in silico interaction of rutin with some immunomodulatory...

Indian Journal of Biochemistry & Biophysics

Vol. 55, April 2018, pp. 88-94

In silico interaction of rutin with some immunomodulatory targets:

a docking analysis

Aditya Ganeshpurkar1,2,3

& Ajay Saluja1,3

*

1Faculty of Pharmacy, Gujarat Technological University, Chandkheda, Ahmedabad-382 424, Gujarat, India 2Shri Ram Institute of Technology – Pharmacy (SRITP), Madhotal, Jabalpur-482 002, Madhya Pradesh, India

3AR College of Pharmacy, Vallabh Vidyanagar, Gujarat, India

Received 24 September 2017; revised 26 March 2018

The use of plant products as immunomodulator has a long history. Forms eternal era, plant, mineral and animal products

are used as drugs for the treatment of various diseases. The present-day synthetic compounds find their leads in natural

products. The process of immunomodulation retunes the immune system of an individual by restoring routine functions.

Research on immunomodulators from natural sources has been extensively studied for modulation of immune system along

with protection and prevention of diseases. Rutin, a flavonoid has been investigated for its potential immunomodulatory effects.

Increased particle clearance, decreased inflammation, increased leucocyte and antibody production was observed with

administration of rutin in rats. The present study is focused on exploring in silico interaction of rutin with some chemokines and

inflammatory targets. In this study, rutin was docked with TNF-α, IL-1β, IL-6, and NOs. Docking studies revealed the excellent

interaction of rutin with these targets. The result of present study provides insight for the discovery of novel molecules for

immunomodulation and treatment of inflammatory disorders. Findings from the present study show that rutin may interact with

several chemokines and inflammatory mediators. Further studies on rutin and associated flavonoids are necessary to develop

and establish QSAR and QSPR studies which may serve a stepping stone for the development of novel and safe

immunomodulator. Rutin therefore, can be considered for development of an immunomodulatory agent.

Keywords: Docking, Immunomodulatory, Inflammation, Interleukin, Nitric oxide, Prostaglandin

Immune system comprises some immune cells that

include lymphocytes, killer cells, macrophages and

neutrophils that can prevent body against invading

infections. In such event, various interconnected

biomolecular pathways lead to inflammation,

destruction of microbes and promotion of wound

healing process. Innate immunity can be regarded as

the first line of defense that comprises of various

physical, biochemical and cellular components.

Adaptive immunity comprises production of antibodies

that protect the body against pathogen invasion (e.g.,

opsonisation)1. During inflammation, acute phase aids

to fight against the early phase of infection. Chronic

inflammation sometimes may lead to progression of

autoimmune diseases. In chronic inflammation, over

expression of inflammatory cytokines (associated with

abnormal gene expression) is observed. Thus,

inhibition of these over expressed pro-inflammatory

cytokines seems to be beneficial in autoimmune

diseases (viz. rheumatoid arthritis)2. Immuno-active

chemicals produced from macrophages play a vital role

in inflammation. Macrophages when coming in contact

with bacterial endotoxin lipopolysaccharide produce an

array of proinflammatory cytokines which chiefly

include interleukins (IL-1β, and IL-6), tumor necrosis

factor (TNF-α) and other inflammatory mediator viz.

prostaglandin E2 (PG-E2) and nitric oxide (NO)3. In the

case of overactive secretion of these mediators,

sometimes, cardiovascular diseases, autoimmune

diseases, and cancer progression may be observed4.

Nitric oxide (NO) is a pro-inflammatory mediator that

leads to the prognosis of inflammation. Its

overproduction is observed in macrophage (stimulated

due to lipopolysaccharides) and its metabolites are

harmful to cellular integrity5.

Pharmaceuticals from natural source have been used

by humanity since eternal era to treat various diseases.

Chemotherapeutic agents used today have

immunosuppressive and cytotoxic effects that prove

detrimental to the health of individuals. Thus, it seems

to be imperative to search for immunomodulatory

agents from natural origin. In addition to

immunomodulation, these compounds have antiradical

—————

*Correspondence:

Phone: 91-2692-230788

E-mail: [email protected]; [email protected]

GANESHPURKAR & SALUJA: IN SILICO INTERACTION OF RUTIN WITH IMMUNOMODULATORY TARGETS

89

and antioxidant properties6. Flavonoids are

polyphenolic compounds found abundantly in plants.

They are known to exert analgesic, antidiabetic,

antimicrobial and cytotoxic effects7. Rutin is

ubiquitous flavonoid in plants. The term rutin comes

from the plant Ruta graveolens L., which is one of the

most abundant sources of rutin. Buckwheat, raspberry,

blackberry, tomato, fenugreek, tea, and grapes are rich

sources of rutin. Rutin is an antioxidant8,9

and

demonstrated various pharmacological effects on living

systems10

. There are various reports about analgesic

and anti-inflammatory effects11,12

. Recently, rutin was

evaluated for its possible immunomodulatory effects13

whereby protective effects of rutin on cellular, and

humoral effects were seen. Rutin proved itself to be a

novel immunomodulator as the treatment with rutin

caused a significant increase in humoral antibody titer,

increased carbon clearance, and increased leukocyte

count. The present study aims to explore underlying

mechanism of immunomodulation. In this study,

in silico interaction of rutin with various

immunomodulatory cytokines was analysed. Along

with this, the molecular basis of interaction of rutin

NOs was determined.

Materials and Methods

Software

Python 2.7-language was downloaded from

www.python.com, Molecular graphics laboratory

(MGL) tools and AutoDock4.2 was downloaded from

www.scripps.edu, Discovery Studio visualizer 4.1

was downloaded from www.accelerys.com.

Docking

The three-dimensional crystalline structures of

4 proteins were obtained from Protein Data Bank

(http://www.rcsb.org/). These protein were TNF-α

(PDB ID: 2AZ5), IL-1β (PDB ID: 2NVH), IL-6 (PDB

ID: 1P9M) and NOs (PDB ID: 5UO1). The structurally

refined protein .pdb files were converted to .pdbqt files

using grid module of autodock tools 1.5.6. Charges

were assigned to the ions to the proteins manually

wherever necessary. The 2D and 3D chemical

structures of rutin was retrieved (http://pubchem.ncbi.

nlm.nih.gov/). These .sdf and .mol files obtained from

PubChem were converted into .pdb files using Marwin

Sketch (http://www.chemaxon.com/marvin/ sketch/

index.jsp). These .pdb files were converted to .pdbqt

using ligand preparation module of autodock tools

1.5.6. The docking analysis of rutin was carried out

using the Autodock tools (ADT) v1.5.4 and autodock

v4.2 programs. Rutin was docked to all the target

protein complexes with the molecule considered as a

rigid body. The search was carried out with the

Lamarckian Genetic Algorithm; populations of 100

individuals with a mutation rate of 0.02 have been

evolved for ten generations. The remaining parameters

were set as default. The docked structure was then

visualised using Discovery Studio 2016 for obtaining

the binding interactions

Results The four crystal structures of proteins were retrieved

from protein databank. A docking program was

performed to simulate the binding mode to identify the

precise binding sites on various immunomodulatory

targets. Docking studies were carried out on active sites

of five target proteins 2AZ5, 1ITB, 1P9M and 5UO1

with rutin. Figure 1-4 indicates the interaction of rutin

with the active pocket of immunomodulatory targets

which demonstrated minimum binding energy with

targets via non-covalent interaction. The docking of

rutin with TNF-α showed that it showed hydrogen

bonding with the backbone at chain A: Gly-121 while

the sugar moiety also showed hydrogen bonding with

Chain B: Gly121 and Tyr-121. Beside these major

interactions, Flavanol moiety also showed π-amide

interaction with Chain B: Gly-121, Gly-122, and π-π

interaction with Chain A: Tyr-59. The other minor

alkyl and π-alkyl interactions were observed with

ChainB: Leu-57, Leu-94, and Phe-124 respectively

(Fig. 1). Its binding affinity was −5.13 Kcal/mole.

Rutin also showed interaction with IL-1β. Its

polyphenolic flavanol nucleus, as well as sugar units,

showed hydrogen bond interaction with Arg-4, Phe-46,

Gln-48and Lys-103. The sugar provides quite strong

interaction with Arg-4 with three hydrogen bonds. The

Glu-42 also demonstrated π-anion interaction flavanol

nucleus (Fig. 2). The presence of such high number of

hydrogen bonds makes it potential inhibitors of IL-1β.

Further, the docking of rutin with IL-6 showed various

H-bond interactions with helix A and D. These helices

interact with IL-1α receptors. The Asp-34, Ser-37, Lys-

171 and Gln-175 residues have demonstrated multiple

H-bond interactions. Apart from these interactions,

Leu-33, Arg-30 showed π-σ interactions. Asp-30

showed π-cation and Leu-178 demonstrated π-alkyl

interaction (Fig. 3).

Rutin shows various interactions with NOs which

include H-bond interaction with Ser-339, Trp-683,

Val-685, Asp-601 and Hem-801, π-π stacking with

Trp-683, π-σ interaction with Met-341 and π-alkyl

interaction with Met-341 (Fig. 4).

INDIAN J. BIOCHEM. BIOPHYS., VOL. 55, APRIL 2018

90

Discussion Molecular docking is a valid practice which aids to

envisage the principal binding modes of the ligand with

the protein having known three-dimensional structure.

The study focused on binding modes which were

necessary for major structural interaction, and provide

useful information for designing the inhibitors.

Molecular docking is one of the widely explored

techniques which is used to discover potential ligands

for known targets. These compounds can be screened

based on the estimation of free energy binding. The

value of free energy binding signifies the affinity of

drug towards the target. Similarly, lowest inhibition

constant represents the potential compound14

.

Fig. 1 — Molecular docking studies of rutin against TNF-α [(A) 2D-interactions; & (B) 3D-interactions]

Fig. 2 — Molecular docking studies of rutin against IL-1β[(A) 2D-interactions; &(B) 3D-interactions]


91

In the present study, TNF-α (PDB ID: 2AZ5), IL-1β

(PDB ID: 1ITB), IL-6 (PDB ID: 1P9M), and NOs

(PDB ID: 1NSI) were analysed for possible interaction

with a flavonoid rutin. Cytokines demonstrate a key

role in the development of inflammation and tissue

destruction leading to progression of inflammatory

diseases. Necrosis factors and interleukins are two

major classes of cytokines involved in the progression

of hyperalgesia. In the present study, TNF-α, IL-1β,

and IL-6 were selected for docking analysis15

. With

Rutin, inhibitory constant for TNF-α was 25.62 µM,

61.42 µM with IL-1β and IL-6 12.35 µM. TNF-α is a

Fig. 3 — Molecular docking studies of rutin against IL-6[(A) 2D-interactions; & (B) 3D-interactions]

Fig. 4 — Molecular docking studies of rutin against NOs [(A) 2D-interactions; & (B) 3D-interactions]


92

key regulator of inflammation. Overactivity of this

chemokine is responsible for various inflammatory

diseases like rheumatoid arthritis, ankylosing

spondylitis, inflammatory bowel disease16

. TNFR1

contains a death domain (DD) motif lying towards its

C-terminal involved in death signalling while in case of

TNFR2 no such death motif is present and works

through apoptotic mechanism17

. TNFR2 have a greater

affinity for TNF ligand is colocalised with TNFR1. It

slowly passes ligand to TNFR1 and gets shredded off

the membrane while TNFR1 get internalised showing

its further action. So, the binding of TNF ligand with

TNFR is the critical step. Cunningham et al. showed

the complex between the small molecule TNF inhibitor

and the intact TNF-α trimer results in a 600-fold

accelerated trimer dissociation. The XRD study

showed small molecule inhibitor displaces a subunit of

the trimer to form an inhibitor bound TNF- α dimer

complex. We also docked the rutin on the similar site.

The docking study revealed that rutin mimic similar

interaction to that of 6,7-dimethyl-3-[(methyl

{2-[methyl({1-[3-(trifluoromethyl)phenyl]-1h-indol-3-

yl}methyl)amino]ethyl}amino)methyl]-4h-chromen-4-

one present in actual pdb18

. The study showed that the

rutin at the molecular level might inhibit the trimer

assembly of TNF-α which may be its molecular

mechanism to prevent inflammation. In a study, rutin

pretreatment significantly reduced levels of TNF-α in

LPS stimulated animals19

. Similar protective effects of

rutin on glial cells were observed due to rutin

pretreatment20

. Polygala paniculata L. extract (rich in

rutin) demonstrated IL-1β inhibition21

. Similar

inhibitory effects were seen in present docking studies.

IL-1β along with IL-6 and TNF-α produces

hyperalgesia 22

. During injury, IL-1β promotes

expression of Interleukin-623

.Drugs against TNF-α and

proinflammatory interleukins (IL-1β and Interleukin-6)

seems to be useful against psoriasis, Crohn s disease,

rheumatoid arthritis. In a study, rutin demonstrated

hepatoprotective effects primarily due to suppression of

IL-6 in rat hepatocytes in CCl4 intoxicated animals 24

andhigh-cholesterol-diet-fed rats25

. In the present

study, docking results revealed affinity of rutin towards

IL-6 for the inhibition of later.

Interleukin-1 is a proinflammatory cytokine that

induces acute as well as chronic inflammation through

expression of the various gene. Interleukin-1 has two

isoforms, i.e. IL-1α and IL-1β. It is observed that IL-1β

gene spontaneously expressed. So, inhibiting expressed

IL-1β may manifest to prevent and cure many

inflammatory conditions. IL-1β shows its action by

binding through the Interleukin-1 receptor. The Arg-11

and Gln-15 forms contact with domain two while

His-30 and Gln-32 form contact with domain

1-2 junction of Interleukin-1 receptor26

. The seven

discontinuous residues, i.e. Arg-4, Leu-6, Phe-46, Ile-

56, Lys-93, Lys-103, and Glu-105 together constitute a

binding site in IL-1β which bind to Interleukin-1

receptor27

. Thus, binding of Rutin with some of the

crucial residues of this site may describe one of its

anti-inflammatory mechanism of action.

IL-6 (IL-6), comprises of four-helix bundle linked

by loops with a mini-helix28

. IL-6 is a secreted from

T cells and macrophages which bind to two cell surface

receptors, IL-6Rα and gp130. It is involved in several

chronic inflammatory diseases. The IL-6 forms

association with IL-6α-gp130 leading to the ternary

complex which activates the JAK family (tyrosine

kinases) and the downstream STAT3 transcription

factor29

. The helices A and D, forms N-terminus to

C-terminus, interact with domain-2 and 3 of the

IL-6Rα. The interaction of rutin with domain A and D

leads to a possible inhibitory effect on its action

mediated through, IL-6Rα receptor. Such inhibition

over IL-6 is c by previous study 24

Nitric oxide synthase is an essential class of enzyme

that synthesizes nitric oxide from l-arginine30

. The

Nitric oxide synthase is responsible for NO production

from Arginine. Its activity is controlled by Ca2+

concentration through Ca2+-calmodulin complex. The

expression of TNF-α, IL-1 follows NOs creation. This

enzyme is abundantly found in macrophages and

monocytes at the site of infection or inflammatory

disease 31

. Thus, due to the biosynthesis of nitric oxide,

bacterial growth is prevented, and inflammation is

retarded due to suppression of proliferation of T cells32

.

However excessive production of nitric oxide and

generation of reactive nitrogen intermediates´ is

involved in the progression of many inflammatory

diseases33

. Thus, inhibition of this enzyme may serve a

vital role in the prevention of inflammation. Several

natural products have been studied for inhibition of

inducible nitric oxide synthase; for instance flavonoids

from Tanacetum microphyllum DC, especially Ermanin

and 5,3 -dihydroxy-4 -methoxy-7-methoxycarbonyl

flavonol were most potent inhibitors of this enzyme34

.

In NOs, the arginine urea group forms a bi-dentate

interaction with Glu-377 adjacent to the active site. It is

a site of larger competitive inhibitors that may inhibit

i-NOs35

. Rutin retained all the primary interaction as


93

shown by the co-crystallized NOs inhibitor. This

porphyrin ring plays an important role in catalytic

enzyme mechanism. In the present study, in silico

docking studies revealed inhibition of NOs. Thus the

docking interaction of NOs with rutin confirms the

previous results whereby rutin exerted protective

effects, probably by inhibiting NOs in ischemic

reperfusion injury.

Conclusion In the present study, we carried out docking studies

on rutin to various inflammatory and immuno-

modulatory targets, with the purpose to study and

analyze in a silico interaction of former on later. The

results obtained from docking and study of the

interactions of the rutin suggests the ability of rutin to

bind to multiple targets involved in inflammation and

immunomodulation. Rutin interacted with various

chemokines and inflammatory mediators viz. TNF-α,

IL-1β, IL-6, and NOs. With each target, rutin

demonstrated a noteworthy affinity for binding.

Findings from the present study show that rutin may

interact with several chemokines and inflammatory

mediators. Further studies on rutin and associated

flavonoids are necessary to develop and establish

QSAR and QSPR studies which may serve a stepping

stone for the development of novel and safe

immunomodulator.

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