Evaluation of novel selective MAO-B inhibitors using immobilized enzymes on magnetic beads

Abstract

Monoamine oxidase A (MAO-A) and B (MAO-B) are critical enzymes involved in regulating neurotransmitters such as dopamine, serotonin, and norepinephrine. Dysregulated MAO activity is associated with neurological disorders, including Parkinson's disease, depression, and cardiovascular diseases, making MAO-A and MAO-B important therapeutic targets. Here, we present a novel assay using MAOs immobilized on magnetic beads (MB) in an immobilized enzyme reactor configuration (MAO-MB) to screen for selective MAO-A and MAO-B inhibitors. The immobilization of both enzymes proved efficient, as no protein was detected in the supernatant after the process by the Bradford assay. Enzyme activity was quantified using HPLC-MS, and kinetic parameters were evaluated, with apparent Michaelis-Menten constants (K_mapp) of 19.36 ± 0.71 µM for MAO-A-MB and 38.85 ± 1.70 µM for MAO-B-MB. The assay was qualified with toloxatone and safinamide as reference inhibitors, yielding IC₅₀ values of 3.42 ± 0.11 µM and 33.38 ± 1.38 nM, respectively. Additionally, a collection of terpenyl-benzohydrazides was screened, identifying PQM-244 (5c) as a selective MAO-B inhibitor (IC₅₀ = 4.17 ± 0.082 µM) with a non-competitive inhibition mechanism (Ki = 2.28 ± 0.38 µM). These findings suggest that 5c (PQM-244) has significant potential to contribute to developing selective MAO-B inhibitors for Parkinson's disease therapy.

Keywords: Immobilized enzymes; Inhibitors screening; MAO inhibitors; Monoamine oxidase.