Cytotoxicity Evaluation of Inhibitors

To evaluate cytotoxic effects of the studied inhibitors, cell viability assays were done as described (15 (link), 26 (link), 27 (link)) using the CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA). VeroE6 or A549-hACE2 cells, plated the previous day at 10,000 cells per well in 96-well flat-bottom plates (Thermo Fisher Scientific, Roskilde, Denmark), were treated with the specified concentrations of inhibitors, including three replicate wells per concentration and 10 nontreated control wells. Following incubation for 46 to 50 hours at 37°C and 5% CO₂, CellTiter 96 AQueous One Solution Reagent was added at 20 μl per well followed by incubation for 1.5 to 2 hours at 37°C and 5% CO₂ and determination of absorbance at 492 nm using a FLUOstar OPTIMA 96-well plate reader (BMG LABTECH, Offenburg, Germany). For calculation of cell viability in %, absorbance values from individual treated wells were related to the mean absorbance of nontreated control wells. Data points are means of triplicates with SEMs. Sigmoidal concentration-response curves were fitted, and 50% cytotoxic concentration values were calculated using GraphPad Prism 8.0.0 with a bottom constraint of 0 applying the equation Y = Top/[1 + 10^{(log10EC50 − X)*HillSlope}].