M5e plate reader

The sensitivity of the cell lines to the compounds was examined using the 3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)-based tetrazolium reduction CellTiter 96 Aqueous Non-Radioactive cell proliferation assay (Promega G5430). The compounds were initially tested at 10 and 50 μM final concentrations. Each plate also contained DMSO alone, medium alone, and an inhibitory compound, 6. DENV replicon or Huh-7 cells were plated at a density of 1,500 or 8 × 10³ cells, respectively, per well in 96-well plates containing 100 μl of culture medium overnight. Compounds were added to triplicate wells in culture medium and incubated for an additional 72 h. MTS reagent was then added to each well and incubated at 37°C in a humidified 5% CO₂ atmosphere. The plates were read at various time points at a wavelength of 490 nm using a Molecular Devices M5e plate reader. Mean values of triplicate wells were determined and compared to the mean value for the wells that received DMSO alone. For compounds selected for dose-response experiments, the CC₅₀ was determined by comparing cell viability for eight serial dilutions of the compound and DMSO treated cells using GraphPad Prism software. The CC₅₀ value was defined as the compound concentration resulting in a 50% reduction readout compared with the DMSO.

Compounds were evaluated for antiviral properties using BHK cells containing a DENV-2 viral replicon. 1.5 × 10³ replicon-containing cells per well were plated in white opaque 96-well plates in the absence of antibiotic selection and the next day, compounds dissolved in DMSO were added to triplicate wells in culture medium. The compounds were initially tested at 10 and 50 μM final concentrations and each plate also contained DMSO alone, medium alone, and 6. Three days later, medium was replaced with a 1:1000 dilution of ViVi-Ren Live Cell Substrate (Promega) in DME minus phenol red and 10% FBS. Luminescence was measured with a Molecular Devices M5e plate reader. Mean values of triplicate wells were determined and compared to the mean value for the wells that received DMSO alone. For compounds selected for dose response experiments, the concentration of compound that reduced luciferase activity by 50% was defined as the 50% effective concentration (EC₅₀). The EC₅₀ was determined by comparing luciferase activity for eight serial dilutions of the compound and DMSO treated cells using GraphPad Prism software.