Volume 4, Issue 10, October – 2019 International Journal of Innovative Science and Research Technology

ISSN No:-2456-2165

IL-6: A Potent Target for the Treatment of

Rheumatoid Arthritis by In-silico Approach
Sneha Bharti 1, Prachi Agnihotri 1, Archana Tiwari 2, Sagarika Biswas 1*
1*
Department of Genomics & Molecular Medicine, CSIR- Institute of Genomics & Integrative Biology, Mall Road, Delhi-110007
2
School of biotechnology, Rajiv Gandhi Technological University, Bhopal- 462033, Madhya Pradesh, India

Abstract:- Rheumatoid Arthritis (RA) is an and less popular. Therefore, there is a necessity of
autoimmune disease affecting life globally. It is alternative biological agent to overcome the related health
characterized by synovial inflammation and joint issues of these drugs. The early diagnosis and treatment of
destruction, eventually inducing severe disability and RA is still giving challenges to the medical field [7].
joint deformity. Many of the research have been done
towards the treatment of RA. Since it is inflammatory The plant comprises of various bioactive compounds
disease, verities of inflammatory cytokines are involved
in RA. IL-6 and TNF-α are most prominently causing that can be considered as a therapeutic agent for RA [8]. In
RA. In this study we are looking for an inhibitory this study, we are approaching towards the bioactive
compound of natural origin which can be used to target compounds of Aloevera and Murraya koenigii (commonly
the above two inflammatory factors. Two plants namely known as curry leaves) as these plants possess different
Aloevera and Murraya koenigii were selected to find out medicinal properties such as antioxidant, anti-microbial and
the activity of their active compounds to inhibit the anti-inflammatory that accelerates wound healing and burn,
target interleukin 6 (IL-6). The protein-ligand docking immunity, itching, blood disorders, improves digestion and
plays an important role in structural based drug skin eruptions [9, 10, 11]. IL-6 is the key cytokine in the
designing. Various molecular docking tools namely pathogenesis of RA, its role in synovitis and joint damage
SwissDock, PatchDock, Argus Lab 4.0.1, UCSF makes it the potential target. The bioavailability of the
Chimera-Autodock Vina and Hex 8.0 were used to find bioactive compounds of these two plants used to target the
the best scoring function of protein ligand interaction. IL-6 by molecular docking and was checked with
Molecular docking followed by ADME/Tox to limit the ADME/Tox tool.
study of molecular docking was used on the basis of
Lipinski rule of five. The results of these tools showed The various pro-inflammatory cytokines entailed in
that out of 10 active components of both the plants only articular cartilage destruction during disease. The present
two have potential to be an inhibitor of the IL-6. The research targeted the IL-6, to obtain a compound of the
aloe emodin of Aloevera and phebalosin of Murraya natural origin which can be further used as predictive drug
koenigii have spontaneous binding with IL-6 protein. treatment. To achieve this, different steps and
These active compounds have potential which can be bioinformatics tools were used for the validation and
used as drug to treat the inflammatory condition of RA. screening of the compounds on the basis of their properties.

Keywords:- Autoimmune Disease, Molecular Docking, TNF II. MATERIAL AND METHODS
-α, SwissADME, Lipinski Rule.
A. Protein Preparation
I. INTRODUCTION The crystallographic structure of IL-6 bearing PDB
(Protein Data Bank) ID: 1ALU retrieved from the RCSB
RA is a chronic inflammatory autoimmune disease (Research Collaboratory for Structural Bioinformatics)
resulting into mortality, severe disability and bones protein databank (www.rcsb.org). The ribbon model of IL-6
deformity globally [1, 2]. It is characterized by the pannus shown in figure 1 and the stereochemistry of the protein
formation in synovial membrane, deformity in affected structure was analysed by the PROCHECK tool
joints, leading to dysfunction of the interior milieu of (http://services.mbi.ucla.edu/PROCHECK/) for assessing
joints [3]. Women are largely affected then men that the quality on the basis of Ramachandran Plot [12].
degrade the quality of health and increase the
difficulties because it increases vividly around the time B. Ligand Preparation
The bioactive compound structures of Aloevera are
of menopause [4]. Since 1998, lot of advancement has aloin, campesterol, β-sitosterol, lupeol and aloe emodin and
been achieved for the treatment of RA such as Disease Murraya koenigii are quercitin, catechin, epicatechin,
Modifying Anti-Rheumatic Drugs (DMARDS) [5], NSAIDS phebalosin and mukonicine. The structures were taken
and steroids to control the disease activity and they have from the NCBI PubChem compound database
become anchor for the treatment of RA [6]. The high (www.pubchem.ncbi.nlm.nih.gov/), drawn with the help
cost and adverse effects of drugs makes it unaffordable of Marvin Sketch and were saved as mol2 format and

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Volume 4, Issue 10, October – 2019 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
PDB format. The pharmacokinetics evaluation was of Aloevera and phebalosin of Murraya koenigii attained
performed using the Swiss ADME/Tox based on the the satisfactory binding energies (Table 3) and graphically
Lipinski rule of 5[13]. The prepared ligand structures were represented in figure 3. Binding energy is the energy
analysed by SwissADME on the basis of Lipinski rule of utilised by the ligand to dock with the receptor. The more
five. Lipinski rule of five helped to determine the negative the energy the more stable the conformation. The
physicochemical, lipophilicity, water solubility, medicinal ligands ranked according to the binding energy they
chemistry, pharmacokinetics and drug likeliness of the used to reach the stable conformation. Aloe emodin and
selected compounds. For drug likeliness molecular Phebalosin has been proved to be the appropriate ligand
weight, number of hydrogen bond donor, number of that binds to the protein molecule with maximum binding
hydrogen bond acceptor and total polar surface should come energy.
under Lipinski rule of five.
Interactions among IL-6 and other proteins using
C. Molecular Docking STRING database provides the description of various
Protein–ligand docking plays significant role in interacting partner of IL-6 such as interleukins like IL-4
predicting the orientation of the ligand on binding with which participates in the B-cell activation process, IL-10
the protein. The docking of bioactive compounds with IL-6 inhibits the synthesis of a number of cytokines like IFN-
protein were performed using 5 different tools namely gamma, IL-2, IL-3, TNF and GM-CSF pathway, IL-13
SwissDock, PatchDock, Argus Lab 4.0.1, UCSF inhibits inflammatory cytokine production and SOCS
Chimera-AutoDock Vina and Hex 8.0. SwissDock and (Suppressor of cytokine signalling 3) which is involved in
negative regulation of cytokines that signal through the
PatchDock are online servers [14, 15] whereas, Argus Lab JAK/STAT pathway.
4.0.1, UCSF Chimera-AutoDock Vina and Hex8.0 are
offline tools used for flexible docking [16, 17, 18]. IV. DISCUSSION

D. Protein-Protein Interaction RA is an autoimmune disease affecting joints and

The interaction within the proteins is helpful to leads to disability. The disease can be targeted by different
know about the novel therapeutic approaches. STRING available drugs targeting the various biological
Database (Search Tool for Retrieval of Interacting pathways. It is reported that IL-6 is the most
Genes/Proteins) (https://string-db.org/) has been used to significant cytokines causing synovitis deformities in the
identify the interaction between IL-6 and other proteins. joints of the RA patients [2]. Present research aims to target
The study will enable us to analyse metabolic and signal the prominent cytokine IL-6 by the naturally occurring
transduction and also the pharmacogenetics research [19]. compound through in-silico approach. Earlier, Aloevera has
been reported for the treatment of RA by not extracting the
III. RESULT bioactive compounds but by preparing the extract of leaves
for inhibiting the inflammatory factors; but this study
The bioactive compound structures of Aloevera are focuses basically on the bioactive compounds present in
aloin, campesterol, β-sitosterol, lupeol and aloe emodin and plant, to find out the basically which bioactive compound
Murraya koenigii are quercitin, catechin, epicatechin, responsible for this inhibitory action [20]. In this study two
phebalosin and mukonicine properties were analysed on the plants Murraya koenigii and Aloevera having anti-
basis of Lipinski rule of five (Table 1). Lipinski rule inflammatory properties, makes them more suitable to
of five helped to determine the physicochemical,
lipophilicity, water solubility, medicinal chemistry, target the IL-6 [8, 10]. The in-silico approach gives the
pharmacokinetics and drug likeliness of the selected prediction of the analysis before approaching towards
compounds. For drug likeliness molecular weight, number laboratory experiments. The bioactive compound of
of hydrogen bond donor, number of hydrogen bond acceptor Aloevera are aloin, campesterol, β-sitosterol, lupeol and
and total polar surface should come under Lipinski rule of aloe emodin and Murraya koenigii are quercitin,
five using SwissADME and their structures were drawn catechin, epicatechin, phebalosin and mukonicine were
using Marvin sketch (Table 2). selected for the molecular docking followed up by the
Procheck and swissADME analysis. It enables to validate
The IL-6 protein was validated, using PROCHECK the protein and the compound for the flexible molecular
tool. According to the standard percentage, the most docking [11]. The docking study with the different tools
favoured region should consist of more than 90%. The results the scoring function of ligand with the receptor
Ramachandran plot of IL-6 interprets that the total binding site. Two bioactive compounds aloe emodin and
number of residues are 157 and residues under most phebalosin have the potential to act as a drug for inhibition
favoured region are 139 i.e. 95.2% (Figure 2). of IL-6. The binding energy of the Aloe emodin resulted in
5 molecular docking tools namely SwissDock,
Ligands, aloe emodin and phebalosin and the PatchDock, Argus Lab 4.0.1, UCSF Chimera- AutoDock
receptor 1ALU were subjected to the docking procedure Vina and Hex 8.0 were -7.73, -3.748, 8.478, -8.98 and -
with the SwissDock, PatchDock, Argus Lab 4.0.1, 192.16, and of phebalosin was -6.98, -3.87, -7.884, -6.48
UCSF Chimera-Autodock Vina and Hex 8.0 (Figure 4). and -223.35 Kcal/mole. The binding energy of these two
On the basis of obtained binding energies, the aloe emodin compounds was higher among all the 10 selected

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Volume 4, Issue 10, October – 2019 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
compounds. The more negative the energy, the more stable [8]. Amirghofran Z, Herbal medicines for
the conformation of the docked compound. Furthermore, the immunosuppression, Iran J Allergy Asthma
protein protein interaction analysis performed using Immunol,11, 111-19, 2011.
STRING Database, provides the base to study about [9]. Pulok KM, Neelesh KN, Niladri M, Phytochemical
pharmacogenetics research, metabolic and signal and therapeutic profile of Aloevera, J Nat Med,
transduction pathway. Above study suggests aloe emodin 14(1), 2320-3358, 2014.
and phebalosin as the potential compound out of 10 selected [10]. Tanwi C, Shubhnagee S, Pramod K, Extraction and
compounds to target the protein for anti- inflammation Identification of Bioactive components from Aloe
which further needs to be validated by in vitro and in vivo barbedensis Miller, J Pharmacon Phytochem, 2, 14-23,
approaches. 2013.
[11]. Hanan AH, Uma MI, Sarah A, The Antibacterial
V. CONCLUSION effect of Curry Leaves (Murraya Koenigii), Eur J
Pharm Med Res, 3(10), 382-367, 2016.
Compounds derived from natural origin are capable to [12]. Tamiris MA, Daiana TM, Teodorico CR, In Silico
treat RA as novel therapeutic agents. Formulations of these Study of Leishmania donovani- Tubulin and
compounds are mostly preferred by the traditional system Inhibitors, J Chem, 1-8, 2014.
of medicine. This study shows that the aloe emodin and [13]. Antoine D, Olivier M, Vincent Z, SwissADME: a
phebalosin are potential inhibitor of IL-6 and have better free web tool to evaluate pharmacokinetics,
activity than other compounds as these compounds shows druglikeliness and medicinal chemistry friendliness of
more stable conformation. The anti-inflammatory property small molecules, Nat Sci Rep, 7, 42714, 2017.
of these two compounds further needs to be validated by in [14]. Raphael TA, Tayo AA, Babatunji EO, Structural
vitro and in vivo approaches. Studies of Predicted Ligand Binding Sites and
Molecular Docking Analysis of Slc2a4 as a
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arthritis, Scand J Rheumatol, 79, 67-96, 1989. [19]. Damian S, Annika LG, David L, STRING v11:
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college of Rheumatology 2008 recommendations for [20]. Subhashis P, Tanmoy D, Tapas KC, Soumen B,
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Volume 4, Issue 10, October – 2019 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165

FIGURE LEGENDS:

Fig 1:- Ribbon model view of IL-6 protein using Discovery studio 4.0.

Fig 2:- Ramachandran plot of IL-6 protein.

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Volume 4, Issue 10, October – 2019 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165

Fig 3:- Representation of the binding energy (Kcal/mole) of the ligands resulted from the docking tools; Aloe emodin and
Phebalosin are the two potent compounds.

Fig 4:- Docking images of a) Aloe emodin and b) Phebalosin using SwissDock server.

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Volume 4, Issue 10, October – 2019 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165

Figure 5: IL-6 interaction with other proteins using STRING Database.

Table Legends:

Compound Molecular H-bond H-bond Donor Rotatable

Name Weight(g/mol) Acceptor Bonds

Aloin 418.39 9 6 3
Aloe emodin 270.24 5 3 1
β-Sitosterol 414.71 1 1 6
Lupeol 426.72 1 1 1
Campesterol 400.68 1 1 5
Quercitin 302.24 7 5 1
Phebalosin 258.27 4 0 3
Mukonicine 323.39 3 1 2
Catechin 290.27 6 5 1
Epicatechin 289.14 6 5 2
Table 1:- Molecular weight, hydrogen bond donor, hydrogen bond acceptor and rotational bond of different ligands using
SwissADME software.

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Volume 4, Issue 10, October – 2019 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
Ligand Name Ligand Structure
Aloin
(10S)-1,8-dihydroxy-(hydroxymethyl)-10- [(2S,3R,4R,5S,6R)-
3,4,5-trihydroxy-6- (hydroxymethyl) oxan-2-yl]-10H-
anthracen-9- one

Aloe emodin
1,8-dihydroxy-3-(hydroxymethyl)anthracene-9,10-dione

β-Sitosterol
(3S,8S,9S,10R,13R,14S,17R)-17-[(2R,5R)-5- ethyl-6-
methylheptan-2-yl]-10,13-dimethyl-
2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-
cyclopenta[a]phenanthren-3-ol

Lupeol
(1R,3aR,5aR,5bR,7aR,9S,11aR,11bR,13aR,13 bR)-
3a,5a,5b,8,8,11a-hexamethyl-1-prop-1-en-2-yl
1,2,3,4,5,6,7,7a,9,10,11,11b,12,13,13a,13b-
hexadecahydrocyclopenta[a]chrysen-9-ol

Campesterol
(3S,8S,9S,10R,13R,14S,17R)-17-[(2R,5R)-5,6-dimethylheptan-
2-yl]-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-
dodecahydro-1H-cyclopenta[a] phenanthren-3-ol

Quercitin
2-(3,4-dihydroxyphenyl)-3,5,7- trihydroxychromen-4-one

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ISSN No:-2456-2165
Mukonicine
8,10-dimethoxy-3,3,5-trimethyl-11H- pyrano[3,2-a] carbazole

Phebalosin
7-methoxy-8-[(2R,3R)-3-prop-1-en-2- yloxiran-2-yl]chromen-
2-one

Catechin
(2R,3S)-2-(3,4-dihydroxyphenyl)-3,4-dihydro-2H-chromene-
3,5,7-triol

Epicatechin
(2R,3R)-2-(3,4-dihydroxyphenyl)-3,4-dihydro-2H-chromene-
3,5,7-triol

Table 2:- Ligands structure drawn using Marvin Sketch 19.8.

Ligands Binding Energy (Kcal/Mole) with 1ALU

Selected Active Argus SwissDock Hex8.0 UCSF PatchDock
Plants Compounds -8.478 -7.73 -192.16 -8.98 -3.748
Aloevera Aloe emodin
Murrraya Lupeol -4.656 -6.68 -306.47 -3.45 -5.458
Campesterol -4.805 -7.18 -276.46 -5.1 -4.72
β- sitosterol -4.267 -2.56 -254.22 -4.23 -5.074
Aloin -4.696 -7.31 -247.31 -6.56 -4.232
Quercitin -4.556 -7.49 -254.18 -6.2 -4.56
koeinigii Catechin -4.615 -6.73 -250.66 -5.9 -3.674
Phebalosin -7.884 -6.98 -223.25 -6.48 -3.87
Mukonicine -3.592 -6.91 -208.54 -5.9 -2.904
Epicatechin -4.600 -7.32 -250.82 -5.6 -3.564
Table 3:- Binding energy (Kcal/mole) of different ligands docked with IL-6 protein (PDB ID:1ALU).

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