4.2. Inhibitory activity (IC50) value determination by dose response curve

Structures of the initial hit compounds from the HTS were examined, cherry-picked and reanalyzed by continuous kinetic assay by hand for confirmation. For those that showed over 50% inhibition at 50 µM concentration in the confirmation assay, IC₅₀ values were measured using the same assay conditions as the primary screen by hand in triplicate. A series of increasing concentrations (0–200 µM final concentration at 2-fold serial dilution) in 100% DMSO were prepared in a 384-well plate. 7 µL of 600 nM (3X) enzyme solution was distributed into wells, and 7 µL of varying concentration of 3X compounds were added and incubated for 5 min. The enzyme reaction was initiated by adding 7 µL of the 150 µM (3X) substrate, and its activity was continuously monitored for at least 10 min. The IC₅₀ values were calculated by fitting the data with the Hill equation (1), using 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 to four independent assays.