DPP4 inhibitory assay and kinetic analysis

DPP4 inhibitory activity was measured using a DPP(IV) inhibitor screening assay kit (Cayman Chemical Company) according to the manufacturer's protocol, with minor modifications. Briefly, the enzyme solution (120 µl) was dissolved in 480 µl DPP assay buffer [20 mM Tris-HCI (pH 8.0), 100 mM NaCl and 1 mM EDTA] and was used as the enzyme solution. The substrate, 5 mM H-Gly-Pro conjugated with aminomethylcoumarin (AMC), was prepared in the same buffer. The assay procedure was performed according to the manufacturer's protocols, and is briefly described as follows: Diluted assay buffer (30 µl) and diluted enzyme solution (10 µl) were added to the 96-well plates containing 10 µl solvent (blank) or solvent-dissolved test compounds (0-75 µM). The reaction was initiated by adding 50 µl diluted substrate solution, the reaction was measured using fluorometric determination (excitation wavelength, 350 nm; emission wavelength, 450 nm) using a plate reader (Tecan Group, Ltd.) every 30 sec for 20 min at 37˚C. Sitagliptin, which was included in the kit, was used as a positive control. Various concentrations (0-75 µM) of the enzyme inhibitor (tested compounds) and substrate were used in the reactions, the initial rate of reaction (v) based on free AMC, and the release rate were calculated using Lineweaver-Burk [1/v vs. 1/(substrate)] or Dixon plots [1/v vs. (inhibitor)].