associated increase in cleaved caspase 3, a marker of tissue 17 apoptosis and damage, in K18-hACE2 mice infected with Beta variant ( Figure S13F ). The SARS-CoV-2 pandemic necessitates the development of antiviral and anti-inflammatory 4 therapeutics that can be rapidly moved into the clinic. Herein, we demonstrate that Mito-MES 5 has in vitro and in vivo antiviral, antiapoptotic and anti-inflammatory effects on SARS-CoV-2 6 infected epithelial cells. Unlike vaccines, attenuation of detrimental host responses that 7

propagate viral replication may be efficacious even in the setting of mutant strains of SARS-8

CoV-2 to which viral-targeted therapeutics and vaccines may be less effective. Mito-MES had 9 nanomolar antiviral potency against the Beta and Delta SARS-CoV-2 variants as well as MHV. inflammatory effect of Mito-MES was mostly seen against IL-6 and NK cell infiltration in 20 combination of an FDA-approved drug (DMF) with Mito-MES could easily be repurposed and 1 tested in clinical trials to establish a potent novel combined antiviral and anti-inflammatory 2 therapeutic strategy in COVID-19 patients. Our data suggest that Mito-MES may have multiple 3 favorable therapeutic effects in COVID-19 ( Figure S16 ). 4

Although host-targeted antivirals may be toxic, the safety profile of Mito-MES is well 5 established in humans. Mito-MES is currently available as a dietary supplement (10 mg orally 6 daily) and its safety for up to one year in doses as high as 80 mg orally daily has been validated 7 in independent clinical trials for oxidative damage-related diseases such as Parkinson's, hepatitis 8 C, as well as vascular dysfunction (Rossman et al., 2018; Smith and Murphy, 2010) . Thus, Mito-9 MES could easily be repurposed and tested in clinical trials as a small molecule inhibitor of 10 SARS-CoV-2 replication and inflammation-induced pathology for outpatient treatment of mild 11 to moderate acute COVID-19 and for post-exposure prophylaxis against SARS-CoV-2 in high-12 risk exposures. 13

To date, there is no safe, efficacious oral antiviral that is effective against SARS-CoV- 2 14 variants, has anti-inflammatory activity and can also be given long term in humans. Both Mito-15 MES and CoQ10 are nutraceuticals that can be very useful as preexposure prophylaxis in high with SARS-CoV-2 and treated as in Figure 1 . In all panels, data are representative or mean ± 8 SEM of at least two experiments in 3 replicates. Unless otherwise stated, statistical comparison 9 was done between the Ctrl and each shown experimental group by using two-tailed Mann-10 Whitney (***p < 0.001). Summary (means ± SEM) or representative data of at least three experiments in duplicates and 23 triplicates are shown. Each data-point represents one biological sample. Unless otherwise stated, 1 statistical comparison was done between the Ctrl and each shown experimental group by using 2 two-tailed Mann-Whitney (*p < 0.05, **p < 0.01, ***p < 0.001). (100 µg/ml) (1X P/S). HEK293-ACE2 cells were maintained at 37 °C and 5% CO2 in MEM 20 supplemented with 10% (v/v) FBS and hygromycin. Primary mouse lung cells that express 21 human ACE2 were isolated from uninfected K18-hACE2 mice and were maintained at 37 °C and 1 5% CO2 in DMEM supplemented with 10% (v/v) FBS and 1X P/S. Interface cultures. 20 24-well 6.5 mm transwells with 0.4 μm pore polyester membrane inserts were coated with 1 collagen type I dissolved in cell culture grade water at a ratio of 1:10. 100 μl was added to each 2 transwell and allowed to air dry. ABSCs were seeded at 100,000 cells per well directly onto 3 collagen-coated transwells and allowed to grow in the submerged phase of culture for 4-5 days 4

with 500 μl media in the basal chamber and 200 μl media in the apical chamber. ALI cultures 5

were then established and cultured with only 500 μl media in the basal chamber, and cultures 6

were infected with SARS-COV-2 as indicated. Media was changed every other day and cultures 7 were maintained at 37 °C and 5% CO2. allowed free access to irradiated standard rodent diet (Tecklad 2914C) and sterilized water. 5

Littermates of the same sex were randomly assigned to experimental groups and all animal 6 studies included both male and female mice. Mice were anesthetized with a mixture of ketamine/xylazine before each intranasal infection. 9

Additional doses of Mito-MES (n=10) or vehicle (n=10) were given on each day for up to 5-7 10 dpi. The body weight of mice was measured each day. 5-7 dpi animals were humanely 11 euthanized. Whole left lungs were harvested and were processed for histopathology studies, 12 single cell suspension for flow cytometry or protein lysates for immunoassays. 13 14 Cohort C included 20 male K18-hACE2 mice between 4 to 8 weeks of age (16-25 g). Mice were 15 treated via gavage with either vehicle control (normal saline 10% DMSO; Ctrl) (n=10) or Mito-16 MES 20 mg/kg/day (n=10) for at least 20 hrs before the infection (dose 1). As source of Mito-17 MES the actual diet supplement capsule (5 mg Mito-MES per capsule) that is given in humans 18 was utilized. Each capsule was opened and was dissolved in saline 10% DMSO solution before 19

given via gavage to mice. Next day (Day 0), mice were then given a second dose of vehicle or 20 given on day 1, day 2 and day 3 post infection. The body weight of mice was measured each day. 1 On 3 dpi animals were humanely euthanized. Whole left lungs were harvested and were 2 processed for single cell suspension for flow cytometry or viral titration via TCID50. were rinsed with DBPS, cut in 0.5 cm size pieces that were placed in 7 ml 10 screwcap dissociation tubes with ceramic beads (Precellys) filled with 2.5 ml prewarmed (37 °C) 11 digestion medium (2 mg/ml Collagenase, DNAse 0.1 mg/ml, 1% FCS). Tissues 12 were mechanically dissociated at power 3,000 rpm for one 10-second cycle using a Precellys 24 13 homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France), followed by a 30-minute 14 incubation at 37 °C and another 10-second dissociation cycle (3,000 rpm). The homogenate 15 was filtered through a sterile 40 μm nylon filter and cells were then processed for flow 16 cytometry. The entire left lung was processed immediately for viral titer. Lung tissues were 17 placed in 2 ml screwcap dissociation tubes with 60 X 1.4 mm ceramic beads at power 5,000 rpm 18 using a Precellys 24 homogenizer and were mechanically dissociated in 0.5 ml DPBS for 90 sec. 19

Homogenates were centrifuged at 16,000 g for 10 min and supernatants were then cryopreserved 20 at -80 °C for viral titer in Vero-E6 cells. For tissue lysates that were used for protein 21 measurements, 50-100 mg of lung tissue samples were placed in 2 ml 22 screwcap dissociation tubes with ceramic beads (Precellys) and were mechanically dissociated in 23 T-PER tissue protein extraction Reagent at power 5,000 rpm leaving samples to cool on ice 1 between 1-2 repeated 20-second cycles as previously described(Daskou et al., 2021). 

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