3.6. MAO Inhibition Assay

Enzyme activity assays were performed according to the procedure reported by Matsumoto et al. [54] with slight modifications. In order to prevent the destructive effect of light on MAO enzymes, the assays were performed in 96-well black plates. Phosphate buffer solution (0.1 M, pH = 7.4) was used for preparation of stock solutions (5 mg/mL) of both human recombinant MAO-A and MAO-B enzymes. Enzyme stock solutions were further diluted using assay buffer to achieve a final concentration of 0.006 mg/mL for MAO-A and 0.015 mg/mL for MAO-B. Kynuramine was dissolved in sterile deionized water to prepare stock solution (25 mM) and then diluted using assay buffer to get final concentration of 40 μM for MAO-A enzyme and 20 μM for MAO-B enzyme. Synthesized compounds and reference drugs, which were moclobemide for MAO-A and selegiline for MAO-B, were diluted to 10⁻³ M and 10⁻⁴ M concentrations (100 μL/well) using 2% DMSO. The addition of the test compound solution was followed by addition of either MAO-A or MAO-B enzyme (50 μL/well) solutions. After 10 min of incubation at 37 °C, kynuramine (50 μL/well) was added and enzyme-substrate reaction was initiated. The plate was incubated for 20 min at 37 °C and then reaction was terminated by addition of 2 N NaOH (75 μL/well). The fluorimetric reads from the top was performed at 310 nm excitation and 380 nm emission wavelengths.