Investigation of the Anti-Inflammatory Properties of Bioactive Compounds from Olea europaea: In Silico Evaluation of Cyclooxygenase Enzyme Inhibition and Pharmacokinetic Profiling

Abstract

In a landmark study, oleocanthal (OLC), a major phenolic in extra virgin olive oil (EVOO), was found to possess anti-inflammatory activity similar to ibuprofen, involving inhibition of cyclooxygenase (COX) enzymes. EVOO is a rich source of bioactive compounds including fatty acids and phenolics; however, the biological activities of only a small subset of compounds associated with Olea europaea have been explored. Here, the OliveNet^TM library (consisting of over 600 compounds) was utilized to investigate olive-derived compounds as potential modulators of the arachidonic acid pathway. Our first aim was to perform enzymatic assays to evaluate the inhibitory activity of a selection of phenolic compounds and fatty acids against COX isoforms (COX-1 and COX-2) and 15-lipoxygenase (15-LOX). Olive compounds were found to inhibit COX isoforms, with minimal activity against 15-LOX. Subsequent molecular docking indicated that the olive compounds possess strong binding affinities for the active site of COX isoforms, and molecular dynamics (MD) simulations confirmed the stability of binding. Moreover, olive compounds were predicted to have favorable pharmacokinetic properties, including a readiness to cross biological membranes as highlighted by steered MD simulations and umbrella sampling. Importantly, olive compounds including OLC were identified as non-inhibitors of the human ether-à-go-go-related gene (hERG) channel based on patch clamp assays. Overall, this study extends our understanding of the bioactivity of Olea-europaea-derived compounds, many of which are now known to be, at least in part, accountable for the beneficial health effects of the Mediterranean diet.

Keywords: Olea europaea; anti-inflammatory; cyclooxygenase enzymes; oleocanthal; oleohydroxypyretol; olive phenolics.

Figure 1

Chemical structures of bioactive compounds from Olea europaea. This includes the phenolic compounds (A) OLC, (B) OLE, and (C) OLP, as well as the fatty acid (D) LA. OLC is a known inhibitor of the COX-1 and COX-2 enzymes.

Figure 2

Relative inhibition (%) of the COX-1 and COX-2 enzymes by olive-derived compounds. The inhibitory activity of the phenolic compounds OLP, OLC, HT, HTA, OLE, TYR, and HVA, as well as the fatty acids OA, PA, and LA, against COX-1 and COX-2 can be seen. The data presented denotes the mean ± SD from duplicate assays. ** p ≤ 0.01 and *** p ≤ 0.001 quantified using a 2-way ANOVA with Šídák’s multiple comparison test.

Figure 3

Molecular dynamics (MD) simulations of COX-1 and COX-2 homodimers bound with olive-derived compounds. (A) Structure of COX-1 and (B) COX-2 homodimers. Each monomer consists of an epidermal growth factor domain (green), a membrane-binding domain (brown), and a catalytic domain. The heme cofactor is shown in van der Waals representation, and the binding pocket is shown in surface representation in yellow. (C) Root mean square deviation (RMSD) of the protein backbone of COX-1 and (D) COX-2 with respect to its initial structure. (E) Radius of gyration (Rg) of the protein backbone for COX-1 and (F) COX-2. (G) Solvent-accessible surface area of the protein surface for COX-1 and (H) COX-2. Data is shown as an average of three runs, with the ligand-free protein (APO) shown in gray, and the enzymes bound with OLC in purple and OLP in green.

Figure 4

Root mean square fluctuation (RMSF) of the protein backbone for COX-1 and COX-2 bound with olive-derived compounds. (A) RMSF for COX-1 backbone and (B) COX-2 is shown as an average of three runs following equilibration of the trajectory. (C) Difference in RMSF of protein backbone with APO values are subtracted from OLC- and OLP-bound COX-1 and (D) COX-2. The vertical dashed line indicates the residues that form part of chain A and chain B in the dimeric structure.

Figure 5

Dynamics of olive-derived compounds bound to the active site of COX-1 and COX-2. (A) RMSD of OLC and OLP bound to the catalytic site of each chain of COX-1 and (B) COX-2 with respect to its initial structure. (C) Number of hydrogen bonds between olive compounds and COX-1 and (D) COX-2. (E) Number of pairs within 0.35 nm between olive compounds and COX-1 and (F) COX-2. Data is shown as mean ± SD.

Figure 6

Per-residue contributions to binding energy of olive-derived compounds to COX-1 and COX-2. (A) Heatmap of key residues contributing to binding energy for OLC and OLP bound to each chain of COX-1 and COX-2 homodimers. Energy contributions are shown in kcal/mol as an average of three independent binding free energy calculations using MM-PBSA. The asterisk* indicates amino acid difference between COX isoforms. (B) OLC and OLP binding to COX-1 and (C) COX-2, with key residues highlighted in stick representation. Oxygens atoms are red, nitrogen atoms are blue, and hydrogen atoms are white.

Figure 7

Membrane permeability of olive-derived compounds. (A) Mean force profile for OLC and OLP passing through a DOPC membrane along the membrane normal (z). Data is shown as the mean ± SD of ten independent pulling simulations. (B) Permeation of OLP through the DOPC bilayer. (C) Symmetrized potential of mean force (PMF) profile for OLP passing through DOPC membrane. Oxygens atoms are red and hydrogen atoms are white.