Gly pro amc

DPP-4 inhibition was assessed, as previously described [42 (link),43 (link)], 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.

Total circulating DPP4 was assessed by a specific enzyme-linked immunosorbent assay (ELISA) (R&D Systems, DuoSet, lot P185646), using a 1:1 dilution (1 part serum to 1 part saline) as sample and performed according to the manufacturer's instructions.
DPP4 serum enzymatic activity was determined as the rate of cleavage of 7-amino-4-methylcoumarin (AMC) from the fluorogenic substrate Glycyl-Prolyl-AMC (Gly-Pro-AMC; Sigma, USA) in a 96-well microplate in SpectraMax^® M5 ROM v3.0.22 Molecular Devices spectrofluorometer (USA) at 360 nm excitation and 460 nm emission for 15 min and in duplicate. The rate of liberated fluorescence was calculated per minute (U/min). All enzymatic reactions were performed with 10 µL of serum sample in activity buffer (25 mM HEPES, pH 7.5) in the presence of 20 µM of the substrate in a final volume of 100 µL (30 (link)). To confirm the specificity of DPP4 activity in these assays, a highly selective DPP4 inhibitor (Sitagliptin; Sigma) was incubated with some serum samples for 15 min before proceeding to hydrolysis of the substrate, in triplicate, with Sitagliptin at a final concentration of 1 µM.

DPP4 activity was determined by the cleavage rate of 7-amino-4-methylcoumarin (AMC) from the synthetic substrate H-glycyl-prolyl-AMC (Gly-Pro-AMC; Sigma). Briefly, 5 μl of sample was mixed with 35 μl of assay buffer (25 mM HEPES). After 5-min preincubation at room temperature, the reaction was initiated by the addition of 40 μl of assay buffer containing 0.1 mM substrate Gly-Pro-AMC. After incubation for 20 min, fluorescence was determined using a spectrofluorometer (excitation 380 nm/emission 460 nm). The standard curve of free AMC was generated using 0–50 mM AMC (Sigma). DPP4 activity in plasma was expressed as the amount of cleaved AMC per minute per ml (nmol/min/ml).