4.2. In Vitro MAO Inhibition Assay

The in vitro enzyme-inhibition assay was designed to measure the effect of primaquine and NPC1161 enantiomers on catalytic activity of rhMAO-A and -B. The kynuramine oxidative deamination assay was performed in white flat-bottom 96-well plates as previously described, with minor modifications [63,70]. A fixed concentration of kynuramine substrate and varying concentrations of the test compounds were used to determine the IC₅₀ values. Kynuramine concentrations for MAO-A and -B assays were 80 μM and 50 μM, respectively. The concentrations of PQ and NPC1161 enantiomers varied from 0.1 μM to 200 μM, for the rhMAO-A and -B enzyme activity inhibition. The test compounds were dissolved in DMSO, diluted in the buffer solution just before the assay, and pre-incubated with the enzyme (MAO-A (5 μg/mL) or -B (12.5 μg/mL)) for 10 min at 37 °C. The final concentration of DMSO in the enzyme–assay reaction mixtures (total volume 200 μL) did not exceed 1%. The enzymatic reactions were initiated by the addition of the substrate (kynuramine 80 and 50 μM, for MAO-A and -B, respectively) and incubated for 20 min at 37 °C. The enzyme reactions were terminated by the addition of 78 μL of 2N NaOH to each well. The formation of 4-hydroxyquinoline (the enzyme reaction end product) was recorded fluorometrically on a SpectraMax M5 fluorescence plate reader (Molecular Devices, Sunnyvale, CA, USA) with 320 nm excitation and 380 nm emission wavelengths, using the Soft MaxPro-6 program. The inhibition of enzyme activity was calculated in terms of product formation as a percentage of corresponding controls (enzyme–substrate reaction without inhibitors). Assay controls, to define the interference of the test compounds with the fluorescence measurements, were set up simultaneously, and the enzyme or substrate was added after stopping the reaction.