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Assay Method Information

Assay Name:  Inhibition Enzyme Activity Assay
Description:  a. White 384-well plate (Perkin Elmer, Catalog No. 607290/99)b. HEPES buffer: using 1M HEPES buffer (Invitrogen, Catalog No. 15630-080) to prepare 50 ml of 0.5M HEPES buffer by following the steps of taking 25 mL of 1 M HEPES buffer, adding an appropriate amount of ddH2O (re-distilled water), adjusting the pH to 7.8 with NaOH, and finally adding ddH2O to 50 mL.c. Rat plasma: taking blood samples from rat orbit, adding heparin for anticoagulation, centrifuging for 10 minutes at 4000 rpm, taking supernatant plasma as an enzyme source of DPP-IV.d. H-Gly-Pro-AMC (glycine-proline-7-amino-4-methylcoumarin) as the enzyme reaction substrate of DPP-IV, which was synthesized by one of the applicants, was dissolved in DMSO to form 100 mM mother solution.e. 1M MgCl2f. 1.5M NaClg. 10% BASh. DMSO (dimethylsulphoxide)i. ddH2Oj. Test compounds: Omarigliptin as a positive control compound and the compound represented by Formula I of the present application.Following the Sequence Below:1. DPP-IV enzyme reaction buffer was prepared (50 mM HEPES (pH=7.8), 80 mM MgCl2, 150 mM NaCl, 1% BSA), and stored on ice for use:2. The test compounds were diluted with DMSO from 10 mM to 1 mM (100-fold final working concentration), and then diluted gradiently 3 folds in a 96-well plate to obtain 11 concentrations; DMSO was added to the twelfth well as a blank control, and then diluted 25 folds with the enzyme reaction buffer to 4-fold final working concentration for use;3. The DPP-IV enzyme reaction substrate H-Gly-Pro-AMC was thawed and diluted to 160 μM (4-fold working concentration) with the enzyme reaction buffer, and then stored on ice for use:4. The rat plasma was thawed and diluted 100 folds (2-fold working concentration) with the enzyme reaction buffer, and then stored on ice for use;5. 5 μL of the test compounds (4-fold concentration) were added to a 384-well plate, and then 10 μL of the rat plasma (2-fold working concentration) was added, centrifuged and mixed well:6. 5 μL of the enzyme reaction substrate H-Gly-Pro-AMC (4-fold working concentration) was added, centrifuged and mixed well, and then the 384-well plate was sealed with a film:7. The resulting mixture was incubated in an incubator (22-23 C.) for 1 hour:8. The fluorescence signal was determined using FlexStation13 (Molecular devices) microplate reader (excited at 380 nm, and the emission spectrum was determined at 460 nm wavelength);9. IC50 values of the test compounds in inhibiting DPP-IV enzyme activity were determined, i.e., calculating the IC50 values of the compounds using GraFit6 software.
Affinity data for this assay
 

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Last update November 1, 2007
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