Identification and Evaluation of Olive Phenolics in the Context of Amine Oxidase Enzyme Inhibition and Depression: In Silico Modelling and In Vitro Validation

Abstract

The Mediterranean diet well known for its beneficial health effects, including mood enhancement, is characterised by the relatively high consumption of extra virgin olive oil (EVOO), which is rich in bioactive phenolic compounds. Over 200 phenolic compounds have been associated with Olea europaea, and of these, only a relatively small fraction have been characterised. Utilising the OliveNet^TM library, phenolic compounds were investigated as potential inhibitors of the epigenetic modifier lysine-specific demethylase 1 (LSD1). Furthermore, the compounds were screened for inhibition of the structurally similar monoamine oxidases (MAOs) which are directly implicated in the pathophysiology of depression. Molecular docking highlighted that olive phenolics interact with the active site of LSD1 and MAOs. Protein-peptide docking was also performed to evaluate the interaction of the histone H3 peptide with LSD1, in the presence of ligands bound to the substrate-binding cavity. To validate the in silico studies, the inhibitory activity of phenolic compounds was compared to the clinically approved inhibitor tranylcypromine. Our findings indicate that olive phenolics inhibit LSD1 and the MAOs in vitro. Using a cell culture model system with corticosteroid-stimulated human BJ fibroblast cells, the results demonstrate the attenuation of dexamethasone- and hydrocortisone-induced MAO activity by phenolic compounds. The findings were further corroborated using human embryonic stem cell (hESC)-derived neurons stimulated with all-trans retinoic acid. Overall, the results indicate the inhibition of flavin adenine dinucleotide (FAD)-dependent amine oxidases by olive phenolics. More generally, our findings further support at least a partial mechanism accounting for the antidepressant effects associated with EVOO and the Mediterranean diet.

Keywords: Olea europaea; hydroxytyrosol; lysine-specific demethylase 1; monoamine oxidase; oleocanthal; oleohydroxypyretol; olive phenolics.

Figure 1

Chemical structures of key compounds from Olea europaea. The structures of the phenolic compounds (A) HT, (B) HTA, (C) HVA, (D) OLP, (E) OLC, and (F) OLE are shown.

Figure 2

Molecular docking results for LSD1. (A) The crystal structure of LSD1 in complex with CoREST (PDB ID: 5YJB) is depicted. The FAD co-factor is labelled and predicted ligand-binding sites are coloured brown. 4-[5-(piperidin-4-ylmethoxy)-2-(p-tolyl)pyridin-3-yl]benzonitrile was used as the positive control inhibitor and was docked to the substrate-binding cavity of LSD1. The binding affinity was predicted to be −9.5 kcal/mol. (B) The phenolic compounds OLP, OLC, HTA, and HT were screened against the substrate-binding cavity and the binding affinities are provided (kcal/mol). Key residues are labelled, with those predicted to form hydrogen bonds and π–π stacking interactions italicised.

Figure 3

Protein–peptide docking results for histone H3 and LSD1. (A) Crystal structure of LSD1-CoREST in complex with the N-terminal residues of the histone H3 peptide. The FAD co-factor is coloured brown. Blind protein–peptide docking was performed to examine the preferential binding site of the histone H3 peptide in the presence of ligands bound to the substrate-binding cavity. The histone H3 peptide was docked to the crystal structure of LSD1-CoREST in the (B) absence and (C) presence of phenolic compounds bound to the substrate-binding cavity. The results are shown for OLC. (Aii–Cii) The protein–peptide interactions were evaluated using PDBePISA. The residues of the LSD1 substrate-binding cavity that were predicted to form salt bridges with R2 and R8 (underlined) of the histone H3 peptide are labelled.

Figure 4

Molecular docking results for MAO-A. (A) The crystal structure of MAO-A (PDB ID: 2Z5X) is depicted. The FAD co-factor is labelled and predicted ligand-binding sites are coloured brown. Harmine was used as the positive control inhibitor and docked to the active site of MAO-A. The binding affinity was predicted to be −8.5 kcal/mol. (B) The phenolic compounds OLP, OLC, HTA, and HT were screened against the active site and the binding affinities are provided (kcal/mol). Key residues are labelled, with those predicted to form hydrogen bonds and π–π stacking interactions italicised.

Figure 5

Molecular docking results for MAO-B. (A) The crystal structure of the MAO-B dimer (PDB ID: 2V5Z) is depicted. The FAD co-factor is labelled and predicted ligand-binding sites are coloured brown. Safinamide was used as the positive control inhibitor and docked to the active site of MAO-B. The binding affinity was predicted to be −9.9 kcal/mol. (B) The phenolic compounds OLP, OLC, HTA, and HT were screened against the active site, and the binding affinities are provided (kcal/mol). Key residues are labelled, with those predicted to form hydrogen bonds and π–π stacking interactions italicised. The results are shown for chain A of MAO-B.

Figure 6

Inhibitory activity of phenolic compounds against LSD1. (A) The results of the control compound TCP and the phenolic compounds HT, HTA, and OLP from the direct enzymatic assays are provided. The data presented denote the mean ± SEM from duplicate (TCP, HTA, and OLP) and triplicate (HT) assays (representative results from n = 3 independent experiments). (B) A separate set of experiments were performed by Reaction Biology Corporation using the positive control inhibitor HCl 489479 and the phenolic compounds OLP, HT, HTA, and OLC. The demethylase activity (%) of LSD1 was measured for the control compound HCl 489479 and the phenolic compounds at concentrations ranging from 0–10 μM and 0–100 μM, respectively.

Figure 7

Potent inhibition of MAO-A by phenolic compounds. (A) Inhibition (%) of MAO-A by the control compound TCP and the phenolic compounds HT, HTA, OLP, and OLC. The data presented denote the mean ± SEM from duplicate (TCP, OLC, OLP, and HT) and triplicate (HTA) assays. (B) Inhibition (%) of MAO-B by the control compound TCP and the phenolic compounds HT, HTA, and OLP. The data presented denote the mean ± SEM from duplicate (TCP) and triplicate (HT, HTA, and OLP) assays. (C) The BJ cells were incubated with normal growth medium (−DEX) or 100 μM DEX (+DEX) for 7 days and treated with 50 μM OLP, 50 μM OLC, or 5 μM TCP for 24 h. The data obtained are represented as the mean ± SEM from duplicate assays. ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001 quantified using a 2-way ANOVA with Tukey’s post-hoc multiple comparisons test.

Figure 8

Inhibition of MAO activity by phenolic compounds within hESC-derived neurons stimulated with ATRA. hESC-derived neuron cultures were incubated with NBM medium or 1 μM ATRA for 24 h before treatment with 50 μM OLP, 50 μM OLC, or 5 μM TCP for a further 24 h. The total protein concentration was determined through a Bradford assay where the cells were then directly assayed. Through fluorometric kinetic detection, MAO-A (A), MAO-B (B), and the total MAO activity (C) were measured. The data obtained are represented as the mean ± SEM from duplicate assays. ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001 quantified using a 2-way ANOVA with Tukey’s post-hoc multiple comparisons test.