Inhibition of rat VAP1 expressed in CHO cells using [14C]-benzylamine as substrate incubated for 30 mins prior to substrate addition measured after 1 hr by scintillation spectrophotometric analysis
Inhibition of rat VAP1 expressed in CHO cells using [14C]-benzylamine as substrate incubated for 30 mins prior to substrate addition measured after 1 hr by scintillation counting analysis
Inhibition of rat VAP-1 expressed in CHO cells using [14C]-benzylamine as substrate preincubated for 30 mins prior to substrate addition measured after 1 hr by scintillation spectrometric analysis
Inhibition Assay: A rat VAP-enzyme 1 (SSAO) activity was measured by a radiochemistry-enzymatic assay using 14C-benzylamine as an artificial substrate. An enzyme suspension prepared from CHO (Chinese Hamster Ovary) cells stably expressing a rat VAP-enzyme 1 (SSAO) was preincubated with the compound of the present invention in a 96-well microplate at room temperature for 30 minutes. Subsequently, the enzyme suspension was incubated with 14C-benzylamine (a final concentration of 1×10−5 mol/L) to a final volume of 50 mL at 37° C. for 1 hour. The enzymatic reaction was stopped by the addition of 2 mol/L (50 μL) of citric acid. The oxidation products were extracted directly in a 200-μL toluene scintillator, and the radioactivity was measured with a scintillation spectrometer.
Inhibition of rat VAP-1 expressed in CHO cells using 14C-benzylamine as substrate preincubated for 20 mins followed by substrate addition measured after 1 hr by scintillation spectrometric analysis
Recombinant SSAO/VAP-1 Inhibition Assay in Rat Fat: Abdominal fat from BALB/c mice, Wistar or Sprague Dawley rats, which are tissues enriched with SSAO/VAP-1-were surgically removed. For every gram of animal abdominal fat tissue, 1 mL of 0.1 M NaPO4 buffer (pH 7.4) was added. Tissues were homogenized using IKA Ultra-Turrax T 10 homogenizer for 3 min, homogenate was centrifuged for 15 min at 3000×g. The middle layer (clear supernatant) was removed without disturbing the top layer (high fat content) or the debris on the bottom of the tube. SSAO/VAP-1 activity was determined by checking the fluorescent signal. Km/Vmax values were determined and the fat homogenate was aliquoted and stored at −80 °C until assays were performed. Assay was performed in a similar fashion as for human SSAO/VAP-1 (Example 5) except, the substrate (benzylamine) concentrations used for mouse fat homogenate and rat fat homogenate were 80 μM and 30 μM respectively: Briefly, in a standard 96 well plate assay 50 μL of purified human SSAO/VAP-1 (0.25 μg/mL) in 0.1 M NaPO4 buffer (pH 7.4) was added into each well. Test compounds were dissolved in DMSO and tested in a Concentration Response Curve (CRC) with 4-9 data points, typically in the micromolar or nanomolar range after incubation with human SSAO/VAP-1 for 30 min at 37 °C. After 30 min incubation, 50 μL of the reaction mixture containing 600 μM benzylamine (Sigma Aldrich), 120 μM Amplex Red (Sigma Aldrich) and 1.5 U/mL horseradish peroxidase (Sigma Aldrich) prepared in 0.1 M NaPO4 buffer (pH 7.4) were added to the corresponding well. The fluorescence unit (RFU) was read every 2.5 min for 30 min at 37 °C. excitation 565 nm and emission 590 (Optima; BMG labtech). The slope of the kinetics for each well was calculated using MARS data analysis software (BMG labtech) and this value was used to deduce the IC50 value (Dotmatics).
Inhibition of rat VAP-1 expressed in CHO cells preincubated for 20 mins followed by [14C]-benzylamine addition measured after 1 hr by scintillation spectrometric analysis
Inhibition of rat VAP-1 expressed in CHO cells using 14C-benzylamine as substrate preincubated for 20 mins followed by substrate addition measured after 1 hr by scintillation spectrometric analysis