Polarstar optima microplate reader

For initial inhibitor test, 10 mM stock solutions of the 71 compounds (Life Chemicals and synthesized) were prepared in 100% DMSO. 7 μL of 3× ZIKV NS2B-NS3^pro (final 100 nM) was distributed to a 384-well plate, and 7 μL of 3× compounds were added followed by 10 min incubation at room temperature. Enzyme reaction was initiated by adding 7 μL of 3× substrate (final 50 μM), and fluorescence intensity was continuously monitored for at least 6 min with a POLARstar OPTIMA microplate reader (BMG LABTECH). All compounds were tested in triplicate.
IC₅₀ values were measured in the same concentration of enzyme and substrate as in the initial screening with a series of compound concentrations (0 to 200 μM). The enzyme reaction was initiated by adding fluorogenic substrate, and its activity was continuously monitored for at least 10 minutes. The IC₅₀ values were calculated by fitting with the three parameter Hill equation,
$y=V_{\mathit{\text{max}}}\left(\frac{x^n}{{{\mathit{\text{IC}}}_{50}}^n+x^n}\right)$ , with Sigmaplot v12.0 where y is percent inhibition, x is inhibitor concentration, n is the slope of the concentration–response curve (Hill slope), and V_max is maximal inhibition from three independent assays.