Vol 3, Issue 2, 2021 (61-70)
http://journal.unpad.ac.id/idjp
*Corresponding author,
e-mail : abdulkaharumar@gmail.com (A. K. Umar)
https://doi.org/10.24198/idjp.v3i2.35849
© 2021 A. K. Umar et al
Structure-Based Virtual Screening and Molecular Dynamics of Quercetin and Its
Natural Derivatives as Potent Oxidative Stress Modulators in ROS-induced Cancer
Abd. Kakhar Umar1,*, James H. Zothantluanga2
1Department of Pharmacy, Faculty of Math and Natural Sciences, Universitas Tadulako, Palu
94148, Indonesia
2Department of Pharmaceutical Sciences, Faculty of Science and Engineering, Dibrugarh
University, Dibrugarh 786004, Assam, India
Submitted : 29 July 2021, Revised : 12 August 2021, Accepted : 13 August 2021, Published : 20 August 2021
Abstract
Quercetin derivatives are known to have significant anticancer activity. The activity
is strongly influenced by the type and position of the substituent group. By studying
the structural pattern of quercetin and its impact on their binding affinity, the
development of quercetin-based drugs can be optimized. The study aimed to
determine the impact of 3D structure, type, and position of quercetin moiety on its
activity against ROS-modulating enzymes that play a role in the induction and
growth of ROS-induced cancer. The 23 natural quercetin derivatives were docked
to 7 ROS-modulating enzymes using Autodock Vina to determine their binding
affinity and interaction. The interaction stability was further studied through
molecular dynamics simulation using the CABS Flex 2.0 server. Determination of
crucial amino acid targets of the quercetin group was determined using DockFlin.
Finally, the toxicity of each test ligand was determined using the pkCSM server.
The highest binding affinity for SOD and NOX was produced by quercetin 3'-
glucoside with the binding energy of -10.2 and -12.8 kcal/mol. Quercetin 3,4'-
diglucoside had the highest binding affinity for CAT and GR at -11.5 and -10.5
kcal/mol, respectively. Routine produced the highest binding affinity at LOX (-
10.9). Quercetin 3-O-xyloside and quercetin 3-O-rhamnoside-7-O-glucoside had
the highest binding affinity in XO with a value of -10.4 kcal/mol. The glucose and
prenyl groups are beneficial for quercetin in interacting with all ROS-modulating
enzymes except XO. In contrast, the methoxy group negatively affects all
interactions of quercetin with receptors. The perfect fit between the binding pocket
and the 3D structure of the ligand greatly benefits the ligand in accessing more
amino acids in the binding pocket. Their interaction stability and toxicity show that
quercetin 3'-glucoside, quercetin 3,4'-diglucoside, and rutin are potent oxidative
stress modulators in treating ROS-induced cancer.
Keywords: ROS-induced cancer, Quercetin derivates, Oxidative stress modulator,
ROS-modulating enzymes
A. K. Umar et al / Indo J Pharm 3 (2021) 57-70
61
1. Introduction
The imbalance between the number
of free radicals (ROS) and reactive
metabolites (antioxidants) in the body
causes oxidative stress. High amounts of
ROS can accelerate the oxidation process in
normal cells, leading to cell damage.
Premature aging and the onset of chronic
diseases such as cancer are the most
prominent outcomes. It is widely
acknowledged that oxidative stress
promotes tumor genesis and growth by
causing genetic instability [36]. As a result,
focusing on redox-sensitive pathways and
transcription factors has significant
potential for cancer prevention and
treatment [10, 27]. Lipoxygenase (LOX),
NADPH oxidase (NOX), and xanthine
oxidase (XO) are enzymes regulating ROS
generation. The inhibition of these three
enzymes significantly impacts the
suppression of ROS generation and cancer
progression [5, 10, 16, 22, 25, 31].
Likewise, induction of the catalase (CAT),
glutathione reductase (GR), glutathione
peroxidase (GPx), and superoxide
dismutase (SOD) can reduce ROS levels
and prevent cell damage [3, 7, 11, 12, 14,
34].
One of the powerful natural
antioxidants is quercetin. Quercetin has
been shown to effectively prevent and
inhibit cancer growth via the regulation of
ROS [33, 35, 37]. Quercetin was reported
to increase catalase activity up to 28.6% in
3-NP treated animals [29]. In addition,
quercetin also inhibits the activity of
several pro-oxidant enzymes such as LOX,
NOX, and XO [4, 9, 17, 18, 28, 30, 37]. The
activity is strongly influenced by the
structure and position of functional group
on quercetin (see Figure 1). The
substitution of functional groups of
quercetin impacts its biochemical and
pharmacological properties [20, 26, 33].
Therefore, this research was conducted to
study the impact of 3D structure, type, and
position of quercetin moiety on its activity
against ROS-modulating enzymes to
develop more optimal quercetin-based
drugs in treating ROS-induced cancer. The
23 natural quercetin derivates are present in
fruit, seeds, tubers, and honey [21]. In this
study, 23 natural quercetin derivatives were
docked on 7 ROS-modulating enzymes,
and then a molecular dynamics study was
conducted to determine the stability of the
ligand-protein interactions. Toxicity
studies were also carried out to assess the
safety of the test ligands.
Figure 1. Quercetin basic structure.
2. Methods
2.1 Ligand Preparation
The test ligands were quercetin and
its derivates found in plants [21]. Some
ligand structures were downloaded from
PubChem, and the rest were drawn using
ChemDraw Pro 12.0 (PerkinElmer
Informatics, PerkinElmer Inc, USA).
Structure errors were checked using the
"Check Structure" feature, and then the
structures were cleaned using the "Clean
Up Structure" feature in ChemDraw. The
default geometry of each ligand was
removed using the "Clean Geometry"
feature in Discovery Studio 2021 Client
(DS) (BIOVIA, San Diego, CA, USA).
62
Energy minimization (MM2) was
performed using Chem3D. Each ligand was
then optimized using AutoDockTools 1.5.6
(ADT) (TheScripps Research Institute,
USA) to add Gasteiger charges, set
rotatable bonds, and TORSDOF.
Furthermore, the ligands are stored in
PDBQT format.
2.2 Protein Preparation
The structure of the proteins was
obtained from the Protein Data Bank (PDB)
website. The code for each protein used is
lipoxygenase (3O8Y), NADPH oxidase
(5O0X), xanthine oxidase (3BDJ), catalase
(1DGF), glutathione reductase (1XAN),
glutathione peroxidase (6ELW), and
superoxide dismutase (2C9V). Native
ligand and protein were separated using the
DS. The protein was optimized using ADT
to remove water, regulate the charges
(Kollman charges), and add polar
hydrogen. The protein was then stored in
PDBQT format. The grid position was
arranged based on the active site attached
by the native ligand. The grid dimension
was set to 40 x 40 x 40 magnification with
a spacing of 0.375Å. Gridbox parameters
can be seen in Table 1.
Table 1. The ROS enzymes’ gridbox
parameters.
Enzymes
Active sites
Coordinate (Å)
Lipoxygenase
4,976 x 21,401 x
0,286
NADPH Oxidase
65,738 x 0,875 x
62,590
Xanthin Oxidase
17,175 x -17,782 x
16,527
Catalase
14,877 x 15,793 x
78,537
Glutathione
Reductase
82,170 x -5,827 x
36,154
Glutathione
Peroxidase
42,474 x 14,099 x -
18,061
Superoxide
Dismutase
95,611 x 47,146 x
112,872
2.3 Molecular Docking
Molecular docking was performed
using DockFlin software (ETFLIN,
Indonesia). This software is a tool for
systematically scheduling multi-ligand and
multi-protein docking processes by the
Autodock Vina. Ligands and proteins were
added to the respective list panel, then the
docking parameters per protein were loaded
in the order in the grid list panel. The
docking parameters used were energy range
of 4 and exhaustiveness of 8. The operating
system used was Windows 10 Home Single
Language 64 bit with AMD Ryzen 5
3500U, Radeon Vega Mobile Gfx 2.10
GHz, and RAM of 8 GB.
2.4 Molecular Dynamics Study
The protein's stable structure was
studied using the CABS Flex 2.0 server,
based on coarse-grained simulations of
protein motion [15]. Distance restraints
generator mode was SS2 with minimal
restraint length of 3.8 Å and maximal
restraint length of 8.0 Å. The number of
cycles and trajectory frames was set to 50,
with a global weight of 1.0 and a
temperature of 1.4. The distance restraints
generator was set to default values. The
output of this step is ten structural models
for each enzyme based on their flexibility.
Each model of each enzyme was then re-
docked with potent ligands obtained from
the molecular docking results. The
fluctuation of the binding energy of each
ligand-protein interaction is presented in
63
the form of a line graph. This test aimed to
see whether the ligand-protein interaction
remains stable during attachment without
losing binding energy on all models [2].
2.5 Determination of Crucial Amino
Acid
This test was carried out to determine the
most active amino acid residues at the
binding site of the proteins based on the
habit of a group of compounds interacting
with the protein. The output file of the
docking process (Autodock Vina) produced
nine ligand-protein interaction models for
each ligand. The active amino acid of each
protein receptor was determined based on
its occurrence (binding ligands via
hydrogen bonds) in each model and all
ligand-protein complexes. The number of
occurrences has been scored using
DockFlin. If an amino acid has a score of
more than 9, it is easily accessible and
preferred as a target for binding [32].
2.6 Toxicity prediction of quercetin
derivates
Prediction of acute oral toxicity (LD50)
was carried out using pkCSM ADMET to
determine the safety of quercetin and its
derivates. The SMILES string for each
ligand was obtained from a PDB file
converted to SMI format using DS.
3. Results
3.1 Molecular Docking
Based on the molecular docking results, it
was found that quercetin has strong
interactions with antioxidant and pro-
oxidant enzymes, especially with CAT,
LOX, and NOX. Quercetin and its
derivatives produced binding energy in
CAT of -8.9 to -11.5 kcal/mol, GR of -7.6
to -10.5 kcal/mol, GPx of -6.9 to -8.8
kcal/mol, SOD of -7.5 to -10.2 kcal/mol,
LOX of -7.6 to -10.9 kcal/mol, NOX of -9.7
to -12.8 kcal/mol, and XO of -7.4 to -10.4
kcal/mol. Each binding energy of the test
ligands can be seen in Table 2.
Table 2. Binding energy of test ligand on each ROS-modulating enzymes.
Compound Name
Ligand Code
CAT
GR
GPx
SOD
LOX
NOX
XO
Quercetin
Ligand 0
-9.5
-8.8
-7.5
-9
-9.1
-10.8
-9.6
Quercetin 3-O-galactoside
Ligand 1
-9.7
-8.7
-7.6
-8.3
-10.2
-10.4
-8
Quercetin 3-O-glucoside
Ligand 2
-9.5
-8.6
-7.5
-8.2
-10.2
-10.3
-9.3
Quercetin 3-O-rhamnoside
Ligand 3
-9.6
-8.7
-7.8
-8.9
-9.8
-10.8
-7.9
Quercetin 3-O-rhamnozyl-(1→6)-glucoside
Ligand 4
-10.4
-8.2
-8.8
-8.1
-10.9
-10.9
-9.2
Quercetin 7-O- glucoside
Ligand 5
-10.9
-9.3
-8.6
-8.9
-10.4
-11.5
-8.3
Quercetin 3-O-rhamnoside-7-O-glucoside
Ligand 6
-9.5
-8
-7.3
-7.7
-8.7
-9.9
-10.4
Quercetin 6-C- glucoside
Ligand 7
-10.5
-9.9
-8.1
-9
-8.9
-11.1
-8.1
Quercetin 3-(2’’-acetylgalactoside)
Ligand 8
-9
-7.6
-7.5
-7.5
-7.6
-11
-7.4
Quercetin 3-sulfate-7-O-arabinoside
Ligand 9
-10.3
-9.2
-8.7
-9.1
-9.9
-10.1
-8.1
Quercetin 3-O-glucoside-3’-sulfate
Ligand 10
-9.5
-9.2
-7.9
-8.3
-9.8
-10.3
-8
Quercetin 5-methyl ether
Ligand 11
-9.4
-8.5
-7.3
-7.8
-8.9
-9.7
-8.9
Quercetin 7- methyl ether
Ligand 12
-9.4
-7.8
-7.4
-8.8
-8.9
-10.6
-7.7
Quercetin 3’- methyl ether
Ligand 13
-9.4
-8.5
-7.3
-8.8
-8.3
-10.7
-8.7
Quercetin 4’- methyl ether
Ligand 14
-9.4
-8.5
-7.5
-8.5
-8.7
-10.6
-9.5
Quercetin 7-methoxy-3-O-glucoside
Ligand 15
-10
-8.2
-7.6
-8.2
-9.7
-10.7
-8
61
Quercetin 3’- methoxy -3-O-galactoside
Ligand 16
-9.9
-8
-7.5
-8.3
-9
-10.3
-7.9
6,5’-Di-C-prenylquercetin
Ligand 17
-10.3
-9.1
-8.4
-9.4
-10.6
-11.7
-9.1
Quercetin 3-O-xyloside
Ligand 18
-9.8
-8.2
-7.5
-8.3
-9.5
-9.9
-10.4
Quercetin 3-O-glucuronide
Ligand 19
-9.9
-8.3
-8.4
-8.4
-10.1
-10.9
-9.9
Quercetin 3,4’-diglucoside
Ligand 20
-11.5
-10.5
-8.7
-9.3
-10.6
-10
-8.3
Quercetin 3-O-6’’-acetylglucoside
Ligand 21
-11
-8.4
-7.8
-7.9
-10.3
-11.2
-7.8
Quercetin 3,3’-dimethyl ether
Ligand 22
-8.9
-8
-6.9
-8.6
-8.1
-10.3
-8.8
Quercetin 3'-glucoside
Ligand 23
-11.2
-9.8
-8.8
-10.2
-10.7
-12.8
-8.8
In the XO enzyme, structural modification
of quercetin will generally decrease its
binding energy except for the substitution
of xylose and glucuronate at the C-3 atom.
In other enzymes, the substitution of
glucose, prenyl, arabinose, and glucuronate
groups generally increases the binding
energy of quercetin. However, the binding
energy of quercetin can be decreased if
there is a methoxy group as an alkyl group.
The increasing or decreasing percentage in
the binding energy of quercetin based on its
functional group can be seen in Table 3.
Table 3. Changes in binding energy due to the influence of substituents
Ligand
Code
Moiety position
Differences in binding energy (%)*
R1
R2
R3
R4
R5
R6
R7
CAT
GR
GPx
SOD
LOX
NOX
XO
Average
Ligand 23
OH
OH
H
OH
Glu
OH
H
17.9
11.4
17.3
13.3
17.6
18.5
-8.3
12.5
Ligand 20
Glu
OH
H
OH
OH
Glu
H
21.1
19.3
16.0
3.3
16.5
-7.4
-13.5
7.9
Ligand 17
OH
OH
Pre
OH
OH
OH
Pre
8.4
3.4
12.0
4.4
16.5
8.3
-5.2
6.8
Ligand 5
OH
OH
H
Glu
OH
OH
H
14.7
5.7
14.7
-1.1
14.3
6.5
-13.5
5.9
Ligand 4
Glu
&
Rha
OH
H
OH
OH
OH
H
9.5
-6.8
17.3
-10.0
19.8
0.9
-4.2
3.8
Ligand 19
Gcr
OH
H
OH
OH
OH
H
4.2
-5.7
12.0
-6.7
11.0
0.9
3.1
2.7
Ligand 9
Sul
OH
H
Ara
OH
OH
H
8.4
4.5
16.0
1.1
8.8
-6.5
-15.6
2.4
Ligand 7
OH
OH
Glu
OH
OH
OH
H
10.5
12.5
8.0
0.0
-2.2
2.8
-15.6
2.3
Ligand 21
6-A
OH
H
OH
OH
OH
H
15.8
-4.5
4.0
-12.2
13.2
3.7
-18.8
0.2
Ligand 0
OH
OH
H
OH
OH
OH
H
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
Ligand 2
Glu
OH
H
OH
OH
OH
H
0.0
-2.3
0.0
-8.9
12.1
-4.6
-3.1
-1.0
Ligand 18
Xyl
OH
H
OH
OH
OH
H
3.2
-6.8
0.0
-7.8
4.4
-8.3
8.3
-1.0
Ligand 3
Rha
OH
H
OH
OH
OH
H
1.1
-1.1
4.0
-1.1
7.7
0.0
-17.7
-1.0
Ligand 10
Glu
OH
H
OH
Sul
OH
H
0.0
4.5
5.3
-7.8
7.7
-4.6
-16.7
-1.6
Ligand 1
Gal
OH
H
OH
OH
OH
H
2.1
-1.1
1.3
-7.8
12.1
-3.7
-16.7
-2.0
Ligand 14
OH
OH
H
OH
OH
Met
H
-1.1
-3.4
0.0
-5.6
-4.4
-1.9
-1.0
-2.5
Ligand 15
Glu
OH
H
Met
OH
OH
H
5.3
-6.8
1.3
-8.9
6.6
-0.9
-16.7
-2.9
Ligand 13
OH
OH
H
OH
Met
OH
H
-1.1
-3.4
-2.7
-2.2
-8.8
-0.9
-9.4
-4.1
Ligand 6
Rha
OH
H
Glu
OH
OH
H
0.0
-9.1
-2.7
-14.4
-4.4
-8.3
8.3
-4.4
Ligand 16
Gal
OH
H
OH
Met
OH
H
4.2
-9.1
0.0
-7.8
-1.1
-4.6
-17.7
-5.2
Ligand 12
OH
OH
H
Met
OH
OH
H
-1.1
-11.4
-1.3
-2.2
-2.2
-1.9
-19.8
-5.7
Ligand 11
OH
Met
H
OH
OH
OH
H
-1.1
-3.4
-2.7
-13.3
-2.2
-10.2
-7.3
-5.7
Ligand 22
Met
OH
H
OH
Met
OH
H
-6.3
-9.1
-8.0
-4.4
-11.0
-4.6
-8.3
-7.4
62
Ligand 8
2-A
OH
H
OH
OH
OH
H
-5.3
-13.6
0.0
-16.7
-16.5
1.9
-22.9
-10.4
Note: Glu = glucose, Met = methoxy, Rha = rhamnose, Sul = sulfate, Gal = galactose, Pre =
prenyl, Gcr = glucuronate, Ara = arabinose, 6-A = 6-acetylglucose, 2-A = 2-acetylgalactose,
and Xyl = xylose. *A negative percentage indicates a reduction in binding affinity and a
positive percentage indicates an increase in binding affinity (compared to basic quercetin).
3.2 Molecular Dynamics Study
Molecular dynamics simulations were only
carried out on enzymes that have strong
potential to become targets of quercetin
derivatives, namely CAT, LOX, and NOX.
Based on the molecular dynamics
simulation, it can be seen that the flexibility
of the protein structure does not change the
3D pattern of the enzyme significantly (see
Figure 2). Each enzyme's binding site
retains a similar shape and coordinates to
not interfere with the ligand binding.
Figure 2. Simulation of protein flexibility and ligand-protein dynamics interaction of (A)
quercetin 3,4'-diglucoside and CAT, (B) quercetin 3-O-rhamnozyl-(1→6)-glucoside and
LOX, and (C) quercetin 3'-glucoside and NOX.
After re-docking the potent ligands on each
model of the three target enzymes, it was
found that the interactions of CAT-Ligand
20, LOX-Ligand 4, and NOX-Ligand 23
remained stable without losing binding
energy in any of the models. The average
binding energies of CAT-Ligand 20, LOX-
Ligand 4, and NOX-Ligand 23 were -9.06
± 0.55, -9.02 ± 0.97, and 8.77 ± 0.48
kcal/mol. The fluctuations of the three
interactions can be seen in Figure 2.
3.3 Determination of Crucial Amino
Acid
Based on the scoring results, it is known
that the CAT enzyme has ten crucial amino
acids that were active in forming hydrogen
bonds with compounds from the quercetin
group. In LOX, there are four crucial amino
acids, while in NOX, there are nine crucial
amino acids. The average bond length,
hydrogen bond types, and scores of each
amino acid in each enzyme can be seen in
Table 4.
3.4 Toxicity prediction of quercetin
derivates
Toxicity data of each test ligand can be seen
in Table 5. Ligand 22 has the highest dose
tolerance, while ligand 4 has the lowest
dose tolerance. The predicted dose is
recommended for use in phase I clinical
trials. All tested ligands are non-toxic to the
liver and do not induce skin sensitization.
63
Figure 3. Fluctuations in the binding energy of CAT-Ligand 20, LOX-Ligand 4, and NOX-
Ligand 23 in each model.
Table 4. The crucial amino acid at the binding sites of CAT, LOX, and NOX.
Enzyme
Crucial
Amino
Acids
Average
Bond Length
(Å)
Average Bond Type
Score
CAT
His305
4.638
Conventional Hydrogen
36.91
Arg203
4.475
Conventional Hydrogen
22.64
His305
4.587
Carbon Donor Hydrogen
19.73
Phe446
4.695
Carbon Donor Hydrogen
19.55
Phe198
4.52
Carbon Donor Hydrogen
19.09
Ser201
3.971
Conventional Hydrogen
14.36
Asp202
4.715
Conventional Hydrogen
11.64
His194
4.487
Conventional Hydrogen
11.09
Gln442
4.38
Conventional Hydrogen
9.41
Ala445
5.076
Carbon Donor Hydrogen
9.14
LOX
Asp170
4.672
Conventional Hydrogen
16.50
Val243
4.598
Conventional Hydrogen
16.27
Asp442
4.728
Conventional Hydrogen
9.41
Ser447
4.544
Conventional Hydrogen
9.00
NOX
Thr462
3.74
Conventional Hydrogen
29.05
Arg478
4.09
Conventional Hydrogen
23.14
Pro460
4.41
Conventional Hydrogen
23.14
Phe461
4.44
Carbon Donor Hydrogen
18.68
Thr462
4.66
Carbon Donor Hydrogen
12.18
His459
4.81
Carbon Donor Hydrogen
10.82
Pro460
5.17
Carbon Donor Hydrogen
10.32
Thr484
4.24
Conventional Hydrogen
9.77
Trp695
4.43
Conventional Hydrogen
9.41
6
6.5
7
7.5
8
8.5
9
9.5
10
10.5
11
1 2 3 4 5 6 7 8 9 10
Binding energy (in positif)
Protein models
CAT-Ligand 20 LOX-Ligand 4 NOX-Ligand 23
A. K. Umar et al / Indo J Pharm 3 (2021) 57-70
64
Table 5. Toxicity prediction of quercetin derivates.
Ligand
Max. tolerated
human dose
(mg/KgBW/day)
Hepatotoxicity
Skin Sensitisation
Ligand 0
5.13
No
No
Ligand 1
15.10
No
No
Ligand 2
12.85
No
No
Ligand 3
8.93
No
No
Ligand 4
2.62
No
No
Ligand 5
8.38
No
No
Ligand 6
10.02
No
No
Ligand 7
3.05
No
No
Ligand 8
5.77
No
No
Ligand 9
3.23
No
No
Ligand 10
8.93
No
No
Ligand 11
5.62
No
No
Ligand 12
4.15
No
No
Ligand 13
3.14
No
No
Ligand 14
5.19
No
No
Ligand 15
4.36
No
No
Ligand 16
6.59
No
No
Ligand 17
6.59
No
No
Ligand 18
6.92
No
No
Ligand 19
6.55
No
No
Ligand 20
7.67
No
No
Ligand 21
8.83
No
No
Ligand 22
7.48
No
No
Ligand 23
12.42
No
No
4. Discussion
Quercetin is a powerful natural antioxidant.
Changes in functional groups in the basic
structure of quercetin will have a significant
effect on its pharmacological activity [20,
26, 33]. This study found that the
dimensions, position, and type of
substituent functional groups of quercetin
significantly affect their interactions with
ROS-modulating enzymes. The most
abundant substituent groups in quercetin
derivatives were glucose (9 compounds),
followed by methoxy (7 compounds),
rhamnose (3 compounds), sulfate (2
compounds), galactose (2 compounds),
prenyl, glucuronate, arabinose, 6-
acetylglucose, 2-acetylgalactose, and
xylose. The basic structure of quercetin can
be seen in Figure 1.
Based on the molecular docking results, it
was found that, on average, quercetin and
its derivatives only bind strongly to CAT,
LOX, and NOX enzymes. The mean
binding energies for GR, GPx, SOD, and
XO were -8.67 ± 0.708, -7.85 ± 0.566, -8.56
± 0.61, and -8.67 ± 0.854 kcal/mol,
respectively. Meanwhile, the average
65
binding energy of CAT, LOX, and NOX
were -9.938 ± 0.69, -9,538 ± 0.9, and -
10,688 ± 0.676 kcal/mol, respectively.
Quercetin was reported to have no
significant effect on glutathione reductase
and glutathione peroxidase [8]. SOD
activity is also said to not increase
significantly after being given quercetin
[19]. However, some quercetin derivatives
still provide high binding affinity for all
enzymes, except GPx.
Quercetin 3,4'-diglucoside produced the
highest binding energies for CAT and GR
at -11.5 and -10.5 kcal/mol, respectively.
The highest binding affinity for SOD and
NOX was produced by quercetin 3'-
glucoside with a binding energy of -10.2
and -12.8 kcal/mol. Rutin (ligand 4) had the
highest binding affinity at LOX (-10.9).
Quercetin 3-O-xyloside and quercetin 3-O-
rhamnoside-7-O-glucoside produced the
highest binding affinity in XO with a value
of -10.4 kcal/mol. Based on these values, it
can be seen that all quercetin derivatives
that have a glucose group as a substituent
are potent ligands for all enzymes. The
effect depends strongly on the position of
glucose [38, 39]. Glucose substituents in
rings A and C are the main contributors to
quercetin activity. In Table 3, it can be seen
that the substitution of glucose on the 3-O
atom did not significantly increase the
binding affinity of all enzymes, except
LOX. The glucose group on the C ring of
quercetin (see Figure 1) increases binding
affinity significantly in all enzymes except
XO. Still, it decreases considerably in NOX
if there is another group on the 3-O atom. It
is because other groups on the 3-O atom
affect the position of the ligand entry into
the NOX binding pocket (see Figure 4). The
3-O atom's glucose group appears to bind
the residue outside the binding pocket,
causing the ligand to become stranded
outside. It caused quercetin 3,4'-
diglucoside (see Figure 4B) to lose the four
hydrogen bonds it would have formed if it
had managed to fit snugly into the binding
pocket like quercetin 3'-glucoside (see
Figure 4A).
Figure 4. Quercetin 3’-glucoside (A) and quercetin 3,4’-diglucoside (B) on NOX binding
pocket.
66
Figure 4. Interaction of rutin (A), quercetin 3’-glucoside (B), and 6,5’-Di-C-prenylquercetin
(C) on LOX binding pocket in 2D and 3D.
In the LOX enzyme, glucose and rhamnose
complex (rutin) produced the highest
binding affinity, followed by quercetin 3'-
glucoside and 6,5'-Di-C-prenylquercetin.
This is due to the broader dimensions of the
binding pocket so that ligand with large 3D
volumes can reach and interact more with
amino acid residues. In Figure 5, it can be
seen that rutin forms nine conventional
hydrogen bonds and two hydrogen carbons,
quercetin 3'-glucoside forms seven
conventional hydrogen bonds and one
hydrogen carbon. In comparison, Di-C-
prenylquercetin only includes four
conventional hydrogen bonds and one
hydrogen carbon. In contrast to rutin,
quercetin 3'-glucoside and 6,5'-Di-C-
prenylquercetin have a slender and
elongated structure so that they bind amino
acids that are only in the elongation
pathway. For that, it is more suited to the
extended binding pocket like the NOX.
In addition to the glucose group, the prenyl
group also significantly increased the
binding affinity of quercetin in all enzymes
except XO. The compound 6,5'-Di-C-
prenylquercetin binds strongly to the LOX.
67
The two prenyl groups at the ends of the
quercetin structure act as anchors and
anchors to the LOX binding pocket and
form pi-alkyl bonds with the amino acids
Ala439 and Leu448 (see Figure 5C).
Adding a prenyl group often increases the
pharmacological activity of aromatic
compounds such as quercetin [1, 6, 13].
Prenylation of the flavonoid structure can
also enhance the bioavailability, allowing
the effect to last longer [24].
The 2-acetylgalactose, methoxy, xylose,
and sulphate groups have a terrible effect
on the activity of quercetin. The acetyl
group at the C-2 position prevents the
formation of hydrogen bonds from the
galactose hydroxy group. All ligands with a
methoxy group had their hydrogen bonds
with all enzymes weaken. Research
conducted by Z. Sroka et al. (2017) also
showed decreased activity due to a methoxy
group in ring B of quercetin [30]. The
methoxy group can block the formation of
hydrogen bonds or weaken them [23]. In
XO, all substitutions on atoms other than C-
3 (R3) will decrease the binding affinity of
quercetin. Xylose, rhamnose, and
glucuronate are favorable C-3 substituents
for quercetin and XO interactions.
Crucial amino acids were obtained by
scoring their presence in binding to ligands
in each Autodock Vina docking output
model. Amino acids that get a score of more
than nine can be said to be easily accesible
and form hydrogen bonds in every
interaction model. The more derivatives
used, the more accurate the results for that
group of compounds. Molecular docking
results showed that potent ligands such as
quercetin 3'-glucoside, quercetin 3,4'-
diglucoside, and rutin formed hydrogen
bonds with crucial amino acid residues to
produce stable bonds in each flexible model
of the enzyme. Based on toxicity studies,
quercetin 3'-glucoside and quercetin 3,4'-
diglucoside have a higher dose tolerance
than quercetin, are non-hepatotoxic, and do
not induce skin sensitization.
5. Conclusion
Some quercetin derivatives produce greater
binding affinity than basic quercetin. The
3D volume of the structure, type, and
position of the substituent groups plays a
significant role in determining the
interaction of quercetin and ROS-
modulating enzymes. The glucose and
prenyl groups are beneficial for quercetin in
interacting with all ROS-modulating
enzymes except XO. In contrast, the
methoxy group negatively affects all
interactions of quercetin with receptors.
Based on molecular docking studies,
interaction stability, and toxicity, we
conclude that quercetin 3'-glucoside ,
quercetin 3,4'-diglucoside, and rutin are
potent oxidative stress modulators in
treating ROS-induced cancer with the
binding energy of -12.8 kcal/mol, -11.5 and
-10.5 kcal/mol, respectively.
Acknowledgement
The authors are thankful to Chusnul Nur
Ramadhani for her assistance in finding the
quercetin derivates.
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