INTRODUCTION:

Secreted phospholipases A₂ (sPLA₂) is an enzyme that responsible in the release of arachidonic acid from the cell membrane phospholipids for the synthesis of eicosanoids prostaglandins¹. Mammalian genome encode ten distinct sPLA₂ groups including IB, IIA, IIC, IID, IIE, IIF, III, V, X, XIIA². Among these isoforms, group X (sPLA₂-X) has gain current interest due to its highest ability to hydrolyse phosphatidylcholine, the most abundance phospholipid in the cell membranes. The sPLA₂-X involve in inflammation by driving arachidonic acid metabolism³^-5.

Another enzyme that involve in arachidonic acid metabolism cascade is cyclooxygenase (COX). The COX catalyzes the conversion of arachidonic acid into eicosanoids⁶. This biologically active substance not only involved in normal physiological process, but also in pathological conditions including inflammation⁷. COX exists in two isoforms namely cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2). COX-1 is constitutively expressed which produce cytoprotective prostaglandins in the gastrointestinal tract and prothrombotic molecule thromboxane in blood platelets. In contrast, COX-2 is express in response to pro-inflammatory stimuli such as tumor necrosis factor-α, interleukin-1 and lipopolysaccharide, implying a role for COX-2 in inflammation⁸.

COX-2 activity is associated with several pathophysiological conditions including cardiovascular disease, atherosclerosis, hypertension, obstructive nephrology, angiogenesis and metastasis of cancer cells^9,10. Administration of selective COX-2 drugs has been shown to improve endothelial function in patients with severe coronary artery disease¹¹. Currently, much interest has been attracted to naturally occurring compounds for potential health benefits effect including antioxidant, anti-viral, anti-inflammatory, wound healing, anti-cancer and antibacterial agents¹². The present study investigates the molecular interaction between selected phytochemicals and inflammatory enzymes PLA2 and COX-2. This study provides data on interaction of isolated phytochemicals with amino acids residues of target proteins for potential sPLA₂-X and COX-2 inhibition.

METERIAL AND METHODS:

Phytochemicals:

In this study, ten compounds (Table 1) which were isolated from various Piper species^{13,14,15,16,17,18} were used for Molecular Docking studies. The structure of all the phytochemicals were retrieved from PubChem database.

Table 1: List of ligands and PubChem ID

S. No.	Ligand	Pub Chem ID
1	Eugenol	3314
2	Methyl eugenol	7127
3	Isoeugenol	853433
4	Dillapiole	10231
5	Asarone	636822
6	4-Allyl resorcinol	13898113
7	Piperine	638024
8	Farnesyl acetate	638500
9	trans-Farnesol	445070
10	Cubebin	117443
11	Indomethacin	3715
12	Celecoxib	2662
13	Varespladib	155815

Protein and ligand structure preparation:

The crystal structure of COX-2 (PDB ID: 4COX)¹⁹ and Phospholipase A2 (PDB ID: 4UY1)²⁰ were extracted from the Protein Data Bank (PDB) and used as proteins target for our in silico studies. All bound water, ligands and heteroatoms were removed from the PDB files using Discover Studio Visualizer 4.0 (Accelrys Software Inc., San Diego, CA). AutoDock 4.2 software was used to add structures with polar hydrogen atoms and Kollman partial charges. All phytochemicals with PubChem ID were shown in Table 1. Indomethacin and celecoxib were used as control ligands for COX-2. Varespladib was selected as positive control for sPLA₂-X. The 3D molecular structures of ligands were downloaded from PubChem database and written as .pdb files format using Discover Studio Visualizer 4.0 software.

Docking Procedure:

Docking studies were performed using AutoDock 4.2 software, which allows docking calculation performed under flexible ligand, while the protein is kept rigid. The grid map consist of 90×90×90 grid points with 0.250 Å spacing for COX-2 and 60×60×60 grid points with 0.375 Å spacing for Phospholipase A2 created by Autogrid. Lamarckian genetic algorithm (LGA) was used in AutoDock 4.2 software for predictions of the pose and the score as free binding energy. Other docking parameters settings were applied as default. AutoDock protocol for docking was performed on Cygwin²¹. Successful docking files were analysed for binding energy and inhibition constant using Autodock 4.2. Ligand-protein complex were visualized using Discover Studio Visualizer 4.0. Molecular interaction were analysed by LigPlot+²².

RESULTS:

Binding interaction with COX-2:

The ligand conformations were ranked based on their predicted binding energies using default Autodock 4.2 scoring function. In the present study, cubebin and piperine showed the best docking pose and binding energy. Therefore, both compounds were selected to discover in depth on molecular interaction. Results of the binding energy and inhibition constant for protein-ligand interaction were tabulated in Table 2.

The number of hydrogen bond and hydrophobic interaction between cubebin and COX-2 residues were shown in Table 3. Cubebin showed the most favourable docking pose in the catalytic site with predicted binding energy of −10.68 kcal/mol. The predicted binding energy is comparable to that of reference compound indomethacin and celecoxib. Cubebin formed three hydrogen bond and 14 hydrophobic interactions with COX-2 amino acids as shown in Figure 2. Hydrogen bond interactions involve three different amino acids including His-90, Val-349 and Trp-387.

Table 2: Binding energy and inhibition constant of ligands against COX-2

Ligand	Binding energy (kcal/mol)	Inhibition constant
Eugenol	-6.36	21.63 µM
Methyl eugenol	-6.08	34.70 µM
Isoeugenol	-5.54	87.42 µM
Dillapiole	-6.18	29.45 µM
Asarone	-6.25	26.24 µM
4-allyl rosorcinol	-5.69	67.63 µM
Piperine	-9.33	145.79 nM
Farnesyl acetate	-8.15	1.06 µM
trans-Farnesol	-7.84	1.79 µM
Cubebin	-10.68	14.75 nM
Indomethacin	-10.25	30.76 nM
Celecoxib	-10.37	24.96 nM

The interaction between piperine and COX-2 resulted in binding energy of −9.33 kcal/mol with inhibition constant of 145.91 nM (Table 3). Piperine formed two hydro-gen bond interactions with Arg-120 and Tyr-355 residues similar to indomethacin (Figure 3). In addition, this compound also makes contact with 11 COX-2 amino acids through hydrophobic interactions.

Table 3: Interaction between ligands and COX-2 amino acids

Ligand	Hydrogen bond interaction	Hydrophobic interaction
Indomethacin	Arg-120 and Ser-355	Val-349, Ala-527, Gly-526, Ser-530, Met-522, Leu-384, Trp-387, Tyr-385, Val-523, Phe-518, Ser-353 and Leu-352
Celecoxib	Arg-120, Leu-352, Gln192 and His-90	Ala-527, Phe-518, Met-522, Trp-387, Val-523, Ala-516, Arg-513, Ile-517, Ser-353, Tyr-355, Leu-359, Val-349
Cubebin	His-90, Val-349 and Trp-387	Gln-192, Gly-526, Leu-352, Phe-381, Ala-527, Tyr-385, Met-522, Val-523, Ala-516, Phe-518, Tyr-355, Ser-353, Leu-384 and Ile-517
Piperine	Arg-120 and Tyr-355	Val-523, Ala-527, Met-522, Leu-384, Gly-526, Trp-387, Tyr-385, Leu-531, Val-349, Leu-359 and Val-116

Figure 2: 2D plot of cubebin interactions with the COX-2 active site

Indomethacin showed binding energy slightly lower than cubebin with −10.25 kcal/mol (Table 3). Two hydrogen bond interactions were formed with Ser-355 and Arg-120 residues, similar to that of piperine. In addition, indomethacin also formed a total of 12 hydrophobic interactions with COX-2 amino acids. Selective COX-2 inhibitor, celecoxib, shows binding energy of −10.37 kcal/mol. Celecoxib has the most number of hydrogen bond formed with COX-2 residues as can be seen in Table 3.

Figure 3: 2D plot of piperine interactions with the COX-2 active site

Binding interaction with sPLA₂-X:

Docking results showed that interaction of cubebin and piperine with sPLA₂-X has the lowest binding energy, similar pattern as in COX-2 interaction. Again, both compounds were selected to discover in-depth on molecular interaction. Results of the binding energy and inhibition constant for protein-ligand interaction were tabulated in Table 4. Binding conformation was depicted in Figure 4.

Table 4: Binding energy and inhibition constant of ligands against sPLA₂-X

Ligand	Binding energy (kcal/mol)	Inhibition Constant
Eugenol	-5.68	68.97 µM
Methyl eugenol	-5.42	106.56 µM
Isoeugenol	-5.98	41.54 µM
Dillapiole	-5.73	63.10 µM
Asarone	-5.58	81.91 µM
4-allyl resorcinol	-5.26	139.12 µM
Piperine	-8.38	722.79 nM
Farnesyl acetate	-7.03	7.00 µM
trans-Farnesol	-6.68	12.69 µM
Cubebin	-9.32	147.08 nM
Varespladib	-12.77	437.71 pM

The ligand-dock analysis showed that binding energy of cubebin on sPLA₂-X was −9.32 kcal/mol (Table 4). Cubebin formed a total 3 hydrogen bonds Lys-61, Cys-43 and Asp-47 (Table 5 and Figure 5). There are also hydrophobic interactions were formed as be seen in Table 5. Piperine formed one hydrogen bond with Gly-30 as depicted in Figure 6. Piperine also makes contact with a total of 13 amino acids. These interactions contributed to binding energy of −8.38 kcal/mol.

Table 5: Interaction between ligands and amino acids sPLA₂-X

Ligand	Hydrogen bond interaction	Hydrophobic bond interaction
Varespladib	Cys-43, Asp-47, His-46 and Asp-91	Ala-6, Leu-5, Val-9, Tyr-20, Pro-17, Met-21, Leu-29, Gly-30, Phe-26, Cys-27, Ile-94
Cubebin	Lys-61, Cys-43 and Asp-47	Leu-29, Gly-28, Tyr-20, Val-9, Pro-17, Met-21, Ile-94, Leu-5, Cys-27, His-46 and Phe-26.
Piperine	Gly-30	Leu-5, Leu-98, His-46, Tyr-50, Asp-47, Leu-29, Lys-61, Gly-28, Tyr-20, Ile-94, Cys-43, Val-9 and Pro 17

Figure 5: 2D plot of cubebin interactions with the sPLA₂-X active site

Figure 6: 2D plot of piperine interactions with the sPLA₂-X active site

Our docking analysis showed that varespladib has excellent binding affinity towards sPLA₂-X with 7 hydrogen bonds and 8 hydrophobic interactions. All these interaction resulting lowest binding energy among tested ligands which recorded at −12.77kcal/mol.

DISCUSSION:

Molecular docking is computer-aided technique that elucidate how small molecule (inhibitor, drug candidate or substrate) and the target macromolecule (receptor or enzyme) interact together. This technique is commonly used in developing new drugs candidate and also for the understanding in binding interaction^23,24. The present study explores the binding mode of ten phytochemicals from Piper species for potential anti-inflammatory activity. Docking was performed using AutoDock4 software into the 3D structure of the active site of COX-2 enzyme.

There are distinctive amino acid residues that determine the inhibition selectivity between COX-1 and COX-2. The switch of a Val-523 in COX-2 in place of relatively bulky side chain Ile-523 in COX-1 allowed access to an additional hydrophobic pocket²⁵. His-90 is another important residue in COX-2 side pocket²⁶.

Our present study showed that cubebin formed interaction with His-90, which similar to celecoxib²⁷. Cubebin also formed hydrophobic interaction with Val-523 as found in rofecoxib, a potent selective COX-2 inhibitor²⁸. These interactions indicate the cubebin as a potential selective COX-2 inhibitor. Arg-120 and Tyr-355 are important residues in COX structure which both forms a narrow constriction in the channel towards the bottom of the active site²⁹. Arachidonic acid binds to the active site through interaction between carboxylate group of arachidonic acid and guanidinium group of Arg-120²⁹. Our present study showed that piperine formed hydrogen bond interaction with Arg-120 which may reduce the binding of arachidonic acids and hence, suppressing prostaglandins production.

Previous study has reported that cubebin reduced carageenin-induced paw edema in rats, indicating its anti-inflammatory activity³⁰. The present study enhances the current understanding by providing data on potential anti-inflammatory mechanism of cubebin through binding interaction with COX-2.

The results also support the findings of previous in vitro anti-inflammatory studies. Previous study showed that piperine attenuated lipopolysaccharide-stimulated production of prostaglandin derivatives, PGE₂ and PGD₂, through suppression of COX-2 activity in RAW264.7 macrophages³¹. The sPLA₂-X releases arachidonic acid from membrane glycerophospholipids which later serve as a precursor for protaglandins synthesis by COX. Inhibition of sPLA₂-X expression or activity may reduce inflammation³². The conformation of ligand in the active site is highly dependent on binding interactions including hydrogen and hydrophobic interactions. The present study showed that cubebin has higher number of hydrogen bond interaction as compared to piperine. From the structure point of view, cubebin has a total of 1 hydrogen bond donor and six hydrogen bond acceptors. On the other hands, piperine possesses only 3 hydrogen bond acceptors. The cubebin structural advantage resulted in higher number of hydrogen bond interaction than those of piperine. Cubebin interacts with Gly-28 through formation of hydrogen bond which similar to several potent sPLA₂-X inhibitor including varespladib, 4-benzylbenzamide derivative (AZD2716) and pyrazole derivatives^20,33.

Results from the present study provide potential dual inhibition mechanism of cubebin and piperine on sPLA₂-X and COX-2. Inhibition of both sPLA₂-X and COX-2 could reduce arachidonic acid release and subsequent eicosanoids synthesis, thus provide an important anti-inflammatory effect.

CONCLUSION:

Cubebin predicted to be potential inhibitors against COX-2 and sPLA₂-X which could result in reduced prostaglandins synthesis. This compound also can be used as structural basis in designing new lead compounds in drug discovery.

ACKNOWLEDGMENT:

This work is supported by the Universiti Teknologi Malaysia (UTM) under Research University Grant (Q.J130000.2609.15J20).

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Received on 14.08.2019 Modified on 20.10.2019

Research J. Pharm. and Tech 2020; 13(5): 2181-2186.

DOI: 10.5958/0974-360X.2020.00392.3