CHEMBL5736768

Monoamine Oxidase A (MAO-A) Selectivity Assay: The following reagents were prepared:ReagentsMAO-Glo Assay Kit: Promega, Cat No. V1402MAO-A Enzyme: Promega, Cat No. V1452Clorgyline: Sigma, Cat No. M3778 (50 mg)Assay Plates: 96 well white polystyrene, flat bottom plates, no lid: Fisher, Cat No. DPS-134-050AInhibitors (Test Compounds):10 mM stock diluted to 10, 3, 1, 0.3, 0.1, 0.03, 0.01, 0.003, 0.001, and 0.0003 mM in drug plate, resulting in a concentration of 100, 30, 10, 3, 1, 0.3, 0.1, 0.03, 0.01, and 0.003 uM in the assay.Procedure:1. Dilute MAO substrate 1:50 with MAO A reaction buffer and add 25 μl to each well of a 96 well plate Require 2.8 ml: 56 μl MAO substrate+2744 μl reaction buffer2. Add 0.5 μl test compound serial dilutions, in duplicate3. Add 0.5 μl serial dilutions of Clorgyline (positive control) down column 11 (no duplication)4. Add 25 μl specific MAO enzyme dilution: dilute 1:520 with MAO-A specific reaction buffer (5.3 μl MAO-A+2750.7 μl reaction buffer)5. Add following controls, in duplicate, down column 12: No Luciferin: 25 μl MAO enzyme DMSO: 0.5 μl DMSO+25 μl MAO enzyme Blow Out: 1 μl undiluted MAO-A enzyme+24 μl reaction buffer6. Incubate for 1 hour at room temp on a plate shaker7. Add 50 μl Luciferin detection reagent to each well EXCEPT no luciferin control wells (add 50 μl reaction buffer)8. Incubate for 20 min at room temp on a plate shaker, protected from light9. Measure the luminescence using a plate reader (read mode: luminescence 500 ms/well)Values for the blank (no MAO-enzyme controls) are subtracted from all samples. The DMSO controls are set as 100% activity and samples are calculated as a % of the DMSO control. Data is plotted using Graph pad Prism software, and a non-linear regression line is calculated using a variable slope sigmoidal dose response equation:Y=Bottom+(Top-Bottom)/(1+10{circumflex over ( )}((LogIC50−X)*HillSlope)).Where X is the logarithm of concentration and Y is the response. The IC50 is the concentration of the drug that produces a percentage control fluorescence value midway between the saturation and zero effect plateaus. Two independent assays are usually performed, and the mean IC50 is reported.