| Assay Method Information | |
| | Antiviral Assay using MTT |
| Description: | The antiviral activity of compounds was examined by evaluating the cytopathic effect in Vero-E6 cells grown at 37°C in a 5% CO2 atmosphere for 72 h using 96 multi-well plates (50,000 cells/ well) using 3-(4,5-dimethyl-2- thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT; Sigma- Aldrich) method according to the manufacturer's instructions. Cells were challenged with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.01. The virus was added together with the compound(s) under investigation and incubated in DMEM supplemented with 2% FBS and using 0.1% DMSO with no inhibitor as a control. To assess in vitro antiviral activity, serial dilutions of compounds in (0.1% DMSO in 2% FBS in DMEM media) were made in a concentration range of 0.1 uM to 25 uM. Optical densities were measured at 560/620 nm with a Spectramax Plate Reader. Three independent experiments with triplicate measurements were performed. Data were analyzed by a four-parameter curve-fitting from a dose-response curve using GraphPad Prism (version 7.00) to calculate the EC50 (concentration of the compound that inhibited 50% of the infection) based on the MTT method. Concurrently in this experiment, general cellular cytotoxicity in the absence of virus was determined. |
| Affinity data for this assay | |
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