Assay of human monoamine oxidase (hMAO) inhibitory activity

The effects of FCS005 on hMAO isoform enzymatic activity were evaluated using a MAO kit (Molecular Probes™) using the experimental protocol previously described by Yáñez, et al.30. Briefly, adequate amounts of recombinant hMAO-A (1.1 mg protein; specific activity: 150 nmol p-tyramine oxidized to p-hydroxyphenylacetaldehyde/min/mg protein) or hMAO-B (7.5 mg protein; specific activity: 22 nmol p-tyramine transformed/min/mg protein) were added to 0.1 mL of sodium phosphate buffer (0.05 M, pH 7.4) to obtain the same reaction velocity and FCS005 in various concentrations. This mixture was incubated for 15 min at 37°C in a flat, black-bottomed 96-well microtest plate in a dark fluorimeter chamber. Then, the reaction was started by adding (final concentrations) 200 µM Amplex™ Red reagent, 1 U/mL horseradish peroxidase, and 1 mM p-tyramine. The production of H2 O ₂ and, consequently, of resorufin was quantified at 37°C in a multi-detection microplate fluorescence reader (FLX800TM, Bio-Tek Instruments, Inc., Winooski, VT, USA) based on the fluorescence generated (emission: 590 nm, excitation: 545 nm) over a 15-min period in which the fluorescence increased linearly.

The test drugs were added to solutions containing only the Amplex™ Red reagent in a sodium phosphate buffer to determinate the capacity of FCS005 or the reference inhibitors to modify the fluorescence generated in the reaction mixture due to non-enzymatic inhibition. In the control experiments, the test drugs were replaced with dilutions of the vehicles. In addition, the specific fluorescence emission was calculated after subtraction of the background activity, which was determined from wells containing all components except the hMAO isoforms that were replaced by a sodium phosphate buffer solution.