Evaluation of the oxidation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to toxic pyridinium cations by monoamine oxidase (MAO) enzymes and its use to search for new MAO inhibitors and protective agents

Figures & data

Figure  1.  Bioactivation of MPTP neurotoxin by MAO enzymes with the formation of reactive oxygen species (ROS) (H₂O₂) involved in oxidative damage and pyridinium cations (MPDP⁺ and MPP⁺), which are directly-acting toxicants producing neurotoxicity. Inhibition of MAO enzymes is a target for neuroprotection.

Figure  2. . HPLC chromatogram of the enzymatic oxidation of MPTP neurotoxin by human MAO-B (A) and MAO-A (B). MPDP⁺ is determined at 355 nm, MPP⁺ at 280 nm, and MPTP at 254 nm.

Figure  3.  (A) Concentration of pyridinium cations (MPDP⁺ and MPP⁺) (μM) determined by HPLC and produced from MPTP oxidation in presence of increasing concentrations of recombinant human MAO-B (MPDP⁺, ▪;MPP⁺, o) and MAO-A (MPDP⁺, •; MPP⁺ Δ). Incubations (37°C, 40 min) and MPTP (200 μM). (B) Michaelis-Menten kinetics for the formation of pyridinium cations determined by HPLC, and corresponding to the oxidation of MPTP catalyzed by MAO-B (MPDP⁺, ▪;MPP⁺, Δ) and MAO-A (MPDP⁺, ▴; MPP⁺, x). Incubations (37°C, 40 min) contained MAO enzymes (0.05 mg/mL protein fraction).

Figure  4. . Activity ratio of MAO-B and -A isozymes under the same conditions determined by using the products from two different substrates: kynuramine (formation of 4-hydroxyquinoline) following a previous method (30), and MPTP (formation of MPDP⁺) analyzed by HPLC, as described here. Separate incubations (37°C, 40 min) with enzymes (0.05 mg/mL protein fraction) and 150 μM kynuramine or 150 μM MPTP were carried out.

Figure  5. . Inhibitors of the oxidation of MPTP neurotoxin to toxic pyridinium cations catalyzed by MAO-A and -B.

Figure  6.  Inhibition of the human MAO-B (0.05 mg/mL protein fracion) catalyzed-oxidation of MPTP (300 µM) to give pyridinium cations in the presence of R-deprenyl (A) or the β-carboline norharman (B) (MPDP⁺, ▴;MPP⁺, •). Incubations carried out at 37°C for 40 min.

Figure  7.  Reduction of pyridinium cation (MPDP+) produced in the oxidation of MPTP catalyzed by MAO-B in the presence of several protective agents: control (absence of inhibitor), norharman (5 μM), 5-nitroindazole (5 μM), menadione (5 μM) and deprenyl (1 μM). Incubations (37°C, 40 min) were carried out with MPTP (300 μM) and MAO-B (0.05 mg/mL protein fraction) in the presence or absence of agent.

Figure  8.  Inhibition of the human MAO-A (0.05 mg/mL recombinant protein) catalyzed oxidation of MPTP neurotoxin (300 μM) to give pyridinium cations in the presence of clorgyline (A) or the β-carboline harman (B) (MPDP+, ▪; MPP+, ▴). Incubations carried out at 37°C for 40 min.

Figure  9.  Inhibition of the human MAO-B-catalyzed oxidation of MPTP to toxic pyridinium cations in the presence of cigarette smoke extract (A): MPDP+ + MPP+ (□), MPDP+ (⧫), MPP+ (•), and the inhibition (%) in the presence of the β-carboline norharman isolated from cigarette smoke (B) or coffee (C) as reported previously34,35. Incubations carried out at 37°C for 40 min with MPTP (300 μM) and MAO-B (0.05 mg/mL).