Selective Inhibition of Human Monoamine Oxidase B by Acacetin 7-Methyl Ether Isolated from Turnera diffusa (Damiana)

Abstract

The investigation of the constituents that were isolated from Turnera diffusa (damiana) for their inhibitory activities against recombinant human monoamine oxidases (MAO-A and MAO-B) in vitro identified acacetin 7-methyl ether as a potent selective inhibitor of MAO-B (IC₅₀ = 198 nM). Acacetin 7-methyl ether (also known as 5-hydroxy-4', 7-dimethoxyflavone) is a naturally occurring flavone that is present in many plants and vegetables. Acacetin 7-methyl ether was four-fold less potent as an inhibitor of MAO-B when compared to acacetin (IC₅₀ = 50 nM). However, acacetin 7-methyl ether was >500-fold selective against MAO-B over MAO-A as compared to only two-fold selectivity shown by acacetin. Even though the IC₅₀ for inhibition of MAO-B by acacetin 7-methyl ether was ~four-fold higher than that of the standard drug deprenyl (i.e., Selegiline^TM or Zelapar^TM, a selective MAO-B inhibitor), acacetin 7-methyl ether's selectivity for MAO-B over MAO-A inhibition was greater than that of deprenyl (>500- vs. 450-fold). The binding of acacetin 7-methyl ether to MAO-B was reversible and time-independent, as revealed by enzyme-inhibitor complex equilibrium dialysis assays. The investigation on the enzyme inhibition-kinetics analysis with varying concentrations of acacetin 7-methyl ether and the substrate (kynuramine) suggested a competitive mechanism of inhibition of MAO-B by acacetin 7-methyl ether with Ki value of 45 nM. The docking scores and binding-free energies of acacetin 7-methyl ether to the X-ray crystal structures of MAO-A and MAO-B confirmed the selectivity of binding of this molecule to MAO-B over MAO-A. In addition, molecular dynamics results also revealed that acacetin 7-methyl ether formed a stable and strong complex with MAO-B. The selective inhibition of MAO-B suggests further investigations on acacetin 7-methyl as a potential new drug lead for the treatment of neurodegenerative disorders, including Parkinson's disease.

Keywords: Turnera diffusa; acacetin; acacetin 7-methyl ether; molecular docking; molecular dynamics; monoamine oxidase-A; monoamine oxidase-B; neurological disorder.

Figure 1

Chemical structure of acacetin, acacetin 7-methyl ether and vetulin.

Figure 2

Concentration-dependent inhibition of recombinant human MAO-B by (A) deprenyl, (B) acacetin, and (C) acacetin 7-methyl ether. Each point shows mean ± SD of three observations.

Figure 3

Kinetics characteristics of inhibition of recombinant human MAO-B by (A) deprenyl, (B) acacetin and (C) acacetin 7-methyl ether. Each point shows the mean value of three observations.

Figure 4

Reversibility assay of recombinant human MAO-B with acacetin (0.5 μM), acacetin 7-methyl ether (Aca 7-MeE) (1.0 and 2.0 μM), deprenyl (0.5 μM), and safinamide (0.2 μM). The remaining activity was expressed as % of activity. Each point shows the mean ± S.D. value of three observations.

Figure 5

Time-dependent inhibition of recombinant human MAO-B by deprenyl (0.100 μM), acacetin 7-methyl ether (Aca-7-MeE) (0.500 μM), and acacetin (0.100 μM). Each point represents mean ± S.D. of triplicate values.

Figure 6

Three-dimensional (3D) protein-ligand interactions of acacetin (C magenta, stick model) and acacetin 7-methyl ether (C cyan, stick model) with the X-ray crystal structures of MAO-A and MAO-B. (A) Acacetin with MAO-A, (B) acacetin 7-methyl ether with MAO-A, (C) acacetin with MAO-B, and (D) acacetin 7-methyl ether with MAO-B. FAD (C green, stick model), some crystallographic waters (O red, H gray, stick model), and the important residues of MAO-A and MAO-B (C gray) are also shown. The black dashed lines represent H-bonding.

Figure 7

Root Mean Square Deviation (RMSD) plot of atom locations vs. simulation time of MAO-B (protein) and acacetin 7-methyl ether (ligand) for the molecular dynamics (MD) simulation of their interaction complex.

Figure 8

The Root Mean Square Fluctuation (RMSF) plot based on Cα atoms of MAO-B. Protein residues that interact with the acacetin 7-methyl ether is marked with green vertical bars.

Figure 9

SID (Simulation Interactions Diagram) plots showing the protein-ligand interactions between the amino acid residues of the hMAO-B binding site and acacetin 7-methyl ether during the MD simulation. (A) The stacked bar charts are categorized as follows: hydrogen bonding (green), hydrophobic interactions (violet), and water bridges (blue) formed. (B) A schematic of detailed ligand atom interactions with the protein residues.