Enzymatic Inhibition Assays for MAO, ChE, and BACE1

Recombinant MAO-A and MAO-B were used in MAO inhibitory activity assays using kynuramine (0.06 mM) and benzylamine (0.3 mM) as substrates, respectively [23 (link)]. The MAO-A and MAO-B reactions were carried out at 25 °C and monitored for 30 min at 316 and 250 nm, respectively, in 0.5 mL mixture of 50 mM sodium phosphate (pH 7.2). Clorgyline and toloxatone were the drug references used for MAO-A, and pargyline and lazabemide were used for MAO-B as reference compounds. The IC₅₀ values were determined by measuring the residual activity at different concentrations of the compounds and by using GraphPad Prism software 5.0. The K_m value of benzylamine against MAO-B was measured to be 0.40 mM [24 (link)]. On the other hand, multitarget analysis, ChE (AChE, BChE) inhibitory activity assays were carried out at 25 °C and monitored for 15 min at 412 nm in a 0.5 mL mixture of 100 mM sodium phosphate (pH 7.5). BACE1 inhibitory activities were tested by the BACE1 activity detection kit. The assay method was described previously [25 (link)]. Recombinant human MAO-A and MAO-B, AChE from Electrophorus electricus, BChE from equine serum, BACE1, and their substrates and reference inhibitors were purchased from Sigma-Aldrich (St. Louis, MO, USA).