In-silico Molecular Docking of Coumarin and Naphthalene Derivatives from Pyrenacantha volubilis with the Pathological Mediators of Rheumatoid Arthritis
Department of Pharmacology, College of Pharmacy, Mother Theresa Post Graduate and Research Institute of Health Sciences, Puducherry - 605006, India.
*Corresponding Author E-mail: anjalicology1820@gmail.com; vimalavathini@gmail.com
Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease which mainly targets synovial membrane during its disease pathogenesis. Available therapeutic drugs for the treatment of RA provide only symptomatic relief and are associated with severe side effects. Herbal plants comprise many active biological compounds that cure the disease with minimal adverse effects. Pyrenacantha volubilis is a climber and member of Icacinaceae family. Gas chromatography- mass spectrometry (GC-MS) analysis of ethanolic extracts of leaves of Pyrenacantha volubilis (EEPV) reveals the presence of 2-isopropyl-5-methylcyclohexyl 3-(1-(4- chlorophenyl)-3-oxobutyl)-coumarin-4-yl carbonate and 1-naphthalenepropanol, alpha-ethyldecahydro-5- (hydroxymethyl)-alpha,5,8A-trimethyl-2-methyl phytoconstitutents. Hence these compounds were docked with various pathological mediators of RA using Autodock 4.2. The docking results unveils that these compounds had better binding energy against inflammatory, oxidative stress and receptor for advanced glycation end products (RAGE) mediators that plays a pivotal role in the progression of RA. However, this study warrants further in- vitro and in-vivo studies to be carried out to establish the anti-inflammatory and anti-arthritic activity of selected phytoconstitutents.
KEYWORDS: Rheumatoid arthritis, Pyrenacantha volubilis, anti-inflammatory and anti-arthritic activity.
Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that primarily affects joints1 and eventually results in end organ damages such as skin, eyes, heart, kidneys, and lungs.2 However its pathogenesis still remains obscure. Unknown antigen triggers synovial tissue3 and activates CD4+ T lymphocytes and stimulates monocytes, macrophages and synovial fibroblasts.4 These factors together generates nuclear factor kappa B (NFκB)5 causes the release of proinflammatory cytokines such as interleukin 1 and 6, tumor necrosis factor alpha (TNF α) and matrix metalloproteinases6,7 culminating in erosion and degradation of cartilage and bone.8
RA also occurs due to the production of autoantibodies such as anti-citrullinated protein auto antibodies (ACPAs) and rheumatoid factor (RF).9,10
Reactive oxygen species (ROS) and reactive nitrogen species (RNS) cause oxidative stress which results in release of proinflammatory cytokines including cyclooxygenase-2 at the inflamed joints.11 Free radicals directly destroy joint cartilage by inhibiting the synthesis of proteoglycans.12 In addition, ROS and RNS can damage the components of the extracellular matrix indirectly through the activation and upregulation of matrix metalloproteinases.13,14 Oxidative stress causes somatic mutations to p53 in fibroblasts like synoviocytes and induces synovial hyperplasia that finally leads to the formation of Pannus.15,16 The receptor for advanced glycation end products (RAGE) is a multi-ligand member of the immunoglobulin superfamily being expressed as a cell surface molecule and interacting with a diverse class of ligands.17,18 It is mainly activated by cross-linked advanced glycation end products (AGEs) that causes stimulation of inflammatory signalling cascade19 and gets expressed in macrophages, neutrophils and T cells and participates in maturation of RA. All these factors together put forth to direct cartilage destruction and bone erosions in RA.
Present-day treatment for RA includes non-steroidal anti-inflammatory drugs (NSAIDS), glucocorticoids and disease modifying anti-rheumatic drugs (DMARDS). All these drugs provide only symptomatic relief from the disease and produces severe side effects on chronic use.20 The way to cure the disease is still uncertain. Plant compounds encompass abundant active biological constituents and possess potent effects which are in the limelight for treating and curing RA. Pyrenacantha volubilis is a climber and member of Icacinaceae family. Literature study reveals that Pyrenacantha volubilis possess richest source of camptothecin and its derivatives that has been isolated from the cotyledons, ripened whole fruit, root followed by seed coat, fruit coat, young and mature leaves.21 In-silico docking studies reveals that camptothecin possess good binding energy with inflammatory mediators such as mitogen activated protein kinase (MAPK), NFκB, TNF α, cyclooxygenases 1 and 222 and in-vitro study results suggest that camptothecin inhibits angiogenesis, synoviocyte proliferation and metalloproteinase expression23. GC-MS analysis of the ethanolic extract of Pyrenacantha volubilis shows the presence of 2- isopropyl-5-methylcyclohexyl 3-(1-(4-chlorophenyl)-3- oxobutyl)-coumarin-4-yl carbonate and 1-naphthalenepropanol, alpha-ethyldecahydro-5- (hydroxymethyl)-alpha,5,8a-trimethyl-2-methyl in higher ratio. Hence in-silico docking of these two compounds was carried out with various pathological mediators of RA using Autodock 4.2.
Collection of plant material and extraction: Pyrenacantha volubilis leaves were collected based on good collection practice during the month of July 2019 from Poothurai region- Puducherry. The collected leaves were authenticated by Dr. N. Ayyappan, Researcher, French Institute of Pondicherry. The collected leaves were cleaned, dried in room temperature. It was periodically checked for the presence of microbial growth. The leaves which are free from brown or yellow spots was selected and then minced to coarse powder. About 50g of coarse dried powder was taken in a Soxhlet apparatus and extracted with 400ml ethanol and placed on a heating mantle, followed by continuous hot percolation process for 3 successive days. The extract obtained was filtered and concentrated under reduced pressure to a semisolid mass.
Phyto-chemical screening of extract:
Preliminary phytochemical screening of EEPV was carried out to determine the phytoconstitutents present in it. Gas chromatography and mass spectrometry (GC-MS) was carried out at sophisticated instrumentation facility (SIF), School of advanced sciences, chemistry division VIT University, Vellore-632 014, Tamilnadu for GC-MS to identify different bioactive compounds present in the extracts.
Two compounds such as 2-isopropyl-5- methylcyclohexyl 3-(1-(4-chlorophenyl)-3-oxobutyl)- coumarin-4-yl carbonate (ligand 1) and 1- naphthalenepropanol, alpha-ethyldecahydro-5- (hydroxymethyl)-alpha, 5, 8a-trimethyl-2-methyl (ligand 2) was selected based on the results obtained from GC- MS analysis. The ligands was prepared using Chemsketch ACD 2019 version 1.2 and converted into protein data bank using open babel GUI software version 2.3.1.
The various target proteins were fetched from RCSB protein data bank. Proteins with method x-ray crystallography and resolution less than 2Ε was selected for docking process. Organism chosen was Homosapiens that are expressed in E. coli. Hetero atoms present in the proteins were removed.
in-silico docking studies using Autodock 4.2: Autodock is
an automated procedure
for predicting the interaction
of
ligands
with bio macromolecule targets.24,25 The target proteins were added with polar atoms
and charges to generate proper
optimization. pdbqt files was created that contains atomic
partial charges and atom types. The active sites for the protein
were determined from PDBsum EMBL-EBI. Grid parameters were generated by embedding the protein in three-dimensional grid (60*60*60). The spacing was fixed in the range of 0.3-0.4Ε
and the gpf files
were developed. Docking
parameters was prepared
by using Lamarckian genetic algorithm (4.2) conformation. The number of evaluations was set to 25 and dpf was also created. Autogrid and autodock was carried
out such that the glg and dpf files were obtained. Analysis
of results was performed based on their ranking order and the interaction between
ligand and protein was predicted
from the root mean square deviation (RMSD)
table.
1. Table 1 shows that the ligand 1 exhibited maximum binding energy when docked against metalloproteinase (MMP)-13, reactive nitric oxide synthase (RNOS), MMP-10 and MAPK which are considered as potential mediators of RA. MMP-8 had shown maximum number of hydrogen bonding with the ligand than other target proteins and also had better inhibition constant.
Table 1: Docking of 2-isopropyl-5-methyl cyclohexyl 3-(1-(4-chlorophenyl)-3-oxobutyl)-coumarin-4-yl carbonate (ligand 1)with various pathological mediators of RA
|
PROTEIN |
CODE |
BINDING ENERGY (kcal mol-1 ) |
INHIBITION CONSTANT (nM)=ki |
H-BONDS |
BONDING |
|
MAPK |
5mtx |
-11.53 |
3.56 |
2 |
5mtx: A: ASP168:OD1: 2: LIG1:O 5mtx: A: LYS53:HZ1: 2: LIG1:O |
|
TNF-α |
5m2j |
-10.23 |
31.89 |
2 |
5m2j: A: GLN47:HE22 5m2j: A: LYS: HZ3 |
|
MMP-3 |
4dpe |
-10.75 |
13.24 |
1 |
4dpe: B: GLN243:HE22:1 |
|
MMP-7 |
2y6c |
-9.64 |
86.34 |
- |
2y6c: A: LEU:181:HN |
|
MMP-8 |
2oy4 |
-9.83 |
62.54 |
4 |
2oy4: A: THR224:2: LIG1:O 2oy4: A: ASN184:2: LIG1:O 2oy4: A: GLU108:2: LIG1:O 2oy4: A: ASN184:2: LIG1:O |
|
MMP-10 |
3v96 |
-11.7 |
2.63 |
1 |
3v96: A: VAL102:O: 2: LIG1:O |
|
MMP-13 |
1xuc |
-12.94 |
0.325 |
1 |
1xuc: B: TYR195:HN |
|
RNOS |
4d1o |
-12.61 |
0.572 |
1 |
4d1o: A: ARG365:HH12: 2: LIG1:O |
|
NADPH |
6sz5 |
-7.94 |
1510 |
- |
2: LIG1:O 2: LIG1:O |
|
RAGE |
3cjj |
-8.27 |
867.56 |
1 |
3cjj: A: GLY153:HN: 2:LIG1:O |
Figure 2: Molecular interaction of ligand 1 with RAGE
Figure 1: Molecular interaction of ligand 1 with NADPH
The ligand also showed better binding energy with RNOS and nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) which induces oxidative stress in RA. Inhibition constant was high for NADPH (figure 1) and RAGE (figure 2).
2.
The
binding energy ligand 2 with various proteins of RA has been tabularized (table 2). It was observed that this ligand exhibited good binding energy
and maximum inhibition constant with
proteins such as MMP-8 (figure 3), RAGE (figure 4), MMP-7, NADPH,MMP-13 and TNF-α. MMP-8 had also shown topmost
number of hydrogen bonding with the ligand 2 than
other target proteins.
Table 2: Docking of 1-naphthalene propanol, alpha-ethyl decahydro-5-(hydroxy methyl)-alpha, 5, 8a-trimethyl-2-methyl (ligand 2) with various mediators of RA
|
PROTEIN |
CODE |
BINDING ENERGY (kcal mol-1) |
INHIBITION CONSTANT (nM)=ki |
H- BONDS |
BONDING |
|
MAPK |
5mtx |
-10.08 |
41.2 |
1 |
5mtx: A:ASP168:HN: 1: LIG1:O 1 |
|
TNF-α |
5m2j |
-8.81 |
347.21 |
1 |
5m2j:D: LY559:HZ11 |
|
MMP-7 |
2y6c |
-8.52 |
571.89 |
1 |
2y6c: A: LEU:181:HN1 |
|
MMP-8 |
2oy4 |
-9.83 |
62540 |
4 |
2oy4: A: THR224:2: LIG:O 2oy4: A: ASN184:2: LIG:O 2oy4: A: GLU108:2: LIG:O 2oy4: A: ASN184:2: LIG:O |
|
MMP-10 |
3v96 |
-10.29 |
28.6 |
2 |
3v96: A: VAL102:HN 3v96: A: SER161: HG |
|
MMP-13 |
1xuc |
-8.96 |
271.4 |
1 |
1xuc: B: TYR195:HN |
|
RNOS |
4d1o |
-10.5 |
20.08 |
2 |
4d1o: A: SER102:O: 1: LIG1:O 4d1o: A: ARG:365: HH12: 1: LIG1:O |
|
NADPH |
6sz5 |
-6.94 |
8180 |
1 |
6sz5: A: MET146:O: 1: LIG1:O |
|
RAGE |
3cjj |
-6.0 |
39820 |
2 |
3cjj: A: GLY56:HN: 1: LIG1:O 3cjj: A: ARG:179HH22: 1: LIG1:O |
Figure 3: Molecular interaction of ligand 2 with MMP-8
Figure 4: Molecular interaction of ligand 2 with RAGE
RA is a multifactorial disease whose pathogenesis still stands as heterogeneous. Conventional treatment for RA is connected with threatening side effects. Available synthetic metalloproteinase inhibitors such as collagen peptidomimetics, non-peptidomimetics had showed unsatisfactory results in treating RA at clinical trials.26 There is a need to develop drugs that cures the disease with lesser side effects. Evidence based interventions depicts that medicinal plants are the most potent source of bioactive compounds, which potentially serve as new life-saving drugs for many diseases.27-29
Since MMPs plays a crucial role in joint destruction, they have been considered as useful biomarkers and therapeutic targets for treating RA. When these enzymes get activated MMP-3, MMP-10 that is found in osteoblast activates pro MMP-1, 8 and 13 that destructs collagen.30 MMP-13 which is present in deep layers of cartilage cause articular damage. Both the ligands had shown better binding energy against these MMPs that can lowers cartilage destruction.
Autoimmune phenomena and connective tissue destruction within the synovium occur due to oxidative stress that is stimulated in the inflammatory joint.31 Reactive nitrogen species and reactive oxygen species represent the mediators and effectors of cartilage damage.32 No mediates signal transduction, mitochondrial function and apoptosis at the site of synovial inflammation.33 Therefore NO act as important mediator for the proliferation of RA. In this study both the ligands revealed maximum binding energy against reactive nitric oxide synthase.ROS are also generated from mitochondria and catalysed by the enzyme complex NADPH oxidase (NOX).34,35 Both the ligands represent lesser binding energy against NADPH. Inhibiting all these factors can lower the level of oxidative stress that prevents cartilage destruction in RA.
Proinflammatory ligands of RAGE such as 100A8/A9 complex (calprotectin), S100A8 and S100A12 are found in RA serum and synovial fluid.36 S100A12 is expressed by infiltrating granulocytes that initiate inflammatory processes in the synovium resulting in chronic arthritis.37 Activated granulocytes binds to RAGE on myelomonocytic cells and endothelium and exhibits pro- inflammatory functions via NFκB activation.38 Henceforth it is necessary to down regulate its expression. Ligand 1 showed fair binding energy but exhibited highest inhibition constant than other mediators of RA, whilst ligand 2 manifested lesser binding energy and better inhibition constant when docked against RAGE.
Pyrenacantha volubilis being a richest source of camptothecin that had been isolated from various parts of the plant exhibited better activity against the pathological mediators of RA. It is a known fact that coumarin possess anti-inflammatory activity and antioxidant activity in both in-vitro methods and in-vivo animal models.39 Naphthalene derivatives had known to possess anti-inflammatory40 and antioxidant property.41 These reasons together with docking studies may account for the in-silico anti-inflammatory and anti- arthritic effects of selected phytoconstitutents.
Thus our in-silico docking studies suggest that the selected ligands such as 2-isopropyl-5-methylcyclohexyl 3-(1-(4-chlorophenyl)-3-oxobutyl)-coumarin-4-yl carbonate and 1-naphthalenepropanol, alpha- ethyldecahydro-5-(hydroxymethyl)-alpha,5,8a-trimethyl- 2-methyl exhibited significant anti-inflammatory and anti-arthritic effect by acting on MAPK, NFκB, TNF-α, metalloproteinases, RNOS, NADPH and RAGE. However meticulous studies using in-vitro and in-vivo models are in need to establish the above said effects.
The authors are thankful to the sophisticated instrumentation facility (SIF), School of advanced sciences, chemistry division VIT University, Vellore 632 014, Tamilnadu for GC-MS analysis.
CONFLICT OF INTEREST:
The authors declare that there are no conflicts of interest.
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Received on 02.09.2020 Modified on 24.09.2020
Accepted on 08.10.2020 © RJPT All right reserved
Research J. Pharm. and Tech. 2021; 14(10):5121-5125.
DOI: 10.52711/0974-360X.2021.00892