Fig 2. In vitro validation of the antiviral activity of clofoctol.

A, Clofoctol inhibits the genomic replication of SARS-CoV-2. Vero-81 and Vero-81-TMPRSS2 cells were infected for 6h at an MOI of 0.25 in the presence of increasing concentrations of clofoctol. Then, total RNA was extracted and viral RNA was quantified by RT-qPCR and normalized by the amount of total RNA. Results are presented as the percentage of the viral load of the control and represent the average of seven independent experiments performed in duplicates. Error bars represent the standard error of the mean (SEM). B, Clofoctol is not cytotoxic in cell culture at concentrations below 40 μM. Vero-81 cells and Calu-3 cells were cultured in the presence of given concentrations of clofoctol. Cell viability was monitored using the MTS-based viability assay after 24 hours of incubation. C, Clofoctol inhibits the production of progeny virions. Vero-81 and Vero-81-TMPRSS2 cells were infected with SARS-CoV-2 at a MOI of 0.25. After 1h, the inoculum was removed and the cells were washed with PBS prior treatment with clofoctol. Cells were then further incubated for 16h. Thereafter, supernatants were collected and the amounts of secreted infectious virus were quantified. The limit of detection was 1.5TCID₅₀/mL. These data represent the average of three independent experiments (N = 3). Experiments were performed in duplicate for each condition. D, Clofoctol inhibits SARS-CoV-2 replication in Calu-3 cells. Calu-3 cells were infected at a MOI of 0.25 in the presence of increasing concentrations of clofoctol for 24h. Then, total cellular RNA was extracted and viral RNA was quantified by RT-qPCR. Results are presented as the percentage of the viral load of the control and represent the average of three independent experiments performed in duplicates. Error bars represent the standard error of the mean (SEM).