3.4. Kinetic Analyses

For the IC₅₀ evaluation, different concentrations of each compound were tested on NAPRT activity at 0.4 mM PRPP, 0.1 mM NA by using the NADH assay. The IC₅₀ value was determined by plotting the rates versus inhibitor concentration and fitting to the equation: V_i = V₀/(1 + [I]/IC₅₀) using GraphPad Prism 7. V₀ and V_i represent initial rates in the absence and presence of the inhibitor at concentration [I], respectively.

For K_i determination, the NADH assay was used to analyze the effect of different concentrations of each compound on the enzyme’s activity at different concentrations of NA, maintaining PRPP at 0.4 mM. K_i values were calculated from the straight line deriving from the replotting of the slopes of the reciprocal plot against the concentrations of the inhibitor (secondary plot). In particular, the straight line intersects the x-axis at [I] = −Ki [28].