SARS-CoV-2 is a new type of coronavirus capable of rapid transmission and causing severe clinical symptoms; much of which has unknown biological etiology. It has prompted researchers to rapidly mobilize their efforts towards identifying and developing anti-viral therapeutics and vaccines. Discovering and understanding the virus’ pathways of infection, host-protein interactions, and cytopathic effects will greatly aid in the design of new therapeutics to treat COVID-19. While it is known that chloroquine and hydroxychloroquine, extensively explored as clinical agents for COVID-19, have multiple cellular effects including inhibiting autophagy, there are also dose-limiting toxicities in patients that make clearly establishing their potential mechanisms-of-action problematic. Therefore, we evaluated a range of other autophagy modulators to identify an alternative autophagy-based drug repurposing opportunity. In this work, we found that 6 of these compounds blocked the cytopathic effect of SARS-CoV-2 in Vero-E6 cells with EC50 values ranging from 2.0 to 13 μM and selectivity indices ranging from 1.5 to >10-fold. Immunofluorescence staining for LC3B and LysoTracker dye staining assays in several cell lines indicated their potency and efficacy for inhibiting autophagy correlated with the measurements in the SARS-CoV-2 cytopathic effect assay. Our data suggest that autophagy pathways could be targeted to combat SARS-CoV-2 infections and become an important component of drug combination therapies to improve the treatment outcomes for COVID-19.
New anti-viral drug repurposing opportunities are necessary for pre-clinical and clinical evaluation for treating COVID-19. In this work we have identified several autophagy inhibitors that can protect against CPE of SARS-CoV-2 in Vero-E6 cells. ROC-325 and clomipramine display autophagy inhibitor activity that can completely prevent SARS-CoV-2 CPE without any significant inherent cytotoxicity. Hycanthone, an FDA-approved schistosomicide and oxidative metabolite of lucanthone, and mefloquine both showed moderate levels of activity against SARS-CoV-2 CPE, but did exhibit drug-induced cell toxicity at the highest drug concentration tested (up to 30 μM). The autophagy inhibitor verteporfin, a benzoporphyrin derivative used in the clinic as a photosensitizer, did not inhibit CPE of SARS-CoV-2, and was not tested in follow-up autophagy assays. To confirm whether CPE protecting compounds interrupted cellular autophagy and lysosomal function, we examined their effects on autophagy marker LC3B, along with late endosome and lysosomes as visualized with LysoTracker dye. We found that the activities of autophagy inhibition as measured by LC3B spot counts correlated well with inhibition of SARS-CoV-2 measured in the CPE assay for ROC-325, clomipramine, hycanthone, and mefloquine. To our knowledge, this is the first report showing that ROC-325 and hycanthone are efficacious against SARS-CoV-2.
The 72 h SARS-CoV-2 CPE assay measures the phenotypic consequence of viral infection and replication in cells. SARS-CoV-2 can induce cell death after 48 to 72 h of infection, and thus cell viability is a surrogate measure of viral replication in vitro. However, there are limitations to the CPE assay including its dependence on the host response and the fact that it is an indirect measurement of SARS-CoV-2 infection and replication. The phenotypic outcome can also vary depending on culture conditions and viral multiplicity of infection (MOI), number of virions that are added per cell during infection. The potencies of drug protection against virally-induced cell death can be lower than in other assays that directly measure viral load. Nevertheless, this study confirms that SARS-CoV-2 infection in Vero-E6 cells results in cell death similar to other reports, and that CPE can be suppressed by blocking autophagy with small molecule inhibitors to the same extent as positive control remdesivir. Recently, a drug-repurposing screen of FDA-approved compounds, using a similar CPE assay with SARS-CoV-2 in Vero-E6 cells, found clomipramine (IC50 5.93 μM; CC50 >30 μM) and mefloquine (IC50 7.11 μM; CC50 >18.5 μM) to be active with low toxicity. The same study found HCQ to be more active than CQ with an IC50 of 9.21 μM and 42.03 μM, respectively. Mefloquine was also found to be active in another SARS-CoV-2 CPE screen using Caco-2 cells with an IC50 of 14.1 μM. In our study, the SI was calculated using the ratio of the EC50, the half-maximal effective concentration, and the CC50, the half-maximal cytotoxic concentration. Between the CPE and the autophagy assays there was good correspondence in the cytotoxicity measurements by CellTiter-Glo and nuclei counts, respectively. The SI is an important measure for future preclinical development, as it provides insights into the potential clinical safety of a compound at a cellular level. From this work, we show that CQ, HCQ, clomipramine, and ROC-325 were less than 50% cytotoxic at all concentrations, whereas mefloquine and hycanthone were cytotoxic at the highest concentrations with mefloquine being the most cytotoxic.
Evolution has endowed many viruses with the ability to escape autophagic degradation by using the autophagosome membrane for the formation of viral double membrane vesicles (DMVs), although the precise mechanism is still unclear. It has also been reported that some coronavirus proteins such as open reading frame protein 8b (ORF-8b), directly contribute to cell death following viral infection.
Interestingly, ORF-8b causes the induction of autophagosome formation accompanied by damaging effects on lysosomal function and autophagy flux. ORF-8b also forms aggregates in cells that caused ER stress and lysosome malfunction, which could be responsible for reduced clearance of viral particles by autophagic flux. The nonstructural protein 6 (NSP-6) of the infectious bronchitis virus (IBV), an avian coronavirus, significantly increased the number of autophagosomes in host cells. The SARS-CoV accessory protein ORF-3a has three transmembrane domains that insert into the lysosomal membrane causing lysosome function dysregulation and necrotic cell death. Recently, Benvenuto and colleagues analyzed 351 available SARS-CoV-2 gene sequences and discussed that the mutations in NSP-6 may modify the virus’ activity for inducing autophagy, though experimental data was not presented. It appears paradoxical that viral infection inhibits autophagic clearance while autophagy inhibitors, also known to block autophagosome to lysosome fusion, suppress viral infection. Our data, combined with the reported mechanism of action for CQ as an antiviral, suggest that these autophagy inhibitors may interrupt the early steps in the viral life cycle, namely the fusion of the viral endosomes with the lysosome, thereby reducing viral replication and protecting cells from viral induced cell death. The effect of altering endosomal pH among other mechanisms appears to make compounds like HCQ and CQ highly effective against SARS-CoV-2 and other viruses. However, more work is needed to elucidate the exact mechanism of action for these autophagy inhibitors in relation to SARS-CoV-2 and the impact at the different stages of the viral life cycle. Other host targets for viral inhibition include the point of entry with clathrin-mediated endocytosis of the virus, p38 MAPK involved in viral replication, post-translational processing of viral proteins in the Golgi apparatus, and budding of the virus from the infected cell.
ROC-325 was originally developed as an orally available inhibitor of autophagy designed to incorporate the chemical motifs of HCQ and lucanthone, with the goal of both improved autophagic inhibition and consequent single-agent anticancer activity. ROC-325 is a preclinical candidate with low in vitro and in vivo toxicity and strong anti-cancer properties. Our study shows that it may also be a candidate for repositioning as a treatment for COVID-19. Clomipramine, a centrally acting, FDA-approved, tricyclic antidepressant used for the treatment of obsessive-compulsive disorder, panic disorder, major depressive disorder, and chronic pain may also be an interesting preclinical candidate with its existing regulatory status easing a path towards use in the clinic, although the human Cmax does not cover the CPE EC50. Because most of these compound EC50 values were higher than their human plasma concentrations at the clinically efficacious doses, they likely will not be efficacious as single agents for the treatment of COVID-19. Indeed, toxicity with CQ and HCQ has been reported and caution has to be taken with its clinical application because of potential cardiotoxicity. Furthermore, a large observational trial did not find a reduction in death of patients taking HCQ, which suggests that large randomized clinical trials are needed to assess the true benefit to patients with regard to decreased mortality rate and duration of hospitalization. However, the sum of this work indicates that targeting steps of the viral life cycle in cells with molecules similar to CQ, focusing on their anti-autophagic properties, could be a valid drug discovery strategy for combating SARS-CoV-2. The compounds described here also have value as research tools to better understand the interplay between host autophagy pathway and viral live cycle.
Because such compounds target host cells to suppress SARS-CoV-2 CPE, they have potential to be combined with other drugs that directly target viral proteins for treatment. This type of combination therapy has certain advantages including synergistic activity from different mechanisms of action and reducing the development of viral drug resistance due to the involvement of a host cell target. Furthermore, individual drug concentrations can be lowered in combination therapies to prevent the toxicity seen at higher doses when treating with a single drug. Further tests of the drug combination therapy using SARS-CoV-2 animal models will be needed to confirm the therapeutic usage of these compounds.
Reference & source information: https://www.biorxiv.org/
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