2.4. Determination of Enzyme Selectivity

To determine selectivity, the compounds were tested for activity against the related FAD-dependent amine oxidase enzymes LSD1, MAO-A, and MAO-B. LSD1 activity was assessed using a commercially available assay kit (#700120, Cayman Chemical, Ann Arbor, MI, USA), in which the enzyme activity was determined by the horseradish peroxidase-catalyzed oxidation of 10-acetyl-3,7-dihydroxyphenoxazine to the highly fluorescent resorufin in response to H₂O₂ production. Monoamine oxidase activity was determined using recombinant human MAO-A and MAO-B (M7316-1VL & M7441-1VL, Sigma-Aldrich, St. Louis, MO, USA) and a commercially available assay kit (#V1401, MAO-Glo, Promega, Madison, WI, USA), which utilizes the MAO-catalyzed oxidation of a luciferin derivative coupled with esterase activity to produce luminescence. Fluorescent and luminescent readout was obtained using a SpectraMax M5 plate reader (Molecular Devices) equipped with SOFTmax PRO 7.0.3 software. All the compounds were tested at a concentration of 20 µM and compared to tranylcypromine (TCP) and the vehicle control. Values were reported as mean % inhibition ± SD of triplicate assay wells normalized to background activity and compared to the vehicle control.