Human mao b

The reagents and enzymes (Human MAO-B and Human MAO-A) applied in this experiment were purchased from Sigma-Aldrich. According to the manuals, all components (substrate solution and enzyme solution) are diluted and placed aside for use. The detailed process has been mentioned in our early work³⁴ (link).
To further examine the interaction mode of compound 17d, the type of enzyme inhibition was determined by Michealis–Menten kinetic experiments. The catalytic rates of MAO‐B enzyme were measured at six different concentrations of substrate p‐tyramine (0.05, 0.1, 0.25, 0.5, 1.0, and 1.25 mM) in the absence and in the three different concentrations (33.3, 100, and 300 nM) of compound 17d. The corresponding dose‐response curves and the nonlinear/linear regression analysis were performed using GraphPad Prism version 6 software (Graphpad Software Inc., La Jolla, CA).

MAO-B activity was measured using the MAO assay kit (Cell Biolabs, San Diego, CA, USA) according to the manufacturer’s instructions. Human MAO-B (50 μg/mL; Sigma-Aldrich) was incubated with 1 mM WY dipeptide or 1 mM tryptophan for 30 min. The substrates for MAO-B were added and the amount of hydrogen peroxide in the reaction was measured.

All solvents and fine chemicals were purchased from Sigma–Aldrich (unless specified otherwise) and were used without further purification. For the MAO inhibition studies, recombinant human MAO-A and human MAO-B (5 mg protein/mL) from Sigma–Aldrich were used. Analytical thin layer chromatography was carried out on precoated silica gel 60 F254 aluminum plates purchased from Merck and visualized by UV. Melting points were measured on a Stuart SMP40 automatic melting point apparatus and are uncorrected. For all of the prepared compounds, infrared (IR) spectra were recorded with a Perkin Elmer Spotlight 400 Fourier transform infrared (FTIR) imaging system. GC-MS was performed on a Thermo instrument (ISQELTL). Proton (¹H) and carbon (¹³C) NMR spectra were recorded on JEOL 600 MHz using CDCl₃ as a solvent at the Central Lab Unit, Qatar University and a Bruker Avance III spectrometer (700 MHz) at the King Abdullah Institute for Research & Consulting Studies, King Saud University, Riyadh, Saudi Arabia. The chemical shifts were reported as parts per million (δ) relative to the solvent peak and coupling constants are given in hertz (Hz). The original spectra are provided in the Supporting information. Purity of compounds were confirmed with C, H and S analysis performed on Thermo Scientific FLASH 2000 CHNS/O analyzer.