Topical Perspectives
In silico characterisation of olive phenolic compounds as potential cyclooxygenase modulators. Part 1

https://doi.org/10.1016/j.jmgm.2020.107719Get rights and content

Highlights

  • Oleocanthal from olives is known to inhibit inflammatory cyclooxygenase (COX) enzymes.

  • Molecular docking study of phenolic compounds from the OliveNet™ Library to COX-1 and COX-2 enzymes.

  • 1-oleyltyrosol and ligstroside derivative 2 show strongest binding to COX-1 and COX-2.

  • MM-PBSA analysis identified key residues for potential inhibition of COX enzymes.

  • Part 2 of this study further examines dynamic behaviour of COX and lead olive ligands.

Abstract

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used to reduce pain. These target cyclooxygenase (COX) enzymes which produce inflammatory mediators. Adverse effects associated with the use of traditional NSAIDs have led to a rise in the development of alternative therapies. Derived from Olea Europaea, olive oil is a main component of the Mediterranean diet, containing phenolic compounds that contribute to its antioxidant and anti-inflammatory properties. It has previously been found that oleocanthal, a phenolic compound derived from the olive, had similar effects to ibuprofen, a commonly used NSAID. There is an abundance of olive phenolic compounds that have yet to be investigated for their anti-inflammatory properties. In this study, it was sought to identify potential olive-derived compounds with the ability to inhibit COX enzymes, and study the mechanisms using in silico approaches. Molecular docking was employed to determine the COX inhibitory potential of an olive phenolic compound library. From docking, it was determined that 1-oleyltyrosol (1OL) and ligstroside derivative 2 (LG2) demonstrated the greatest binding affinity to both COX-1 and COX-2. Interactions with these compounds were further examined using molecular dynamics simulations. The residue contributions to binding free energy were computed using Molecular Mechanics-Poisson Boltzmann Surface Area (MM-PBSA) methods, revealing that residues Leu93, Val116, Leu352, and Ala527 in COX-1 and COX-2 were key determinants of potential inhibition. Along with part 2 of this study, this work aims to identify and characterise novel phenolic compounds which may possess COX inhibitory properties.

Introduction

Globally, NSAIDs are among the most widely used therapeutics and are well characterised in their ability to inhibit the COX enzymes COX-1 and COX-2, which are responsible for the production of mediators that drive the inflammatory process, such as prostaglandins. COX-1 and COX-2 enzymes are membrane-bound in the endoplasmic reticulum. They are highly conserved and are structurally homologous, sharing a 60% sequence identity [1]. In general, COX-1 is constitutively expressed and present in nearly all tissues, while COX-2 is inducible during inflammation [2]. COX-1 has a role in the production of prostaglandins involved in various physiological functions, such as the maintenance of renal function and mucosal production in the gastrointestinal tract [[3], [4], [5]]. Conversely, COX-2 expression is induced by cytokines and inflammatory stimuli, facilitating the development of pain, inflammation, and fever, as well as being implicated in some cancers [5].

NSAIDs describe a diverse class of drugs that function by competitively inhibiting COX enzymes, resulting in analgesic and antipyretic effects. NSAIDs are widely used and are a recommended therapy for patients with osteoarthritis and rheumatoid arthritis [4,[6], [7], [8]]. While NSAIDs are commonly used for pain relief, there are important adverse side effects associated with long-term use, including gastrointestinal complications, renal toxicity, exacerbated hypertension, and cardiovascular events [4,9]. Interestingly, low dosages of NSAIDs have been known to confer health benefits. In particular, aspirin has been shown to be beneficial for cardiovascular health when administered in low doses [10]. Thus, there has been a surge in the search for alternative interventions in inflammatory processes, with a particular interest in dietary-derived compounds.

Olive oil, obtained from the fruits of Olea europaea, is a key component of the Mediterranean diet. The Mediterranean diet was first identified in the Seven Countries Study as having potential to increase longevity [11]. More recently, the Prevention with Mediterranean Diet study found that a diet supplemented with extra virgin olive oil (EVOO) or nuts was able to reduce the incidence of major cardiovascular events [12,13]. The health benefits from the consumption of EVOO have been attributed to its high phenolic compound content. Phenolic compounds derived from the olive have been extensively studied, shown to possess potent antioxidant activities, scavenging free radicals and removing reactive oxygen species (ROS) to reduce oxidative stress within cells [14].

There is a complex diversity of olive compounds, with more than 700 different compounds found in the olive [15]. We previously curated OliveNet™, a database of compounds associated with Olea europaea, dividing these into 13 chemical classes [15]. Of particular prominence is the class of olive phenolics, of which 222 compounds were identified, which are further divided into 13 subclasses [15]. These compounds contribute to the stability of the oil during storage and possess a range of biological activities, such as positively affecting plasma lipoproteins and possessing anti-inflammatory properties [16].

In the search for naturally derived compounds with pharmacological properties, oleocanthal has recently become a compound of interest. It has been shown to demonstrate anticancer properties, and has been investigated in neurodegeneration in Alzheimer’s disease [[17], [18], [19], [20], [21]]. In the landmark study by Beauchamp et al. oleocanthal was found to share similar pharmacological activity to the non-steroidal anti-inflammatory drug ibuprofen [22]. Like ibuprofen, oleocanthal was shown to non-selectively inhibit COX-1 and COX-2 [22]. With a daily ingestion of 50 g EVOO containing 200 μg per ml of oleocanthal, of which 60–90% is absorbed, it was noted that this would correspond to an intake of up to 9 mg per day, equivalent to approximately 10% of the ibuprofen dosage for pain relief—a low dosage [22].

The discovery of the COX inhibitory potential of oleocanthal provided a link in the mechanism of health benefits attributed to the Mediterranean diet. It is therefore plausible that low, chronic consumption of naturally occurring COX inhibitors, such as those derived from the olive, may reduce inflammation over time, and hence contribute to the reduced development of chronic inflammatory disease.

We hypothesise that olive derived compounds provide a viable basis for the development of therapeutics in inflammatory processes. Therefore, the specific aims are to identify appropriate candidate compounds as inhibitors of target proteins in inflammation, and to examine in silico mechanisms of inhibition involving protein-ligand complexes implicated in inflammatory pathways through molecular simulations. In this work, we performed docking to screen for candidate olive compounds which bind strongly against COX-1 and COX-2. This was followed by performing molecular dynamics (MD) simulations on the complexes, using the Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) method to obtain more accurate binding free energies and determine specific residues which contribute most to possible inhibition. Thus, this work provides testable hypotheses on potential useful compounds which interact strongly, as well as possible vital residues for such interactions.

Section snippets

Homology modelling

The structure of human COX-1 was generated using homology modelling. The amino acid sequence of COX-1 was retrieved from UniProt (ID: P23219). The template structure with the highest sequence identity was selected using the blastp (protein-protein BLAST) algorithm [23]. The template structure (1CQE) was of ovine COX-1, with a resolution of 3.1 Å and 93% sequence identity. The homology model of COX-1 was built with Modeller 9.16, using the partial sequence (PRO 32—PRO 583), with ten models

Homology model generation and optimization of docking protocol

PDB structure 1CQE (ovine ortholog) was selected as the template for the generation of human COX-1, as it had the highest sequence identity (93%) following the BLAST-protein search. Most residues were conserved between the template and target sequence following alignment. The stereochemical quality of the model was examined using Procheck [25], which demonstrated 92.6% of residues were in the most favoured regions, and 7.4% were within the allowed regions of the Ramachandran plot. This analysis

Conclusion

In this study, computational techniques were employed to examine the potential of olive derived compounds in inhibiting COX-1 and COX-2 enzymes. Following the creation of an olive phenolic library, compounds were screened using docking methods to select candidate compounds for further analysis using MD simulations. Molecular docking against the COX isoenzymes yielded two novel phenolic compounds: 1-oleyltyrosol and ligstroside derivative 2.

MM-PBSA elucidated the energy contribution of

Declaration of competing interest

Although there is no direct conflict of interest with respect to this manuscript, the Epigenomic Medicine Program (TCK) is supported financially by McCord Research (Iowa, USA), which has commercial interests in nutraceuticals, some of which may be beneficial for inflammation. The remaining co-authors have no conflicts of interest.

Acknowledgements

We would like to acknowledge intellectual and financial support by McCord Research (Iowa, USA). JL is supported by an Australian Government Research Training Program Scholarship. This research was undertaken with the assistance of resources and services from Melbourne Bioinformatics, the National Computational Infrastructure (NCI) and the Pawsey Supercomputing Centre, which are supported by the Australian Government. We acknowledge the Partnership for Advanced Computing in Europe (PRACE) for

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