4.3. In Vitro DPP-4, DPP-8 and DPP-9 Inhibition Assay

DPP-4 inhibition was assessed, as previously described [42,43], based on a continuous fluorometric assay method in which the substrate Gly-Pro-AMC (Gly-Pro-7-amido-4-methylcoumarin hydrobromide) was split by DPP-4 to release fluorescent aminomethyl coumarin (AMC). The liberated AMC was continuously monitored using an excitation wavelength of 360 nm and an emission wavelength of 460 nm using an EnVision microplate reader (PerkinElmer, Waltham, MA, USA). The reaction mixture consisted of different concentrations of the synthesized hybrids 10a–j, 20 µU/µL of recombinant human DPP-4 (Sigma Aldrich, St. Louis, MO, USA), 10 µM Gly-Pro-AMC (Sigma Aldrich, St. Louis, MO, USA) and assay buffer (150 mM NaCl, 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 0.12 mg/ml bovine serum albumin (BSA)) and pH of 7.5. The assay was carried out in quadruplicate. IC₅₀ was calculated using GraphPad Prism 8 software (San Diego, CA, USA). Similar to the DPP-4 assay, DPP-8 and DPP-9 inhibition assay was performed and the pH of the assay buffer was 8.