CHEMBL3705252

HDAC Inhbition Assay: Assay was done in 96-well black microplate and total volume of the assay was 100 pt. Mouse liver enzyme (10 mg/ml) was diluted 1:6 with HDAC buffer. Enzyme cocktail was made of 10 μL of diluted enzyme and 30 μL of HDAC buffer. 40 μl of enzyme cocktail followed by 10 μL of test compound (1 μM and 10 μM) or buffer (control) was added to each well. The plate was pre-incubated at 37° C. for 5 minutes. The HDAC reaction was started by adding 50 μl of HDAC substrate Boc-Lys (Ac)-AMC (Bachem AG, Switzerland). The plate was incubated at 37° C. for 30 min. The reaction was stopped by adding 100 μL, of Trypsin stop solution and incubating at 37° C. for 15-30 min. Measuring the fluorescence at excitation wavelength of 360 nm and emission wavelength of 460 nm monitored the release of AMC. Buffer alone and substrate alone served as blank.