This protocol used 12 different concentrations of each compound where the maximum and minimum assay concentrations are 10,000 nM and 0.001 nM respectively. Several replicates at different concentration were done for high potent prediction compound 4b (36 data points) and positive control compound 4a (48 data points). The fitted curve for normalized signal denoted by ∆𝐹/∆𝐹𝑚𝑎𝑥 (calculated using HTRF ratio 665 nm/620 nm) versus log[concentration] was used to determine the 50% inhibitory concentrations (IC50) of the compounds (see next section on