The SARS-CoV-2 cytopathic effect is blocked with autophagy modulators

Abstract

SARS-CoV-02 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.

Keywords: SARS-CoV-2; autophagy; coronavirus; cytopathic effect; small molecule inhibitors.

Fig. 1.

Workflow overview for CPE assay. Activities and incubation times are shown in a workflow.

Fig. 2.

CPE activity and Toxicity for ROC-325, clomipramine, hycanthone, and verteporfin. (A) ROC-325, (B) clomipramine, (C) hycanthone, and (D) verteporfin CPE activity (blue curve, left graph) and Toxicity (green curve, right graph) 10 point, 1:2 dilution concentration-response curves starting at 30.0 μM down to 2.29 nM, along with their structure. ROC-325 started at 15 μM down to 1.14 nM. Red dashed line indicates EC₅₀ or CC₅₀ for CPE and Toxicity assays, respectively. Duplicate values shown for each concentration. Curves generated using non-linear regression.

Fig. 3.

CPE activity and Toxicity for chloroquine, hydroxychloroquine, and mefloquine. (A) Chloroquine, (B) hydroxychloroquine, (C) mefloquine, and (D) remdesivir CPE activity (blue curve, left graph) and Toxicity (green curve, right graph) 10 point, 1:2 dilution concentration-response curves starting at 30.0 μM down to 2.29 nM, along with theirs structure. Dashed line indicates EC₅₀ or CC₅₀ for CPE and Toxicity assays, respectively. Duplicate values shown for each concentration. Curves generated using non-linear regression.

Fig. 4.

Autophagy inhibition assay using LC3B immunostaining in Vero-E6 cells. (A) Image montage of DMSO, CQ, HCQ, clomipramine, mefloquine, ROC-325, and hycanthone stained with Hoechst 33342 (cyan) and LC3B (magenta). CQ and HCQ images taken from wells in positive control column 2. Scale bar, 25 μm. (B) 8 point 1:3 dilution concentration-response curves starting at 50 μM down to 0.023 μM for compounds in A Blue curve indicates Efficacy, red curve indicates Cell Counts. Efficacy data normalized to DMSO (0%) and CQ (100%). Cell count data normalized to DMSO (100%) and 0 (no cells 0%). Error bars indicate SD. N = 3 intra-plate replicates. Curves generated using non-linear regression.

Fig 5.

Autophagy inhibition assay using LysoTracker Deep Red staining in Vero-E6 cells. (A) Image montage of DMSO, CQ, HCQ, clomipramine, mefloquine, ROC-325, and hycanthone stained with Hoechst 33342 (cyan), HCS Cell Mask Green (yellow), and LysoTracker Deep Red (magenta). CQ and HCQ images taken from wells in positive control column 2. Scale bar, 25 μm. (B) 8 point 1:3 dilution concentration-response curves starting at 50 μM down to 0.023 μM for compounds in A. Blue curve indicates Efficacy, red curve indicates Cell Counts. Efficacy data normalized to DMSO (0%) and CQ (100%). Cell count data normalized to DMSO (100%) and 0 (no cells 0%). Error bars indicate SD. N = 3 intra-plate replicates. Curves generated using non-linear regression.

Fig. 6.

Illustration of autophagy inhibitors and their blockade of viral infection. (A) Healthy cells have normal autophagic flux and the endocytic pathway is functional. (B) Autophagy inhibitor treatment in healthy cells causes a blockade of normal fusion processes and a buildup of endosomes and autophagosomes. (C) In healthy cells, viral infection through endocytosis leads to the release of viral RNA after endosome lysosome fusion. Similarly, autophagy of viral particles may result in formation of viral autophagosomes but lysosome fusion would be blocked by the virus (orange X). Dotted arrow indicates a possible, but unverified event of viral RNA release from autophagosomes. (D) Autophagy inhibitors can block steps (red Xs) within the viral life cycle including at the early steps of endocytosis, the fusion of endosomes with the lysosome, to prevent the release of viral RNA and subsequent cell death.