2.3. Determination of IC50 values of the inhibitory compounds

ZIKV, WNV, and DENV NS2B-NS3pro constructs (20 nM) were pre-incubated for 30 min at 20 °C with individual compounds at the indicated concentrations in 0.1 ml 20 mM Tris-HCl buffer, pH 8.0, containing 20% glycerol and 0.005% Brij 35. Pyr-RTKR-AMC substrate (20 μM) was added in 0.1 ml of the same buffer, and the remainder of the assay procedure was as described above. IC₅₀ values were calculated as the compound concentrations giving half-maximal inhibition of NS2B-NS3pro activity. GraphPad Prism was used as the curve fitting software.