Hmao a

The hMAO-A and hMAO-B enzymes were purchased from Sigma-Aldrich (St. Louis, MO, USA). The reaction mixture contained hMAO-A (2.5 µg/mL protein final concentration) or hMAO-B (6.25 µg/mL protein final concentration) enzyme and tested compound in final concentration of 1 and 10 µM in 50 mM potassium phosphate buffer with 20% (v/v) glycerol (pH 7.5). The mixture was pre-incubated at 37 °C for 5 min and subsequently substrate kynuramine was added to the final concentration of 60 µM in the case of hMAO-A and 30 µM in the case of hMAO-B. The final volume of reaction mixture was 0.1 mL. The whole reaction mixture was incubated at 37 °C for 30 min. The reaction was stopped by the addition of 200 µL acetonitrile/methanol mixture (ratio 1:1) and cooling down to 0 °C. The sample was then centrifuged (16.500× g) for 10 min. The deamination product of kynuramine formed during the enzymatic reaction 4-hydroxyquinoline (4-HQ) was determined by HPLC–MS on a 2.1 mm × 50 mm, 1.8 µm Zorbax RRHD Eclipse plus C18 column (Agilent) by using a 6470 Series Triple Quadrupole mass spectrometer (Agilent) (electrospray ionisation – positive ion mode). Three MRM transitions were followed for kynuramine (165.1 = > 30.2, 165.1 = > 118.0, 165.1 = > 136.0) and 4-HQ (146.1 = > 51.1, 146.1 = > 77.0, 146.1 = > 91.0). Eluents: (A) 0.1% formic acid in water; (B) 0.1% formic acid in acetonitrile.

Analyses of the 50% inhibitory concentration (IC₅₀) for in vitro MAO-A and MAO-B enzyme activities were performed as described previously [16 (link)]. In brief, human recombinant MAO-A (hMAO-A) and MAO-B (hMAO-B) (Sigma Aldrich) were diluted in 50 mM phosphate buffer (~ 0.3 μg MAO-A protein/well or ~ 2.5 μg MAO-B protein/well), and the test compound was added in DMSO to a final concentration from 0.1 nM to 10 μM. The amount of hydrogen peroxide (H₂O₂) released after addition of enzyme substrate (p-tyramine for MAO-A or benzylamine for MAO-B) was quantified by measuring the absorbance increase at 570 nm on a microplate reader.

The reagent kits of MAO-Glow™ were obtained from Promega Corporation (Madison, WI, USA). The recombinant human monoamine oxidase-A (hMAO-A) and -B (hMAO-B) derived from recombinant baculovirus were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). From the same company, we purchased chemicals including resazurin, TAX, FLUT, PIRL, DEP, SAF, PARG, and CLORG, and solutions, including Hank’s Balanced Salt Solution (HBSS), N-2-hydroxyethylpiperazine-N-ethanesulfonic acid buffer (HEPES), dimethyl sulfoxide (DMSO), and 0.25% Trypsin-EDTA solution (T/E).
Human PCa cell lines, including LNCaP, DU145, and PC3, derived from metastatic sites, were obtained from American Type Culture Collection (ATCC) (Manassas, VA, USA). Cell-culture flasks and other related equipment were obtained from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Dulbecco’s Phosphate Buffered Saline (PBS), 10,000 U/mL penicillin-G sodium/10 mg/mL streptomycin sulfate (P/S) were obtained from Atlanta Biologicals (Atlanta, GA, USA). Cell-culture media of RPMI1640, EMEM, DMEM/F12K gibco^®, and fetal bovine serum (FBS), and other equipment were purchased from VWR Int. (Radnor, PA, USA).

Ampliflu™ Red (10-Acetyl-3,7-dihydroxyphenoxazine), hMAO-A, hMAO-B, peroxidase from horseradish, tyramine hydrochloride, H₂O₂, clorgiline and selegiline were acquired from Sigma-Aldrich (Steinheim, Germany) and retained under the proposed conditions by supplier. A Biotek Precision XS robotic system (USA) was used for all pipetting operations. Measurements were performed with the use of BioTek-Synergy H1 microplate reader (USA) based upon the fluorescence generated (excitation, 535 nm, emission, 587 nm) over a 30 min period, in which the fluorescence increased linearly.
Enzymatic assay was performed according to recent method pronounced by our research group^{17 ,20–22} (link). Control, blank and all concentrations of obtained compounds were tested in quadruplicate and inhibition percent was calculated with following equation:
$\text{\% Inhibition}=\frac{({\mathrm{FC}}_{\mathrm{t}2\mathrm{ }}-{\text{ FC}}_{\mathrm{t}1})\mathrm{ }-\mathrm{ }({\text{FI}}_{\mathrm{t}2} -\mathrm{ }{\mathrm{FI}}_{\mathrm{t}1})}{{\mathrm{FC}}_{\mathrm{t}2\mathrm{ }}-{\text{ FC}}_{\mathrm{t}1}}\mathrm{ }\times \mathrm{ }100$
FCt₂: Fluorescence of a control well measured at t₂ time, FCt₁: Fluorescence of a control well measured at t₂ time, FIt₂: Fluorescence of an inhibitor well measured at t₂ time, FIt₁: Fluorescence of an inhibitor well measured at t₁ time,
The IC₅₀ values were calculated using a dose-response curve achieved by plotting the percentage inhibition versus the log concentration using GraphPad ‘PRISM’ software (version 5.0). The results were showed as mean ± SD.