2.9. Inhibition assay

To assess the in vitro inhibition potency of the selected compounds, the inhibition constants were estimated from the experimental curves. Inhibition curves were obtained by measuring the enzyme activity as a function of compound concentration: monitoring the substrate fluorescence emission as a function of time, fixing the enzyme concentration at 2 μM, the substrate concentration at 20 μM, and varying the compound concentration from 0 to 125 μM, while maintaining constant the percentage of DMSO. The enzymatic activity was quantitated as the initial slope of the substrate fluorescence emission time curve, and was plotted as a function of compound concentration. Non-linear regression analysis employing a simple inhibition model allowed us to estimate the apparent inhibition constant for both compounds, according to this equation:

where v is the initial slope of the enzymatic activity trace at a (free) compound concentration [I], K_m is the Michaelis-Menten constant for the enzyme-substrate interaction, [S] is the substrate concentration, and K_i ^app is the apparent inhibition constant for the compound. If the inhibitor acts through a purely competitive mechanism, the previous equation can be substituted by the following one:

where K_i is the intrinsic (i.e., substrate concentration-independent) inhibition constant.