Enzyme inhibition assays

To determine the inhibition constants and the mechanism by which compounds BB1, 11 and 38 inhibit δ-secretase, the steady-state kinetic parameters for the hydrolysis of the peptide substrate, Z-AAN-AMC, were determined in the presence or absence of increasing concentrations of inhibitor. In these assays, specified concentrations of the inhibitor were pre-incubated with substrate for 10 min at 37 °C, then 50 nM δ-secretase was added to initiate the reaction, which was quenched after 10 min. The RFU values of the reaction product were converted to micromolar values with an AMC standard curve and the final reaction rates were plotted against substrate concentration and globally fit to equations representative of competitive inhibition (equation 1), noncompetitive inhibition (equation 2), mixed inhibition (equation 3) and uncompetitive inhibition (equation 4) using a nonlinear least fit squares approach by GraFit version 5.0.11.

In the equations, K_ii is the intercept K_i, and K_is is the slope K_i. The mode of inhibition was determined by the best fit of the data to equations 1, 2, 3, 4. Visual inspection of the fits, and a comparison of the standard errors, was used to confirm these assignments.