| Assay Method Information | |
| | Biochemical Evaluation |
| Description: | Table 3 and 4: IC50 measurements. Enzyme activities were determined kinetically in 96-well half area plates (Greiner Bio-One) in a final volume of 100 μL for at least 15 min. at 37° C. by measuring the initial velocities of pNA release (405 nm) or AMC release (λex=380 nm, λem=465 nm) from the substrate using an Infinite™ M200 reader (Tecan Benelux). The Magellan software was used to process the data, which was then fitted using a non-linear fit model in GraFit 7. The chromogenic substrate Ala-Pro-paranitroanilide (pNA) (Bachem) was used for DPP4 (25 μM), DPP8 (300 μM) and DPP9 (150 μM) at pH 7.4 (0.05 M HEPES-NaOH buffer with 0.10/Tween-20, 0.1 mg/mL BSA and 150 mM NaCl) and Lys-Ala-pNA (Bachem) was used for DPP2 (1 mM) at pH 5.5 (100 mM NaAc, 10 mM EDTA, 14 μg/mL aprotinin). The fluorogenic substrate Z-Gly-Pro-7-amino-4-methylcoumarine (AMC) (Bachem) was used for FAP (50 μM) at pH 8 (0.05 M Tris-HCl buffer with 1 mg/mL BSA and 140 mM NaCl) and N-succinyl-Gly-Pro-AMC (Bachem) was used for PREP (250 μM) at pH 7.4 (0.1 M K-phosphate, 1 mM EDTA, 1 mM DTT and 1 mg/mL BSA). As a blank, the assay buffer was used instead of the enzyme. Screening measurements were carried out in duplo and IC50 values were determined in triplo with at least eight different inhibitor concentrations as described by Van Goethem et al.None of the compounds inhibited the other DASH proteins DPP4, fibroblast activation protein (FAP), and prolyl oligopeptidase (PREP) at concentrations of 10 mM, and DPP2 at concentrations of 5 μM. |
| Affinity data for this assay | |
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