Antiviral Activity and Cell Toxicity Determination

The evaluation of antiviral activity and cell toxicity was conducted on drugs that showed anti-SARS-CoV-2 potential in the antiviral screen described above. We seeded 2 × 10⁴ Vero cells per well in 96-well plates one day before infection. Serially diluted drugs (100 μM to 0.16 μM) were added to the cells 1 hour prior to infection. The cells were infected with SARS-CoV-2 at an MOI of 2. At 24 h post-infection, cells were fixed with 4%paraformaldehyde and permeabilized with 0.1% Triton X-100 in phosphate-buffered saline. The cells were then stained with anti-dsRNA antibody and Alexa-Fluor 488-conjugated secondary antibody. Their nuclei were counterstained with Hoechst 33342 (Thermo Fisher Scientific, USA, catalog no. H3570). Image acquisition and analysis were performed as previously described [13]. Viral infection was quantified by dividing the number of cells stained with anti-dsRNA antibody by the total number of cells (obtained by counting the nuclei). Infection rate standards were obtained from mock infected cells (0%) and drug-free SARS-CoV-2 infected cells (100%). Infection rates in drug-treated cells were calculated based on these standards. Drug antiviral activity was determined from dose-response curves; half maximal inhibitory concentration (IC₅₀) values were calculated using Prism v8 software (GraphPad Software). To determine the cell toxicity (CC₅₀), similar experiments were performed without addition of the virus; cell viability was measured using MTS solution. CC₅₀ values were calculated using Prism. The selectivity index (SI) was calculated as CC₅₀/IC₅₀.