Hmao b

MAO inhibition measurements were evaluated following the general procedure previously described⁴⁹ (link). Briefly, test drugs and adequate amounts of recombinant hMAO-A or hMAO-B (Sigma-Aldrich, Spain) required and adjusted to oxidise 165 pmol of p-tyramine/min in the control group, were incubated for 15 min at 37 °C in a flat-black-bottom 96-well microtest plate (BD Biosciences, Franklin Lakes, NJ) placed in the dark fluorimeter chamber. The reaction was started by adding 200 mM Amplex Red reagent (Molecular Probes, Inc., Eugene, OR), 1 U/mL horseradish peroxidase, and 1 mM p-tyramine. Then, the production of resorufin was quantified at 37 °C in a multidetection microplate fluorescence reader (FLX800, Bio-Tek Instruments, Inc., Winooski, VT) based on the fluorescence generated (excitation, 545 nm; emission, 590 nm). The specific fluorescence emission was calculated after subtraction of the background activity, which was determined from wells containing all components except the hMAO isoforms, which were replaced by a sodium phosphate buffer solution.

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.

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.