Netw Model Anal Health Inform Bioinforma (2014) 3:66

DOI 10.1007/s13721-014-0066-x

ORIGINAL ARTICLE

Binding affinity analysis and ADMET prediction

of epigallocatechine gallate (EGCG) derivatives for AP-1 protein:
a drug target for liver cancer
Mamta Sagar Rajesh Kumar Pathak
Rishi Kumar Pandey Dev Bukhsh Singh
Neetesh Pandey Manish Kumar Gupta

Received: 2 December 2013 / Revised: 8 July 2014 / Accepted: 10 July 2014

Springer-Verlag Wien 2014

Abstract The hepatitis B virus 9 protein (HBx) of

Hepatitis B virus activates AP-1 protein and causes the
down regulation of PTEN (full name) and p53 leads to the
tumor formation in liver. In this study, the interactions
between DNA and AP-1 have been targeted by docking of
natural compounds epigallocatechine gallate (EGCG),
curcumin, luteoline, genistein, ellagic acid, resveratrol,
lupeol, betulinic acid and lycopene. Green Tea (Camellia
sinensis) possesses anticancer property. EGCG obtained
from green tea shows favourable binding with AP-1 protein
among all natural compounds. It is necessary to target
DNA binding domain which binds at DNA to induce the
expression of p53 and PTEN gene. EGCG have shown
interaction at these positions, which may minimize down
regulation of p53 and PTEN gene. To increase the binding
affinity and bioavailability of EGCG, derivatives have been
designed by mimicking the position of H and OH group.
One of acetylated derivative EGCG15, made by replacing
OH group by OCOCH3 shows better affinity than other
derivatives and binds at amino acid Asp 163(G), Ser
278(F), Lys 282(F), Arg 288(F) with six hydrogen bonds
and -5.60 kcal/mol energy. Affinity of EGCG with AP-1
protein was greatly enhanced in methoxy derivative
EGCG05 which binds at Asp 163, Asp 170, Ser 278, Arg

M. Sagar (&)  R. K. Pathak  R. K. Pandey  N. Pandey 

M. K. Gupta
Department of Bioinformatics, University Institute of
Engineering and Technology, Chhatrapati Shahu Ji Maharaj
University, Kanpur 208024, Uttar Pradesh, India
e-mail: mamta1060@yahoo.com
D. B. Singh
Department of Biotechnology, Institute of Biosciences and
Biotechnology, Chhatrapati Shahu Ji Maharaj University,
Kanpur 208024, Uttar Pradesh, India

281, and Arg 288 with eight hydrogen bonds and

-6.30 kcal/mol energy (energy complex). Substitution of
OH by OCOCH3 increases bioavailability after sequential
addition at various positions in EGCG leads in silico. These
derivatives also show better affinity, but less than methoxy
(OCH3) derivative. Substitution by OCH3 leads to slight
increment of 6 % in oral absorption. Substitution by NH2
group leads to no changes in oral absorption or bioavailability. This may lead to inhibition of AP-1 protein which
down regulates tumor suppressor gene p53 and PTEN.
Keywords Liver cancer  HBV  Activator protein-1 
Natural compounds  EGCG  Curcumin  Green tea

1 Introduction
Clinical treatments with interferon, natural compounds
such as Green tea, curcumin, resveratrol, genistein, ellagic
acid have been reported in all other cancers, such as lung,
prostrate, skin. Chronically infected HBV patients are
suffering from lack of a comprehensive and efficient antiviral treatment. Natural chemo preventive compounds,
their molecular targets and mechanisms of action has
already been reported (Amin et al. 2009), which may help
in further design and conduct of preclinical and clinical
trials. Therefore, it is crucial to ascertain more effective
compounds from natural resources, which reduce the
cytotoxic effects and work as effective anti-HBV drugs and
can be useful to treat hepatocellular carcinoma. AP-1
inhibition as target has been reported by few methods in
literature, but no evidence is available in reference to its
use in drug discovery process.
Anticancer activity of several natural compounds has
been experimentally proved in research study (Imai et al.

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1997). Green Tea (Camellia sinensis) is one of them and is

the most popular beverage consumed in the world; it has
anticancer activity (Yang et al. 2000). The active compounds found in tea are the polyphenols which include
flavanols or catechins. Catechins include (-)-epigallocatechine gallate, which is the most abundant component,
and (-)-epigallocatechin (EGC), (-)epicatechin-3-gallate
(EGC), and (-)-epicatechin (EC) (Yang et al. 2001).
Migration/invasion of melanoma cells is greatly inhibited
by EGCG compared to other catechins or epicatechin
derivatives (Katiyar et al. 1997). Natural compounds may
be potential inhibitors for cancer disease.
Activation of AP-1 protein is repressed by several natural compounds, which modulate its target gene (Amin
et al. 2009). Although amino acids involved in the interactions are not reported. Hepatitis B virus infection is a
leading source of liver cancer. The HBx protein of Hepatitis B virus activates AP-1 protein (Benn et al. 1996), it
causes the down regulation of PTEN and p53 gene (Wang
et al. 1994; Haviv et al. 1998; Chung et al. 2003). Therefore, tumour suppressor proteins are not formed which
cause the tumour formation in liver. HBx protein plays a
regulatory role in HBV replication and is necessary for in
viral infection. It functions by proteinprotein interaction
and activation of transcription factor AP-1 (Bouchard et al.
2006). Activation of AP-1 protein by HBx protein promotes cell division which ultimately results in the formation of tumours in liver.
AP-1 is transcription factor that has been closely linked
with transformation of tumour cells (Karin et al. 1997).
Activation of AP-1 needs phosphorylation of c-jun
through activation of stress-activated kinase JNK (Xia
et al. 1995). Activation of JNK is also linked to cellular
transformation (Huang et al. 1999). Curcumin inhibits the
AP-1 activity provoked by tumour promoters (Huang et al.
1991) and also JNK activation induced by carcinogens
(Bierhaus et al. 1997) confirmed that curcumin-based
inhibition of AP-1 protein due to its direct interaction with
DNA binding domain of this protein. It is reported that
curcumin down regulates AP-1binding activity in
tumorigenic cells (Prusty and Das 2005). EGCG and
possibly other chemopreventive tea polyphenols target
NF-jB and AP-1 and interfere in cellular signal transduction process (Bode and Dong 2004; Barthelman et al.
1998; Stalmach et al. 2009). EGCG also inhibit UVBinduced AP-1 activation as well as NF-jB-dependent
transcriptional activation (Mukhtar and Ahmad 1999).
Treatment of cells with EGCG before and after UVB
irradiation inhibits activity of AP-protein (Lu et al. 2003).
Black raspberry fractions modulate mitogen-activated
protein kinase pathways by inhibiting activation of AP-1,
NF-kB and nuclear factor of activated T cells (NFAT) by
BaPDE as well as their upstream PI-3K/Akt-p70 (S6K)

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Netw Model Anal Health Inform Bioinforma (2014) 3:66

which explore anti carcinogenic effect of Rasberry that

was unknown.
In this study, interactions of natural compounds EGCG,
curcumin, luteoline, genistein, ellagic acid, resveratrol,
lupeol, betulinic acid and lycopene have been studied by in
silico methods. DNA-binding domain interactions of AP-1
have been targeted by docking of natural compounds.
Highest affinity of EGCG was predicted towards AP-1
protein with a free energy complex of -7.97 kcal/mol.
This may be further enhanced by introducing new hydrogen bonds between functional groups of natural compounds
and amino acids of AP-1 protein. This study provides detail
annotation about hydrogen bond interactions of natural
compounds and AP-1 protein and reveals about interacting
amino acids so that natural compounds may be further
modified to increase its inhibitory effect. Substitution of
OH by OCOCH3 group was done to analyse its possibility
of increased affinity. Furthermore, ADMET prediction was
done to analyse potency of designed derivatives EGCG and
other tea catechins that inhibits the release of tumour
necrosis factor alpha, which promotes tumor formation
(Fujiki et al. 2000). EGCG was shown to reduce specific
binding of both the 12-otetradecanoylphorbol-13-acetate
(TPA)-type and okadaic acid-type tumor promoters (the
two major classes of tumor-promoting agents) to their
receptors (Fujiki 1999).
Several experimental studies indicate about indirect
effect of EGCG on Ap-1 activation. EGCC treatment
reduces the level of active AP-1, induced by PMA which
indicate that EGCG suppress MMP-9 and inhibit AP-1
activation in human gastric AGS cells (Kim et al. 2004).
EGCG inhibit EGF or tumor promoter AP-1 dependent
transcription activity and cell transformation in JBS cell.
Inhibition of AP-1 activity by EGCG and theaflavins take
place through inhibition of c-Jun NH2-terminal Idnase and
do not depend on ERK pathway (Dong et al. 1997). In
another study, EGCG reduce the nuclear levels of transcription factors NF-jB and AP-1/c-Jun which was
induced by IL-1 in human chondrocytes. EGCG causes
inhibition of nuclear translocation and activation (Singh
et al. 2002, 2003).

2 Materials and method

2.1 Structure of AP-1 (PDB ID: 1FOS) was downloaded
from PDB database and used for analyzing binding affinity
of EGCG and their derivatives with AP-1 protein which
may be considered as potential target for liver cancer. 3Dstructure files of natural compounds lycopene, betulinic
acid, lupeol, resveratrol, ellagic acid, genistein, luteoline,
curcumin and EGCG were retrieved from pubchem database. IUPAC name of EGCG derivatives were generated

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include in grid generation. Docking constraints of 1 (one)

for ligand atom was taken. Stereoisomers were computed
A
to generate possible states at PH 7.0.
2.5 Ligand protein docking was done using Glide using
binding site residues of AP-1 protein targeted by natural
compounds. One pose was selected out of 10,000 poses as
per one docking run. Energy threshold of 0.5 kcal/mol was
taken for rejecting minimised pose. All the poses were
generated below this energy threshold for generating
10,000 poses (Halgren et al. 2004). The analysis of docking
results was done using XP visualizer Glide. For selecting
one pose, ten (10) best poses per ligand were analysed.
was taken for generating grid and to
Grid center of 12 A
calculate interaction score. These steps were carried out to
include receptor in docking. Ligand sampling was done
using flexible approach (nitrogen inversions and ring conformation). Bias sampling of torsions was done for amide
only and non-planer conformations are panellized (Friesner
et al. 2004, 2006).
2.6 ADME was predicted by QikProp v3.3 tool using
fast Processing Mode. Its output can be used as input for
the QikFitTM and QikSimTM modules. ADME prediction
was performed using QikFit module which uses linear
regression method for experimentally determined molecular properties and predicts the properties of designed
derivatives given as input structural data file format (SDF).
The resulting regression equations is then be integrated
back into QikProp and used to predict the experimental
property of structurally similar molecules. QikProp can be
run either from the Maestro GUI or from the command line
(Jorgensen and Duffy 2000; Colmenarejo et al. 2001).
Monte Carlo statistical mechanics simulations predict
configurational averages for a number of descriptors,
including hydrogen bond counts and solvent-accessible
surface area (SASA) on organic solutes in periodic boxes
of explicit water molecule followed by comparative study
(Jorgensen and Duffy 2002).
2.7 OSIRIS Property Explorer (http://www.organicchemistry.org/prog/peo/) was used to estimate side effects,

by MarvinSketch (http://www.chemaxon.com/products/
marvin/marvinsketch/) (Table 1). All positions ortho, meta
and para on A and D ring (galloyl moiety) of EGCG
structure were chosen for group replacement (OH by OCH3
and OCOCH3).
2.2 Ligand preparation was done by Ligprep module of
Schrodinger maestro. During ligand preparation, atoms
range was set from 1 to 300 atoms and 150 rotatable
bonds for calculating scoring function. Rotatable groups
allowed rotation of receptor, hydrophobic atoms in ligands
and Van der Waals radii were scaled by 0.8 scaling factor
with less than 0.15 partial atomic charges as a threshold.
This was done to introduce flexibility in binding for nonpolar part of EGCG and their derivatives.
2.3 Protein preparation of AP-1 protein (1FOS) was
done by protein preparation wizard using selected amino
acid residues of AP-1 protein. File is imported in prepwizard and processed then bond order, H bonds, disulphide
bonds and cap termini are created. Water molecules are
from (heteroatom) HET groups in next
deleted up to 5 A
step. File is reviewed and modified. G and F chains of
EGCG protein were selected for docking after removing
water molecules at PH 7.03.0. Then, refinement takes
place followed by sampling water orientation and minimization. Optimization was performed using PROPKA at
PH 7.0. Interactive H-bond optimizer use PROPK then
current orientation is included and network is analysed.
Amino acid were analysed to avoid flip in protein. Amino
acids involved in flip were locked. Clusters are resorted to
less strict clashes. RC plot can be viewed and compared
before and after optimisation.
2.4 Receptor grid generation was done by Glide (Knoll
et al. 2004) selecting binding sites of AP-1 protein which is
targeted by natural compounds. To define active site,
centroid of selected residue can be taken from Q site finder
and ligand with XYZ coordinates. DNA-binding region of
AP-1 protein was selected for docking. Then grid is generated using Vander waal interactions and scaling factor
respectively. Ligand is identified to
were 1.0 and 0.25 A

Table 1 Physicochemical properties of natural molecule taken for study

S. no.

Description

EGCG

Lupeol

Resveratrol

Ellagic acid

Genistein

Curcumin

Luteolin

Compound ID

65,064

259,846

445,154

5,281,855

5,280,961

969,516

5,280,445

Chemical formula

C22H18O11

C30H50O

C14H12O3

C14H6O8

C15H10O5

C21H20O6

C15H10O6

Molecular weight (g/mol)

458.37172

426.7174

228.24328

302.19264

270.2369

368.3799

286.2363

XLogP3-AA

1.2

9.9

3.1

1.1

2.7

3.2

1.4

H-bond donar

H-bond acceptor

11

State

Solid

Solid

Solid

Solid

Solid

Solid

Solid

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Netw Model Anal Health Inform Bioinforma (2014) 3:66

3 Results and discussion

identifying their binding affinity. Molecular docking is an

in silico technique which is used to find the orientation of
the lead molecule in the target protein for docking screen
small molecule. Top-ranked molecules may be tested for
binding affinity in vitro, and may become lead compounds,
the starting points of drug development after optimization.

3.1 Study of AP-1 protein interactions with DNA

3.2 Docking of natural compounds with AP-1 protein

Protein DNA interactions were analysed for the selection

of amino acids for docking with natural compounds so that
interactions between AP-1 and DNA can be minimized.
Motif NKMAAKSRNRRELT (red) located at 147158
positions in G Chain of AP-1 protein shows interactions
with DNA (Fig. 1). The present study shows the docking
interactions of natural compounds with AP-1 protein.
Natural compounds play a significant role in lead discoveries. Literature study shows that the HBx protein of HBV
plays a regulatory role in HBV replication and establishment of viral infection, and activation of transcription
factor protein AP-1. Present study shows that natural
inhibitors derivative may play role in modulating AP-1
regulation. Several natural compounds were reported to
suppress the activation of AP-1 protein; here nine (9)
natural compounds were chosen for the study and their
interactions with AP-1 protein were analysed. In the field
of drug designing finding an optimal leads molecules is a
challenging tasks. With the recent development of the
computational techniques and tools, it is now become easy
to identify and analyse the various large biological dataset
elaborately. Computer aided drug designing play essential
roles in drug discoveries process and tested for the interaction with the target molecule computationally in

The pyrogallol-type structure on the B-ring responsible for

apoptosis and has powerful antioxidative activity due to
forming reactive oxygen species by auto-oxidation (Kanwar et al. 2012). The galloyl moiety (D-ring, gallate group)
responsible for inhibition of fatty-acid synthase which
leads to cytotoxicity in human cancer cells (Wang et al.
1994). Both components B and D participate in exertion of
biological activities related to the cell surface 67 kDa
laminin receptor (Mereles and Hunstein 2011). All positions (R1R9) ortho, meta and para on A and D ring
(galloyl moiety) of EGCG structure were chosen for group
replacement (OH by OCH3 and OCOCH3) to order design
derivatives with improved affinity towards AP-1 protein
than EGCG, although EGCG already has good affinity to
AP-1 protein with six (6) H bond among all natural compounds, taken for study (Fig. 1c). No changes have been
made in pyrogallol-type structure on the B-ring which is
responsible for apoptosis.
Three derivatives (EGCG01EGCG03) were made of
substituting OH group by NH2 at R6 position and shuffling
of H and OH group. In next two derivatives (EGCG04
EGCG05), first derivatives was made by substituting H by
OH group at R9 position and second derivative (EGCG05)
was made of substituting OH by OCH3 at R8 position

Fig. 1 AP-1/DNA complex. a Crystallographic structure of the AP-1

heterodimer comprising c-Fos (cyan) and c-Jun (green) complexed
with DNA (brown), DNA binding domain is shown in red colour.

b EGCG05 and EGCG15 bind at the positions (pink colour) which

may interfere with AP-1and DNA interactions

such as mutagenic, tumorigenic, irritant and reproductive

effects and provide score for drug-relevant properties
including drug-likeness and overall drug-score.

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(Fig. 2b). Ten (10) derivatives (EGCG06EGCG15) were

designed by substituting H and OH group by OCOCH3
(Table 2).
Natural compounds derived from Natural sources were
docked with AP-1 protein. Docking was carried out with
natural compounds. The natural compound EGCG has
greater affinity (-7.97 kcal/mol) towards the AP-1 than
other natural compounds (Table 3). Hydrogen bonds
between ECGC and amino acids serine, arginine, aspartic
acid (three hydrogen bonds) have been depicted in Fig. 2c.
Curcumin, luteolin, genistein, ellagic acid and resveratrol
are also showing good affinity, hydrogen bonds have been
visualized in their complex structure with AP-1 protein, but
no significant interactions were predicted in case of lupeol,
betulinic acid and lycopene. Curcumin, luteolin, genistein,
ellagic acid and resveratrol have shown formation of

66

minimum energy complex which indicates proper and

energetically favourable interactions of these natural molecules with AP-1 protein.
Natural compounds derived from natural sources play a
significant role in lead discoveries for cancer. In present
study, the docking result of natural compounds, such as
lycopene, betulinic acid, lupeol, resveratrol, ellagic acid,
genistein, luteoline, curcumin and EGCG shows binding
energy in the range of ?0.29 to -7.97 kcal/mol (Table 3).
EGCG may be a lead compounds for liver cancer which
shows H-bond interactions with ASP 163, ASP 163, ASP
170 (G chain) and SER 278, GLU 284 (F chain) of AP-1
protein with six hydrogen bonds (-7.97) Kcal/mol
(Table 4). SER 278 is one of amino acid which binds at
DNA, but this binding is not sufficient to induce the
expression of p53 and PTEN gene. EGCG have shown

Fig. 2 a Chemical structure of EGCG: Pyrogallol type structure and galloyl moiety. b Modification point shown by labels (R1R9). c Docked
view of EGCG with AP-1 protein showing H-bond interactions with SER 278, ARG 288 (F chain) and ASP 163, ASP 163, ASP 170 (G chain)

Table 2 Given details of

substituted group at various
position of EGCG structure to
make derivatives

Derivatives EGCG06 to
EGCG15 were designed by
substituting acetyl group at
various positions

Derivatives

R1

R2

R3

R4

R5

R6

R7

R8

R9

EGCG

OH

OH

OH

OH

OH

EGCG01

OH

OH

NH2

OH

OH

EGCG02

OH

OH

NH2

OH

OH

EGCG03

OH

OH

NH2

OH

OH

OH

EGCG04

OH

OH

OH

OH

OH

OH

EGCG05

OH

OH

OH

OH

OCH3

EGCG06

OH

OH

OH

OH

OCOCH3

EGCG07

OH

OH

OH

OCOCH3

OH

EGCG08

OH

OH

OCOCH3

OH

OH

EGCG09

OH

OH

OH

OCOCH3

OCOCH3

EGCG10

OH

OH

OCOCH3

OCOCH3

OCOCH3

EGCG11

OH

OCOCH3

OCOCH3

OCOCH3

OCOCH3

EGCG12

OCOCH3

OCOCH3

OCOCH3

OCOCH3

OCOCH3

EGCG13

OH

OCOCH3

OH

OH

OH

EGCG14

OH

OCOCH3

OCOCH3

OH

OH

EGCG15

OCOCH3

OCOCH3

OH

OCOCH3

OH

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Table 3 G and F chain of AP-1

are docked with natural
compounds derived from
natural sources (plants):
illustrates minimum free energy
required for hydrogen bonding
with different amino acids of
AP-1 protein involved in
interactions

S.
no.

Natural
compounds

Natural sources

Energy
(kcal/
mol)

Amino acid residues and

No. of
H-bonds

EGCG

Camellia sinensis (green tea)

-7.97

Luteolin

-5.78

3
4

Curcumin
Genistein

Artichoke, broccoli, celery,

cabbage, spinach, green
pepper, cauliflower
Curcumin longa (turmeric)
Soybeans and soy products

SER 278(F),GLU 284(F), ARG

288(F), ASP 163 (G), ASP 163(G),
ASP 170(G)
ASP 163(G), ASP 163(G)

2
4

Ellagic
acid

-5.33

Resveratrol

-5.03

ASP 163(G), ASP 174(G)

Lupeol

-4.04

No significant interaction

Betulinic
acid
Lycopene

Pomegranate juice, strawberries,

barks of arjun, Lonicera
caerulea
Red wine, grapes (mainly in the
skin), mulberries, peanuts,
vines, pines
Mango, olive, strawberry, red
grapes
Betula spp., Ziziphus spp.,
Syzigium spp.
Tomatoes, guava, papaya

ARG 288(F),ASP 170(G)

LYS 280(F), LYS 282(F), ASP
163(G), ASP 170(G)
SER 278(F), ASP 163(G)

-3.59

No significant interaction

0.29

No significant interaction

-5.67
-5.59

Table 4 Amino acids residues of AP-1 protein involved in interactions with EGCG derivatives, minimum free energy and hydrogen bonds
corresponding to each derivative are given
Derivative

Energy (kcal/mol)

Amino acid residues, position and chain

No. of H-bonds

EGCG01

-7.10

ASP 163(G), ASP 170(G), ARG 288(F), SER 278(F)

EGCG02

-5.70

ASP 163(G), ARG 281(F), GLU 284(F)

EGCG03

-6.00

ASP 163(G), SER 278(F), ARG 281(F), ARG 288(F)

EGCG04

-6.50

ASP 163(G), SER 278(F), ARG 281(F), ARG 288(F)

EGCG05

-6.30

ASP 163(G), ASP 170(G), ARG 281(F), SER 278(F), ARG 288(F)

EGCG06

-5.20

ASP 163(G), ASP 170(G), ARG 285(F), ARG 288(F)

EGCG07
EGCG08

-5.30
-5.70

ASP 163(G), ASP 170(G), SER 178(F), ARG 288(F)

ASP 163(G), ASP 170(G), SER 178(F), ARG 288(F)

5
6

EGCG09

-5.70

ASP 170(G), LYS 277(F), ARG 281(F), GLU 284(F), ARG 288(F)

EGCG10

-5.70

ASP 163(G), LYS 282(F), ARG 288(F)

EGCG11

-5.90

ASP 163(G), LYS 282(F), ARG 288(F)

EGCG12

-5.20

SER 278(F), ARG 281(F), ARG 285(F), ARG 288(F)

EGCG13

-5.70

ASP 163(G), SER 278(F), ARG 281(F), GLU 284(F), ARG 288(F)

EGCG14

-6.00

ASP 163(G), SER 278(F)

EGCG15

-5.60

ASP 163(G), SER 278(F), LYS 282(F), ARG 288(F)

interaction at these positions. Therefore, this can be used to

prevent down regulation of these gene by interfering AP-1DNA.
3.3 Docking of EGCG derivatives with AP-1 protein
shows improved binding
Binding energy of derivative EGCG01 is less than other
derivatives. Although original EGCG molecule shows six
hydrogen bond interactions (Fig. 2c) with ASP 163(G),
ASP 170(G), ARG 288(F), SER 278(F), but it is improved
significantly when EGCG05 was designed by placing

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Fig. 3 Docking of derivatives with AP-1 protein. a Interaction view c

of EGCG05 with AP-1 protein, showing H-bonding with ASP 163,
ASP 170 (G chain) and SER 278, ARG 281, ARG 288 (F chain).
b EGCG09 forms H-bond interaction with ASP 170 (G chain) and
LYS 277, ARG 281, GLU 284, ARG 288 (F chain). c EGCG10
showing H-bond interaction with ASP 163(G chain) and LYS 282,
ARG 288 (F chain). d EGCG11 forms H-bond interaction with ASP
163 (G chain) and LYS 282, ARG 288 (F chain). e EGCG12 forms
H-bond interaction with SER 278, ARG 281, ARG 285, ARG 288 (F
chain). f H-bond interactions (violet) are depicted with ASP 163 (G
chain) and SER 278, LYS 282, ARG 288 (F chain) in docked view of
EGCG15 with AP-1 protein. Colour symbolise negatively charged
(peach); positively charged (blue), hydrophobic (green), polar (cyan),
H-bond backbone (violet solid line), H-bond-side chain (violet dotted
line)

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methoxy group OCH3 at R8 position which shows greater

affinity in docking results, towards AP-1 amino acids
interacting with DNA with -6.30 kcal/mol energy and
showing H-bond interactions with ASP 163, ASP 170, SER
278, ARG 281 and ARG 288 with eight hydrogen bonds
(Figs. 3a, 4). Docking analysis reveals that EGCG05 has
better affinity than other molecules for liver cancer. In
EGCG15, OH group is replaced by OCOCH3 at R2, R4 and
R7 positions which improves affinity of EGCG than other
designed derivative. EGCG15 (-5.60 kcal/mol) binds at
amino acid ASP 163(G), SER 278(F), LYS 282(F), ARG
288(F) with six hydrogen bonds and energy (-5.60) Kcal/
mol (Figs. 3f, 4).
3.4 Role of pyrogallol type structure and galloyl
moiety in interactions of EGCG and its derivatives
with AP-1 protein
No change has been made in pyrogallol type structure on B
ring. H and OH group was substituted by acetyl group on D
ring (galloyl moiety) and A ring. All derivatives were
docked with AP-1 protein. These docked complex shows
different types of binding pattern, but EGCG05, EGCG09EGCG12 and EGCG15 shows binding pattern which are of
our interest. In original EGCG galloyl moiety is oriented
towards basic environment and make one hydrogen bond
with Arg 288 (blue) while pyrogallol type structure is
oriented towards acidic environment (peach) Asp 163 of
AP-1 protein. EGCG05 galloyl moiety is oriented towards
basic environment without making any H-bond. Two OH
group makes two H bond with side chain of Asp 163 and
OH group of A ring make two H bond with side chain of
Ser 278 and Arg 281. Arg 285 of F chain in AP-1 protein
interacts towards nucleus of pyrogallol type structure, but
galloyl moiety of this derivative is oriented towards solvent
exposure area when interacting with AP-1 protein.

Netw Model Anal Health Inform Bioinforma (2014) 3:66

In case of EGCG15, galloyl moiety moves slightly away

from interaction sites but other parts of EGCG structure
interact more strongly by making two H bond with Asp 163
and Arg 281. Docking of EGCG09 with AP-1 shows that
galloyl moiety is oriented towards basic environment and
make 2 (two) H bond with Arg281. EGCG10-AP-1 docked
complex shows that galloyl moiety is oriented towards
basic environment and bonded with Arg 281, pyrogallol
type structure oriented towards acidic environment where
asp163 is present. In EGCG11, nucleus of galloyl moiety
interacts with Arg 282 and make two H bond in different
orientation. EGCG12 galloyl moiety is oriented towards
basic environment, Arg 281 of AP-1 protein and makes
H-bond with galloyl and pyrogallol type structure.
Hydroxyl group (OH) group of EGCG makes two H-bond
with Ser 278 polar amino acid of AP1 protein. In case of
EGCG06 galloyl moiety is oriented towards polar environment (cyan) and interact with positively charged amino
acid Arg 288 and negatively charged amino acid asp 170 (2
H bond; one with backbone and other with side chain) and
Gln 166 (cyan), same OH group of EGCG is making H
bond with Asp and Arg288 while pyrogallol type structure
is oriented towards polar environment by two H-bond with
Ser 278 (Fig. 3).
3.5 ADME analysis
ADME Prediction was done for all fifteen (15), out of these
only EGCH05, EGCG09, EGCG10, EGCG11, EGCG12
and EGCG15 were observed to possess oral bioavailability
of 12.716, 10.559, 23.319, 26.288, 32.713 and 12.600 ng/
ml respectively which is significantly larger than EGCGs
bioavailability 0.0 (Fig. 5). Out of these EGCG12 has
greater bioavailability than other EGCG derivative and
binds at Ser 278, Arg 281, Arg 285, and Arg 288 with six
hydrogen bonds on F chain of AP-protein. It does not

Fig. 4 Docking of derivative with AP-1 protein a docked view of EGCG05 with AP-1 protein, b docked view of EGCG15 with AP-1 protein

123

Netw Model Anal Health Inform Bioinforma (2014) 3:66

interact with Asp163 on G chain. EGCG has poor oral

bioavailability which can be increased by vitamin C.
Therefore, we tried to increase its oral bioavailability by
adding acetyl groups at different positions (Table 2) EGCG
in silico. EGCG has poor membrane permeability, low
chemical stability and rapid metabolism. A series of EGCG
fatty acid mono ester derivatives by lipase-catalyzed
transesterification were shown to have enhanced antiinfluenza virus action of EGCG up to 44-fold. The addition
of long acyl chains (C16C18) acylation also improved
antitumor activities of EGCG in an alkyl chain length
dependent manner in vitro (Matsumoto et al. 2012).
After oral absorption, tea catechins go through
extensive methylation, glucuronidation, and sulfation.
Rapid methylation of EGCG is catalyzed by liver
cytosolic catechol-O-methyltransferase. Hydrophilicity
of catecholic compounds is reduced by methylation;
further sulfation/glucuronidation is usually needed for
the effective elimination of methylated product from the
body (Lorenz et al. 2007). The level of methylation is
influenced by polymorphisms of catechol-O-methyltransferase, but the scope of this effect has not yet been
studied (Kida et al. 2000; Lee et al. 2002). Therefore, in
our study, and we avoid addition of methyl group (CH3)
in design of EGCG derivatives. Only one methoxy
group is added at R8 position of EGCG structure to
make EGCG05 by replacing OH group (Table 2).
EGCG bioavailability is enhanced by different approaches such as encapsulation of EGCG in nanoparticles
and enhancing intestinal absorption through polyphenol
stabilization (Dube et al. 2010), or the design and
semisynthesis O-acyl derivatives of EGCG (Vyas et al.
2007; Tanaka et al. 2007), or the solid-phase synthesis
of EGCG derivatives (Surh 1999). The hydroxyl groups
of (-)-EGCG are undergone biotransformation reactions, including methylation, glucuronidation, and sulfation and gets changed during this reaction. This leads
to reduced biological activities in vivo (Karin et al.
1997; Lu et al. 2003; Li et al. 2011).
More number of hydroxyl groups increase the
hydrophilic character of drugs which makes drug to
flush out from body in shorter duration and decrease the
amount of drug which is required to achieve therapeutic
effect of drug (Table 5). In our study, OH group of
EGCG have been replaced by OCOCH3 to analyse its
effect on their bioavailability, these acetylated derivatives shows better bioavailability than other derivatives
made by addition of other groups in silico (Table 6). In
these derivatives, two, three, four and five OH groups
are replaced by OCOCH3 group at R7 and R8; R6R8;
R4, R6R8 and R2, R4, R6R8 in EGCG 9EGCG12
respectively (Table 2) to increase bioavailability
because OH groups are more in EGCG structure. This

Page 9 of 13

66

gave us thought to remove these OH group while

chemical criteria for possibility of H bond conditions
are maintained. These derivatives show better affinity as
well as bioavailability in silico, but these predictions
needs to be evaluated in experimental conditions. All
though, bioavailability can be enhanced by taking
ascorbic acid with piperine, in vivo, increases up to
highest ever level of EGCG in plasma (purity [94 %)
without caffeine after all night fasting together with
200 mg ascorbic acid and 1,000 mg omega-3 fatty acids
from salmon (Mereles and Hunstein 2011).
Piperine (from black pepper), improved the bioavailability of EGCG in mice (Lee et al. 2002). Bioavailability of the tea polyphenol (-)-epigallocatechin3-gallate is improved by Piperine in mice. Modifications
have been done in order to maximise bioavailability
while maximizing binding affinity towards AP-1 protein. Stability of EGCG is affected by several other
factors, including temperature (Lambert et al. 2004),
oxygen concentration, antioxidant concentration, metal
ions (Hong Byun et al. 2010). Hard water with high
concentrations of Ca2? and Mg2? or drinking milk
together with EGCG leads to its activation (Li et al.
2008). EGCG absorption takes places mainly in the
small intestine and it is broken down to phenolic acids
in large intestine by the action of colonic micro flora
(Auger et al. 2008; Roowi et al. 2010; Nomura et al.
2000).
3.6 Toxicity study
Toxicity studies indicate that EGCG and its derivatives
were predicted as non-mutagenic, non-tumorigenic, and
non-irritant. Literature study reveals that hepatic gluconeogenesis is repressed by EGCG at concentrations that
are not toxic to hepatocytes and this is achieved by
intake of green tea or pure EGCG through 50 -AMPactivated protein kinase, which is mediated by the Ca2?/
calmodulin-dependent protein kinase (Collins et al.
2007). Natural compound resveratrol was predicted to
have mutagenic effect and genistein might have tumorigenic effect. The overall drug score was estimated by
combining outcome of cLogP, LogS, MW, toxicity risks
and drug likeness. Drug score is a measure of compounds potential to assemble the criteria of a probable
drug candidate. The drug-relevant properties predicted by
OSIRIS Property Explorer are given in Table 7. The
program estimated the risks of side effects, such as
mutagenic, tumorigenic, irritant and reproductive effects,
as well as drug-relevant properties. Moreover, the overall
drug score was estimated by combining outcome of
cLogP, LogS, MW, toxicity risks and drug likeness. Drug
likeness and drug score of EGCG05 are 2.36 and 0.71,

123

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Page 10 of 13

Netw Model Anal Health Inform Bioinforma (2014) 3:66

Fig. 5 a ADME prediction for natural compounds: QPlogBB

predicted brain/blood partition coefficient, prediction of binding to
human serum albumin (QPLog Khsa), apparent MDCK cell permeability (nm/s) (QPPMDCK), apparent CaCo-2 permeability (nm/s),
b predicted affinity of natural compounds and derivative in terms of

hydrogen bonding represented by vertical blue bar, c ADME

prediction of EGCG derivatives: QPlogBB predicted brain/blood
partition coefficient, prediction of binding to human serum albumin
(QPLog Khsa), apparent MDCK cell permeability (nm/s) great
(QPPMDCK), apparent CaCo-2 permeability (nm/s)

Table 5 ADME properties of natural compounds

Natural compound

CNS

QPlogBB

Human oral absorption

EGCG

-2

-4,209

% Human oral absorption

0.000

QPlogKhsa
-0.450

QPPMDCK
0.264

QPPCaCo
0.938

Curcumin

-2

-2.256

81.999

-0.026

64.457

151.758

Resveratrol

-2

-1.286

82.317

-0.172

124.988

280.045

Luteoline
Genistein

-2
-2

-1.910
-1.314

3
3

62.050
76.823

-0.205
-0.099

17.333
73.252

45.023
170.821

1.970

3,337.460

5,848.270

-0.663

2.663

7.958

Lupeol
Ellagic acid

1
-2

0.238

-2.396

which are higher than the respective scores of EGCG and

all its derivatives. Derivatives EGCG09- EGCG10,
EGCG11, EGCG12 and EGCG15 have 1.27 drug likeness score. Although one derivative EGCG02 has negative drug likeness score of -0.71 with drug score of 0.51.
One other derivative EGCG08 has lower drug score value
of 0.60 but has 1.02 drug likeness score.

123

100.00
35.420

4 Conclusion
The inhibitory effect of EGCG against AP-1 protein has been
studied by few researchers. EGCG obtained from green tea
can be used as lead molecule to further design potential
candidate drugs for liver cancer by a series of chemical
modification in the functional group of EGCG. In present

Netw Model Anal Health Inform Bioinforma (2014) 3:66

Table 6 ADME predictions on
predicted central nervous
system (CNS) activity on a -2
(inactive) to ?2 (active) scales,
QPlogBB predicted brain/blood
partition coefficient, prediction
of binding to human serum
albumin (QPLog Khsa),
apparent MDCK cell
permeability (nm/s) (\25 poor,
[500 great (QPPMDCK),
apparent Caco-2 permeability
(nm/s) (\25 poor, [500 great)
(QPPCaCo)

These properties were predicted

for all fifteen (15) derivatives

Derivatives

CNS

Page 11 of 13

QPlogBB

Human oral
absorption

% Human oral
absorption

QPlogKhsa

QPPMDCK

66

QPPCaCo

EGCG01

-2

-4.373

0.000

-0.493

0.260

0.925

EGCG02

-2

-4.075

6.090

-0.328

0.474

1.163

EGCG03

-2

-4.672

0.000

-0.483

0.144

0.534

EGCG04

-2

-4.676

0.000

-0.426

0.148

0.549

EGCG05

-2

-3.806

12.716

-0.244

0.909

2.945

EGCG06

-2

-3.827

0.000

-0.330

0.924

2.990

EGCG07

-2

-4.286

0.000

-0.312

0.496

1.680

EGCG08

-2

-4.414

0.000

-0.303

0.391

1.350
10.193

EGCG09

-2

-3.147

10.559

-0.276

3.480

EGCG10

-2

-3.148

23.319

-0.223

2.797

8.327

EGCG11
EGCG12

-2
-2

-3.518
-3.136

2
2

26.288
32.713

-0.227
-0.310

3.419
7.047

10.029
19.579

EGCG13

-2

-4.321

0.000

-0.301

0.386

1.333

EGCG14

-2

-4.569

0.000

-0.219

0.435

1.488

EGCG15

-2

-4.528

12.600

-0.180

0.712

2.348

Table 7 Toxic effects of natural compounds, EGCG and EGCG derivatives

S. no

Compounds

Mutagenic

Tumorigenic

Irritant

Reproductive effect

Drug likeness

Drug-score

Lycopene

Non-mutagenic

Non-tumoriginic

Non-irritant

Confirmed

-2.2

0.11

Betulinic acid

Non-mutagenic

Non-tumoriginic

Non-irritant

Confirmed

-4.44

0.15

Lupeol

Non-mutagenic

Non-tumoriginic

Non-irritant

Confirmed

-8.04

0.13

Resveratrol

Mutagenic

Non-tumoriginic

Non-irritant

No

-3.25

0.16

Ellagic acid

Non-mutagenic

Non-tumoriginic

Non-irritant

Confirmed

-1.6

0.51

Genistein

Mutagenic

Tumoriginic

Non-irritant

No

1.16

0.17

Curcumin

Non-mutagenic

Non-tumoriginic

Non-irritant

Confirmed

-3.27

0.42

Luteolin

Non-mutagenic

Non-tumoriginic

Non-irritant

Confirmed

1.9

0.84

EGCG

Non-mutagenic

Non-tumoriginic

Non-irritant

Confirmed

1.58

0.7

10

EGCG01

Non-mutagenic

Non-tumoriginic

Non-irritant

Confirmed

1.44

0.67

11
12

EGCG02
EGCG03

Non-mutagenic
Non-mutagenic

Non-tumoriginic
Non-tumoriginic

Non-irritant
Non-irritant

Confirmed
Confirmed

-0.71
0.72

0.51
0.63

13

EGCG04

Non-mutagenic

Non-tumoriginic

Non-irritant

Confirmed

0.53

0.62

14

EGCG05

Non-mutagenic

Non-tumoriginic

Non-irritant

Confirmed

2.36

0.71

15

EGCG06

Non-mutagenic

Non-tumoriginic

Non-irritant

Confirmed

0.78

0.58

16

EGCG07

Non-mutagenic

Non-tumoriginic

Non-irritant

Confirmed

0.78

0.58

17

EGCG08

Non-mutagenic

Non-tumoriginic

Non-irritant

Confirmed

1.02

0.6

18

EGCG09

Non-mutagenic

Non-tumoriginic

Non-irritant

Confirmed

1.27

0.43

19

EGCG10

Non-mutagenic

Non-tumoriginic

Non-irritant

Confirmed

1.27

0.37

20

EGCG11

Non-mutagenic

Non-tumoriginic

Non-irritant

Confirmed

1.27

0.31

21

EGCG12

Non-mutagenic

Non-tumoriginic

Non-irritant

Confirmed

1.27

0.26

22

EGCG13

Non-mutagenic

Non-tumoriginic

Non-irritant

Confirmed

1.62

0.63

23

EGCG14

Non-mutagenic

Non-tumoriginic

Non-irritant

Confirmed

1.02

0.52

24

EGCG15

Non-mutagenic

Non-tumoriginic

Non-irritant

Confirmed

1.27

0.37

123

66

Page 12 of 13

study, some natural compounds were taken into consideration and their inhibitory role against AP-1 protein was
evaluated by computational methods. Lupeol, betulinic acid
lycopene do not show good affinity or hydrogen bonding
interactions to AP-1 protein. Derivative EGCG05 shows
greater affinity with ASP 163, ASP 170, SER 278, ARG 281,
and ARG 288 with eight hydrogen bonds towards AP-1
protein and a free energy complex of -6.30 kcal/mol. One of
acetylated derivative EGCG15, made by replacing OH group
OCOCH3 at R2, R4 and R7 position also shows better affinity
than other derivatives and binds at amino acid ASP 163(G),
SER 278(F), LYS 282(F), ARG 288(F) with six hydrogen
bonds and -5.60 kcal/mol energy. Substitution of OH group
by OCOCH3 at various positions of EGCG leads to increase
in bioavailability. Substitution by OCH3 leads to slight
increment of 6 % in oral absorption. Substitution by NH2
group leads to no changes in oral absorption or bioavailability. This may lead to inhibition of AP-1 protein which
down regulates p53 and PTEN gene. These genes are tumour
suppressor gene and their down regulation is fundamental
cause of liver cancer. This study indicates that inhibition of
AP-1 protein by EGCG acetylated derivative may be
achieved by targeting interacted amino acids.

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