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Rationale The recent emergence of a novel coronavirus, SARS-CoV-2, has led to the global pandemic of the severe disease COVID-19 in humans. While efforts to quickly identify effective antiviral therapies have focused largely on repurposing existing drugs, the current standard of care, remdesivir, remains the only authorized antiviral intervention of COVID-19 and provides only modest clinical benefits. Thus, new antivirals targeting SARS-CoV-2 are urgently needed. Methods Artificial intelligence algorithm MediKanren was used to query FDA-approved and late-stage drug compounds for potential interactions with SARS-CoV-2 proteins, coronaviruses, and host cell networks for possible antiviral activity. From this, 157 compounds were further tested in an antiviral screen against live SARS-CoV-2 for reduction in viral growth. Select compounds were further assessed for synergistic activity with remdesivir. Both in vitro and cell free systems identified tocopherol succinate compounds that inhibited the RNA-dependent RNA polymerase (RdRp). Validation of antiviral and synergistic activity was performed in primary human airway epithelial cell cultures against multiple SARS-CoV-2 variants.Results Here we show that water-soluble derivatives of α-tocopherol have potent antiviral activity and synergize with remdesivir as inhibitors of the SARS-CoV-2 (RdRp). Through an artificial-intelligence-driven in silico screen and in vitro viral inhibition assay, we identified D-α-tocopherol polyethylene glycol succinate (TPGS) as an effective antiviral against SARS-CoV-2 and β-coronaviruses more broadly that also displays strong synergy with remdesivir. We subsequently determined that TPGS and other water-soluble derivatives of α- tocopherol inhibit the transcriptional activity of purified SARS-CoV-2 RdRp and identified affinity binding sites for these compounds within a conserved, hydrophobic interface between SARS-CoV- 2 nonstructural protein 7 and nonstructural protein 8 that is functionally implicated in the assembly of the SARS-CoV-2 RdRp. Conclusion In summary, solubilizing modifications to α-tocopherol allow it to interact with the SARS-CoV-2 RdRp, making it an effective antiviral molecule alone and even more so in combination with remdesivir. These findings are significant given that many tocopherol derivatives, including TPGS, are considered safe for humans, orally bioavailable, and dramatically enhance the activity of the only approved antiviral for SARS-CoV-2 infection.
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RATIONALE: SARS-CoV-2 causes COVID-19 disease and infects respiratory epithelial cells, but how it affects ciliated cell function and the mucociliary transport apparatus is unknown. Abnormal mucociliary function could predispose to COVID-19 progression and/or secondary infection. Micro-optical coherence tomography (μOCT) is a novel method to simultaneously visualize and quantify the functional microanatomy of airways. Here, we established a hamster model of COVID-19 and evaluated their tracheas by μOCT. METHODS: Adult golden Syrian hamsters were inoculated intranasally with 3.2 × 105 (high dose, HD, N=4) or 3.2 × 104 (low dose, LD, N=4) plaque-forming units of SARS-CoV-2 (WA/1 strain). Clinical signs were monitored daily, nasal brushes collected intermittently, and hamsters were euthanized seven days (D7) after inoculation. Tracheas were imaged by μOCT, nasal washes and bronchial alveolar lavage fluid (BALF) from right lung lobes were collected for quantitation of viral load by qRT-PCR, and left lungs were inflated with and fixed in 10% neutral buffered formalin for histological analysis. Age-matched hamsters were used as uninfected controls (N=5). RESULTS: SARS-CoV-2 hamsters lost weight through D7 in dose-dependent fashion (-11% in HD vs. -4% in LD, p=0.02) and HD hamsters showed moderate lethargy. Nasal brushes on D4 and nasal washes on D7 contain 105-106 genome copies of virus while BALF on D7 was less than 104 genome copies and intermittently detected in LD. Histology demonstrated patchy and multifocal interstitial pneumonia (type II pneumocyte hyperplasia and mononuclear cell infiltrate), with ∼20% area affected in HD that was more variable in LD. Functional microanatomy of tracheas revealed diminished area of active ciliary beating (control 18 ± 2 vs. LD 7 ± 1%, p=0.0002, control vs. HD 9 ± 1%, p=0.001), reduced ciliary beat frequency (control 10.88 ± 0.70 vs. LD 8.83 ± 0.34 Hz, p=0.01, control vs. HD 8.26 ± 0.33 Hz, p=0.001), and decreased periciliary liquid depth (control 6.41 ± 0.18 vs. HD 5.61± 0.12 μ m, p=0.027). Mucociliary transport rate was diminished (control 0.84 ± 0.19 vs. LD 0.48 ± 0.16 vs. HD 0.37 ± 0.13 mm/min) although not statistically significant. Additional cohorts are in progress. CONCLUSION: SARS-Cov-2 infected hamsters exhibit reduced body weight, high viral load, and histopathological injury through 7 days. SARS-CoV-2 caused functional deficits of the mucociliary transport apparatus, consistent with early findings in COVID-19 patients (see Vijaykumar et al.). Abnormal ciliated cell function is important to SARS-CoV-2 pathogenesis, and may help monitor progression and represent a treatment opportunity for COVID-19.
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Rationale: SARS-CoV-2 is a novel β-coronavirus that causes a severe disease (COVID-19) in humans. Currently, approved antivirals only provide modest benefits to critically ill patients, require intravenous administration, and are in limited supply. Methods: To address the urgent need for new therapeutics, we performed a computational screen using the freely available platform, mediKanren, to identify a list of 120 FDA-approved drugs with purported activity against SARS-CoV-2. To test these compounds in vitro, we developed a mid-throughput assay based on immunohistochemistry compatible with a 96-well microplate to quantifying the antiviral activity of these compounds against virulent SARS-CoV-2 in the Vero E6 cell line. Results: Of the 120 FDA-approved compounds tested, we identified 11 candidates with potent anti-viral activity. Follow up testing showed all but one of these candidates were equally effective against the seasonal coronavirus, OC-43, and five of these candidates were effective at stopping viral propagation when given 24 hours post infection. Using this same assay to test across a range of doses, we determined the dose-response curves for our 11 best compounds. Based on the predicted efficacy of each drug, we then screened for synergy between all pair-wise combinations of our top hits. We found that the combination of remdesivir with another top candidate, DL-alpha-tocopherol polyethylene glycol succinate (TPGS), was nearly ten times more potent than either drug alone. These results were validated by performing curve-shift analysis, which identified another six drugs that combine with remdesivir in an additive fashion. Response-surface analysis revealed that an equipotent mixture of TPGS and remdesivir yielded maximum synergy, but the potency of both drugs can be significantly enhanced by combining one drug with as little as 10% of the dose of the other drug. Finally, these findings were validated in the Calu 3 cell line. Conclusions: In addition to identifying eleven FDA-approved drugs with antiviral activity against SARS-CoV-2, these results are significant given that cheap, widely available drugs can markedly improve the potency of and protect against resistance to the current standard of care for COVID-19.
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RATIONALE: Cigarette smoking is associated with COVID-19 prevalence and severity, but the mechanistic basis for how smoking alters SARS-CoV-2 pathogenesis is unknown. A potential explanation is that smoking could alter expression of angiotensin converting enzyme-2 (ACE2), which functions as the cellular receptor and point of entry. Here we investigated the severity of SARS CoV-2 infection ex vivo and in vitro and using tissue samples and primary ferret and human airway epithelial cells with and without antecedent exposure to cigarette smoke. Methods: ACE2 expression measured by Quantitative PCR (Q-PCR) of ferret lungs exposed to 6 months of cigarette smoke, and findings validated by immunofluorescence (IF). Primary airway cells isolated from airways of ferrets or human non-smokers or COPD subjects were grown until terminally differentiated at air liquid interface. Cells were then exposed to cigarette smoke extract (CSE) or vehicle control, then infected with SARS CoV-2 (3 MOI) or mock control. Viral copy was measured by Q-PCR. Viral infection was quantified by foci forming assay (FFU/mL) using VeroE6 cells. Results: Ferret lungs following 6 months of smoke exposure had increased ACE2 as compared to air controls by Q-PCR (>1.5 fold, P<0.05, N=6), and IF staining. Higher ACE2 expression was also observed in normal ferret airway cells exposed to CSE (>1.5 fold, P<0.05, N=3), normal Human Bronchial Epithelial (HBE) cells exposed to CSE (>2 fold, P<0.001, N=4), and HBE cells from COPD donors as compared to healthy controls (>2 fold, P<0.001, N=4). When ferret airway cells were inoculated with SARS-CoV-2, intracellular viral load of SARS-CoV-2 was increased in CSE exposed cells as compared to vehicle controls (103-104 vehicle Vs CSE 105 106 copy/μL). Viral infection was also increased >2 fold (P<0.01, N=5). Likewise, CSE exposed (105-107 copy/μL, P<0.0001, N=4) and COPD (106-108 copy/μL, P<0.0001, N=4) HBE had increased viral load as compared to controls (103-104 copy/μL, P <0.001, N=4), and 2 to 3-fold increase in viral infection, respectively. TUNEL staining was increased in infected cells, indicating apoptosis. Transcript analysis of HBE cells with and without SARS-CoV-2 by RNASeq to identify differentially expressed genes in CSE exposed cells as compared to controls is in progress. Conclusion: Cigarette smoke and CSE increased ACE2 expression in ferrets, and ferret and human cells respectively. CSE-induction increased viral replication and infection severity, resulting in increased apoptosis. Cigarette smoking likely influences the severity of SARS-CoV-2 infection by altering expression of ACE2, inducing airway cell apoptosis upon infection.