Interdiscip Sci Comput Life Sci (2017) 9:499–511
https://doi.org/10.1007/s12539-016-0157-8
ORIGINAL RESEARCH ARTICLE
Molecular Docking and Molecular Dynamics Simulation Studies
to Predict Flavonoid Binding on the Surface of DENV2 E Protein
Nurul Azira Ismail1 · Siti Azma Jusoh1
Received: 14 September 2015 / Revised: 22 January 2016 / Accepted: 25 February 2016 / Published online: 11 March 2016
© International Association of Scientists in the Interdisciplinary Areas and Springer-Verlag Berlin Heidelberg 2016
Abstract Dengue infections are currently estimated to be
390 million cases annually. Yet, there is no vaccine or
specific therapy available. Envelope glycoprotein E (E
protein) of DENV mediates viral attachment and entry into
the host cells. Several flavonoids have been shown to
inhibit HIV-1 and hepatitis C virus entry during the virus–
host membrane fusion. In this work, molecular docking
method was employed to predict the binding of nine fla-
vonoids (baicalin, baicalein, EGCG, fisetin, glabranine,
hyperoside, ladanein, quercetin and flavone) to the soluble
ectodomain of DENV type 2 (DENV2) E protein. Inter-
estingly, eight flavonoids were found to dock into the same
binding pocket located between the domain I and domain II
of different subunits of E protein. Consistent docking
results were observed not only for the E protein structures
of the DENV2-Thai and DENV2-Malaysia (a homology
model) but also for the E protein structures of tick-borne
encephalitis virus and Japanese encephalitis virus. In
addition, molecular dynamics simulations were performed
to further evaluate the interaction profile of the docked E
protein–flavonoid complexes. Ile4, Gly5, Asp98, Gly100
and Val151 residues of the DENV2-My E protein that
aligned to the same residues in the DENV2-Thai E protein
form consistent hydrogen bond interactions with baicalein,
Electronic supplementary material The online version of this
article (doi:10.1007/s12539-016-0157-8) contains supplementary
material, which is available to authorized users.
& Siti Azma Jusoh
sitiazma@puncakalam.uitm.edu.my; sitiazma@gmail.com
1 anchored onto the viral lipid bilayer. The RNA genome
Department of Pharmaceutical Life Sciences, Faculty of
Pharmacy, Universiti Teknologi MARA, Puncak Alam
Campus, 42300 Bandar Puncak Alam, Malaysia
quercetin and EGCG during the simulations. This study
demonstrates flavonoids potentially form interactions with
the E protein of DENV2.
Keywords Dengue virus Malaysia · DENV2 ·
Flavonoids · Baicalin · Baicalein · EGCG · Fisetin ·
Glabranine · Hyperoside · Ladanein · Quercetin · Docking ·
Molecular dynamics simulations · Envelope glycoprotein E
1 Introduction
Dengue virus (DENV) infection transmitted by infected
Aedes mosquitoes, particularly Aedes aegypti [1, 2], is now
becoming a global health threat estimated to infect 390
million people annually in tropical and subtropical coun-
tries [3, 4]. The virus causes dengue fever and in severe
cases leads to lethal hemorrhagic fever/shock syndrome. In
Malaysia, dengue infection is a serious public health
problem, and a major outbreak occurs every 4 years [5]. In
2007, there were more than forty-eight thousand dengue
infection cases in Malaysia alone. Yet, there is no vaccine
or specific antiviral available.
DENV is a member of Flaviviridae family and catego-
rized in the subfamily of flavivirus. There are four DENV
serotypes (DENV1, DENV2, DENV3 and DENV4), which
are distinct but closely related to clinical manifestations
and epidemiology [6]. Other well-known viruses in the
same subfamily are West Nile virus (WNV), Japanese
encephalitis virus (JEV) and tick-borne encephalitis virus
(TBEV). The mature DENV virion consists of 180 copies
of envelope glycoprotein E and M on the external shell that
encodes a single polyprotein that is co-translationally and
posttranslationally processed into three structural (capsid,
123
500
prM and E proteins) and seven non-structural proteins
(NS1, NS2a, NS2b, NS3, NS4a, NS4b and NS5) [7, 8].
The E protein plays a vital role in the viral entry by
mediating the viral attachment to the host cell receptors
and involves in the fusion process between the viral and the
host membrane [9]. The virus entry process initially starts
in the endosome, and it is triggered by low pH environment
(~6.5 and below), which induces histidine protonation of E
protein. Due to that, the E proteins of DENV undergo
major rearrangement involving the domain I and domain II
[10, 11]. A hydrophobic stretch, also known as the fusion
peptide, that is buried between the domain I and II during
the viral prefusion state is then exposed and reoriented at
one end after the transition to the postfusion trimeric
structure. In this conformation, the fusion peptide attaches
to the target cell membrane and initiates the viral mem-
brane fusion process [12–14]. Thus, targeting the E protein
to design the DENV entry inhibitors is one of promising
strategies to inhibit the virus. Current in silico screenings
of small molecule inhibitors mostly target the β-N-octyl-
glucoside (β-OG) binding site, which is a pocket found to
occupy a detergent molecule in a DENV E protein crystal
structure [15]. There were potential lead compounds dis-
covered [16–18]; however, most of the compounds failed
to proceed to the subsequent experimental stages due to
their poor druggability properties. Antiviral drugs targeting
the entry stage of the virus were already discovered for
HIV-1 [19, 20] and hepatitis B virus (HBV) [21], but for
the dengue virus it is still very challenging.
Flavonoids are one of the most studied natural com-
pounds that possess various pharmacological activities,
such as antioxidative [22], antibacterial [23] and antimu-
tagenic properties [24]. A study by Calland et al. [25]
showed epigallocatechin gallate (EGCG) inhibited the
hepatitis C virus (HCV) by preventing the virus attachment
to the cell surface. Similarly, ladanein is a phenolic com-
pound from Marrubiumperegrinum L (Lamiaceae) also
showed to inhibit HCV during the viral entry stage [26].
Baicalin, a flavonoid purified from a medicinal plant, was
reported to inhibit the HIV-1 infection by interfering with
the interaction of HIV-1 E protein and chemokine receptors
during HIV-1 entry [27]. However, there is no clear study
of flavonoids and DENV interacting during the viral entry
stage. Several studies showed flavonoid inhibitions during
the DENV replication stage. Fisetin, quercetin and baica-
lein are among the flavonoids that were reported to inhibit
the DENV2 replication by targeting the viral RNA [28–30].
In this work, several computational methods were
employed to study the binding potential of flavonoids to the
soluble region of DENV2 E protein. For the purpose of our
interest, we constructed a homology model of the DENV2 E
protein isolated from Malaysia (DENV2-My). Nine flavo-
noids were selected for this study based on their known
123
Interdiscip Sci Comput Life Sci (2017) 9:499–511
biological activities against viruses. We performed molecular
docking method to evaluate the interaction of flavonoids and
predict their binding location on the surface of the E protein.
The flavonoids were intensively docked to the E proteins of
the DENV2-My and the DENV2-Thai structures. Addition-
ally, the flavonoids were also docked to the E proteins of the
other members of the flavivirus (DENV3, TBEV and JEV).
We also performed molecular dynamics simulations for
selected E protein–flavonoid complexes of both DENV2-My
and DENV2-Thai. Our main goal is to identify the consensus
binding site and amino acid interactions of the flavonoids that
may serve as initial data for other DENV structural studies.
2 Materials and Methods
2.1 Sequence Analyses and Structure Preparation
The primary amino acid sequence of DENV2 isolated from
Malaysia; DENV2-My (GenBank ID: ACN94866.1) was
retrieved from GenBank [31]. The crystal structures and
sequences of E protein for DENV2, DENV3, TBEV and JEV
were obtained from Protein Data Bank (PDB) [32]: DENV2
(PDB ID: 3J27), DENV3 (PDB ID: 1UZG), TBEV (PDB ID:
1SVB) and JEV (PDB ID: 3P54). The DENV2-My sequence
was subjected to the BLAST search analysis using the NCBI
BLAST database [33] (www.blast.nlm.nih.gov) to search for
the local similarity region between the target protein and the
crystal structures. The alignment between the target and
template was computed using the CLUSTAL OMEGA
(http://www.ebi.ac.uk/Tools/msa/clustalo/) [34], which
aligned multiple sequences using a progressive pairwise
alignment algorithm. Minor adjustment of the alignments
was performed using the JALVIEW program [35].
2.2 Homology Modeling of DENV2-My
Homology modeling method was used to build the soluble
ectodomain model of DENV2-My. The aligned sequences
between the target protein (DENV2-My) and the template
(DENV2-Thai: PDB ID: 3J27) were used for the model
construction using the MODELLER 9v9 program [36]. The
stereochemical and structural properties of the model were
evaluated using the PROCHECK program [37]. The
sequence-structure compatibility of the models was eval-
uated based on the Z-score using the ProSA web server
[38]. Root mean square deviation (RMSD) was computed
using the DaliLite server [39].
2.3 Molecular Docking
High-resolution structures of the E proteins from DENV2-
Thai, JEV, and TBEV were used for the molecular docking
Interdiscip Sci Comput Life Sci (2017) 9:499–511
to study their potential interactions with the flavonoids. The
docking results were compared among the studied struc-
tures as well as with the model structure of DENV2-My E
protein.
The 2D structures of the ligands were downloaded from
the PubChem server (https://pubchem.ncbi.nlm.nih.gov/)
and converted to a 3D format using the SMILES Translator
(http://cactus.nci.nih.gov/services/translate/) [40]. Flavo-
noids chosen for the docking study are baicalin (PubChem
ID: 64982), baicalein (PubChem ID: 5281605), EGCG
(PubChem ID: 65064), fisetin (PubChem ID: 5281614),
flavone (PubChem ID: 10680), glabranine (PubChem ID:
124049), hyperoside (PubChem ID: 5281643), ladanein
(PubChem ID: 3084066) and quercetin (PubChem ID:
5280343).
Molecular docking was performed using Autodock Vina
1.1 [41]. Autodock Tools (ADT) were utilized to prepare
the input file for the receptors and ligands. Gasteiger
charges were assigned, and nonpolar hydrogen atoms were
merged. All torsions were allowed to rotate during the
docking process. The auxiliary program AutoGrid is used
to generate the grid maps for the protein. Each grid was
centered at the receptors of the corresponding inhibitors. In
the first stage, the blind docking method was performed on
both DENV2-Thai and DENV2-My E protein structures in
order to search for the common binding pocket of flavo-
noids. The grid dimensions used for the blind docking were
covering the whole structure of the E protein. In the second
docking stage, the targeted docking focused on the con-
sensus region identified by blind dockings. The size of the
grid box was set to 30 Å in each dimension. Analysis of the
protein–ligand complexes was based on the binding energy
score, hydrogen bond interactions and orientation of the
docked compound within the binding pocket [42].
2.4 Molecular Dynamics Simulation
Molecular dynamics simulations of E protein–flavonoids
complexes were carried out using GROMACS 4.5.5
package [43, 44] with GROMOS96 43A1 force field to
describe the atoms’ interactions [45, 46]. The starting
protein–ligand structures for the simulations were obtained
from the docking results. The topology and parameter files
for the ligands were generated using the PRODRG server
[47]. Each protein–ligand complex was placed in a triclinic
box solvated with a simple point charge (SPC) type of
water molecules. The distance between protein and box
was set to 1.2 nm, and van der Waals cutoff was 1.0 nm.
The systems were neutralized with counterions, and the salt
concentration of 0.1 M NaCl was added to the system.
Bond lengths involving the hydrogen atoms were con-
strained using LINCS algorithm, and van der Waals
interactions were evaluated with a cutoff distance of
501
1.0 nm. Particle mesh Ewald (PME) was employed to treat
long-range electrostatics interactions with a Coulomb cut-
off of 1.0 nm [48].
A total of 50 000 steps of steepest descent minimization
were applied to the solvated system to allow the solvent to
adjust around the protein–ligand complex. The systems
were equilibrated with NVT and NPT ensemble protocols
for about 100 ps each. The temperature of the simulation
system was set to 300 K. The production run of the
molecular dynamics simulations was performed for a total
of 20,000 ps (20 ns). The protein–ligand, solvent and ions
were separately coupled to a Berendsen temperature
(τT = 0.1 ps) and pressure (τP = 2.0 ps) baths [49] at 300 K
and 1 bar, respectively. Simulations were run with 2-fs
time steps. Trajectory analysis was performed using
GROMACS 4.5.5 tool [43, 44].
3 Results and Discussions
3.1 Sequence and Structure of E Protein DENV2
Multiple sequence alignment was conducted to evaluate the
sequence similarity of the envelope glycoprotein E from
DENV2-My, DENV2-Thai, DENV3, TBEV and JEV
viruses (Fig. 1). The alignment indicates that the soluble
region of DENV2-Thai and DENV2-My E proteins has
~95 % sequence similarity. The sequence similarity of the
DENV2-My E protein to the DENV3, TBEV and JEV E
proteins is 82, 51 and 62 %, respectively. Structural anal-
ysis showed the E protein soluble region of these viruses is
highly conserved. The average RMSD computed for the
target binding site is 1.04 Å. Figure 1 also shows the
sequence region known to form the dimeric fusion peptide
(residues 98–108), which is conserved among the fla-
viviruses. Residues located in the flavonoid consensus
binding pocket are highlighted and discussed in the
molecular docking section.
The search for the homologous structure of the E protein
DENV-My using the NCBI BLASTP database resulted in
the cryoEM structure of E protein from the DENV2-Thai
(PDB ID: 3J27). The E protein DENV2-My model was
developed using the E protein from the DENV-Thai
structure. Structural superimposition between the E protein
DENV2-My model and the template structure shows high
structural similarity (RMSD 0.58 Å) (Fig. 2). The model
quality of the DENV2-My model was also evaluated using
the PROCHECK and ProSA programs (Supplementary
Figure S1). The Ramachandran plot shows the main chain
conformations for more than 88.7 % of amino acid residues
are within the most favored and 1 % in the disallowed
region. In addition, the Z-plot provides the Z-score value of
−7.65, which describes that the model is located within the
123
502 Interdiscip Sci Comput Life Sci (2017) 9:499–511

Fig. 1 Multiple sequence alignment of E protein sequences of Residues involved in the flavonoids potential binding pocket are
DENV2-My, DENV2-Thai, DENV3, TBEV and JEV. Sequence highlighted and compared to the DENV2-My and DENV2-Thai E
similarity between E protein DENV-My and DENV-Thai is ~95 %. proteins

Fig. 2 Superimposition of the DENV2-My E protein model (magenta) to the DENV2-Thai E protein cryo EM structure (cyan) (PDBID 3J27)

123
Interdiscip Sci Comput Life Sci (2017) 9:499–511
conformational space of proteins determined by X-ray
crystallography method (Supplementary Figure S1).
3.2 Docking of Flavonoids
Initially, we used the blind docking method to identify a
consensus binding site on the E protein of both DENV2-
My and the DENV2-Thai structures. Nine flavonoids were
extensively docked into the entire E protein dimeric
structure with several different grid sizes and locations.
Based on this docking result, we observed a consensus
binding site for the flavonoids that located approximately
2.5 Å from Asn153, which is one of the two known gly-
cosylation sites for the DENV2 E protein. The binding
pocket is formed by the residues from domain I of chain B
(residues 4–9, 151–154) and domain II of chain A (residue
98–103, 244–247), which includes the conserved region of
the fusion peptide [13] (Figs. 1, 3). This pocket will be
referred as the flavonoid consensus pocket throughout this
manuscript.
In the second docking stage, the docking area was tar-
geted to the flavonoid consensus pocket that was observed
in the blind docking. This step of docking was applied to
all E protein structures. Interestingly, eight out of nine
flavonoids docked to the same binding pocket on the E
protein surface of DENV2-Thai, DENV2-My, TBEV and
JEV structures (Fig. 3 and Supplementary Figure S2).
Baicalin showed the highest binding affinity to all E protein
structures (−9.6 to −7.6 kcal/mol), followed by glabranine
(−9.0 to −6.7 kcal/mol), baicalein (−8.7 to −6.6 kcal/mol),
quercetin (−8.6 to −6.5 kcal/mol) and EGCG (−8.4 to
−6.4 kcal/mol) (Table 1). Flavone is the only flavonoid that
consistently docked to another pocket, which is located
approximately 13 Å away from the flavonoid consensus
pocket (Fig. 3 and Supplementary Figure S2). In addition,
the binding affinity of flavone to the E protein is the lowest
(−5.9 to −6.5 kcal/mol) among all the flavonoids (Table 1).
Interestingly, flavone was actually reported to act antago-
nistically compared to the other flavonoids that were
known to inhibit the DENV replication. The study showed
flavone increased DENV2 infectivity during the viral
adsorption and intracellular replication [50].
Although the DENV3 E protein has high sequence
similarity to the DENV2 E protein, flavonoids docked to E
protein of DENV3 showed different results compared to
the other E proteins. Only baicalin, glabranine and hyper-
oside docked into the flavonoid consensus binding pocket.
The other three compounds baicalein, EGCG and flavone
docked to another pocket located on another E protein
subunit (chain A). Fisetin, ladanein and quercetin docked
to a region between the E protein dimer interface that was
approximately 11 Å distant away from the flavonoid con-
sensus pocket (Supplementary Figure S2). The explanation
503
to this behavior is highly likely due to the different ori-
entations of the domain I and II of the DENV3 E protein
crystal structure (1UZG.pdb) [51].
3.3 Molecular Dynamics Simulations
Molecular dynamics simulations were performed in order
to evaluate the dynamic behavior of E protein–flavonoid
complexes. We selected four E protein–flavonoid com-
plexes from both DENV2-My and DENV2-Thai based on
the consistent binding mode observed in the docking
results and known experimental data of the flavonoids
interacting with the host cells during the virus entry. Initial
starting structures of the complexes (E protein, baicalein,
quercetin, EGCG and baicalin) were taken from the
docking results. In addition, molecular dynamics simula-
tions were also performed for the unbound E proteins of
both DENV2-My and DENV2-Thai. All simulations were
subjected to 20,000 ps molecular dynamics simulations.
The structural stability of protein–ligand complexes was
examined by the time evolution of root mean square
deviation (RMSD) based on the E protein backbone atoms.
Figure 4a, b shows the RMSD curves of the unbound
proteins for both DENV2-Thai and DENV2-My E proteins
were stable between 0.35 and 0.40 nm after 10 000 ps of
simulation. The RMSD analysis of both DENV2-Thai and
DENV2-My E proteins in complex with the baicalein is
similar to the unbound E proteins, whereas the other E
protein—flavonoid complexes result in higher RMSD than
the unbound proteins.
Radius of gyration (Rg) of the backbone atoms describes
the compactness of the E protein complexes and the
unbound E protein structures. As shown in Supplementary
Figure S3 (a), in the unbound E proteins, the Rg value was
stabilized as early as 1500 ps while the E protein–flavonoid
complexes reach stability at around 7000 ps. Rg for all
complexes decreased along the simulation time, indicating
an increase in the compactness that might be due to the
increase in interactions between the flavonoid and the E
protein. The flavonoid consensus binding pocket is located
in between the two subunits of the E protein dimer.
Therefore, the interactions of the flavonoids to the residues
in the binding pocket brought both E protein subunits
closer to each other. This observation can also be explained
by the reduction in distance between the both subunits of E
protein dimers during the simulations (Supplementary
Figure S3 (b)).
Hydrogen bond analysis was performed for each com-
plex to determine the atomistic interactions between the
flavonoids and specific E protein residues. Table 2 and
Supplementary Table S1–S5 describe hydrogen bond
interactions formed between the E proteins and flavonoids
during the simulations. Detailed description of hydrogen
123
504 Interdiscip Sci Comput Life Sci (2017) 9:499–511

Fig. 3 Docking of the

flavonoids to the E protein of
a DENV2-Thai, b DENV2-My
model, c TBEV and d JEV.
Domain I (red), domain II
(yellow) and domain III (blue).
Potential glycosylation site;
Asn153 is represented as the
gray sphere

bond interactions is explained for each of the complexes
below.
3.4 DENV2 E Protein–Baicalein Complex
In traditional Chinese medicine, Scutellaria baicalensis is
one of the most popular herbs that are being used to treat
common cold and other virus infections. Baicalein (5, 6,
7-trihydroxyflavone) is one of the flavones isolated from
Scutellaria reported to show potent inhibitory activity
against the influenza A virus during the viral entry [52, 53].
We showed in the previous section that baicalein docked to
the same binding pocket of the DENV2, TBEV and JEV E
proteins. The docking results provided high binding affinity
to the E protein of the DENV2-Thai and DENV2-My E
proteins (Table 1).
During the simulations, baicalein molecule was stably
interacting with the E proteins of both DENV2. The 6-OH
and 7-OH groups of baicalein formed hydrogen bonds with
Asp98, Gly100, Ile4, Gly5 and Val151. For the E protein of

123
Interdiscip Sci Comput Life Sci (2017) 9:499–511 505

Table 1 Docking binding affinity of the nine flavonoids to the E interacting throughout the 20,000 ps simulation (Fig. 6).
proteins of flaviviruses The carbonyl oxygen of Asp98 and amino group of Gly100
Flavonoids E proteins of DENV2-Thai E protein formed hydrogen bonds to the
3′-OH group of quercetin (Fig. 6a; Table 2). Meanwhile,
DENV2-Thai DENV2-My DENV3 TBEV JEV
Binding affinity (kcal/mol) the carbonyl oxygen of Gly5 formed hydrogen bonds with
the 4′-OH hydroxyl group; the 7-OH group interacted with
Baicalein −8.7 −8.1 −6.7 −7.6 −6.6 both the amino group of Ile4 and the carbonyl oxygen of
Flavone −6.8 −6.7 −6.5 −7.6 −5.9 Val151. The hydrogen bonds between both the Gly5 and
Fisetin −8.5 −8.1 −7.4 −8.0 −7.3 Val151 and quercetin were consistently formed along the
Quercetin −8.4 −8.6 −7.5 −8.0 −6.5 simulation time (Supplementary Table S2). The hydrogen
Glabranine −8.9 −9.0 −7.0 −7.7 −6.7 bonds between Asp98 and Gly100 and the quercetin
Hyperoside −8.1 −7.7 −7.4 −7.5 −6.8 occurred only after 2000 ps simulation and consistent till
Baicalin −9.2 −9.6 −8.7 −8.5 −7.6 the end of the simulation. The interaction between quer-
EGCG −8.2 −8.4 −6.7 −7.0 −7.3 cetin 7-OH group and Ile4 occurred at the beginning of the
Ladanein −8.9 −8.2 −6.7 −7.3 −6.4 simulation, but disrupted after 12,500 ps of the simulation
(Supplementary Table S2). During the simulation of
DENV2-My E protein–quercetin complex, the carbonyl
DENV2-Thai, the carbonyl oxygen of Asp98 and Val151 oxygen of Ile4 and Gly5 formed a hydrogen bond to the
formed interactions with the 6-OH of the baicalein. How- 7-OH group of quercetin (Fig. 6b; Table 2). At the same
ever, hydrogen bond interactions between the baicalein and time, the carbonyl oxygens of Asp98 and Val151 formed
Asp98 disrupted after 13,000 ps of the simulation (Sup- hydrogen bonds to the 3′-OH and 5-OH groups of quer-
plementary Table S1). Meanwhile, the amino group of cetin, respectively. The amino group of Gly100 formed an
Gly100, carbonyl oxygen of Gly5 and Ile4 formed hydro- interaction to the oxygen atom of quercetin at position 1.
gen bonds with to the 7-OH of the baicalein throughout the The hydrogen bond interactions of Asp98, Gly100 and Ile4
simulation time. For the E protein of DENV2-My, the of the E protein DENV2-My and quercetin were consistent
carbonyl oxygen of Val151 and Ile4 formed hydrogen during the simulation (Supplementary Table S2); however,
bonds with the 6-OH of baicalein. Meanwhile, Gly100 and the interactions of Gly5 and Val151 with quercetin were
Gly5 formed hydrogen bond with 7-OH of baicalein. disrupted before the end of the simulation.
Similar as in the E protein of DENV2-Thai, hydrogen bond
interactions formed by Asp98 with the baicalein were not 3.6 DENV2 E Protein–EGCG Complex
retained after 15,000 ps of simulation time (Fig. 5b;
Table 2 and Supplementary Table S1). Final conformations EGCG (also known as epigallocatechin-3-gallate), a com-
of E protein–baicalein complexes from the both DENV2- pound present in green tea extract, was shown to be a
Thai and DENV2-My obtained at 20,000 ps molecular potent HCV entry inhibitor that blocked the virus attach-
dynamics simulations are shown in Fig. 5a, b. ment to the host cells [25]. Similar to other selected
complexes, EGCG docked to the same flavonoid consensus
3.5 DENV2 E Protein–Quercetin Complex pocket; therefore, we selected the E protein–EGCG com-
plexes for further analysis using the molecular dynamics
Quercetin is a phenolic type of flavonoids commonly found simulations.
in many fruits and vegetables. Although no specific study The E protein–EGCG complexes were stable in all
relates this compound with DENV E protein, however, simulations. Hydrogen bonds between the DENV2-Thai E
there were reports showed that quercetin caused concen- protein and EGCG were observed between the 3,4,5-tri-
tration-dependent reduction in viral infectivity in other type hydroxybenzoate moiety of EGCG and four residues of
of viruses [54]. This suggests potential activity of quercetin DENV2-Thai E protein Gly100, Ile4, Gly5 and Val151 as
during the viral entry. In the molecular docking section, the shown in Fig. 7a and Table 2. We observed the carbonyl
result showed quercetin consistently docked to the con- oxygen of Ile4 formed hydrogen bonds with 5″-OH, and
sensus binding pocket of E proteins of DENV2, JE and both Gly5 and Val151 formed hydrogen bonds with the 4″-
TBEV except for the DENV3. Therefore, the E protein– OH of the 3,4,5-trihydroxybenzoate moiety of EGCG,
quercetin complexes of DENV2-My and DENV2-Thai respectively. The amino group of Gly100 formed interac-
were further investigated using the molecular dynamics tions to the 3″-OH of the same moiety. The hydrogen bond
simulations. interactions between Gly100, Ile4 and Gly5 residues and
During the simulations, quercetin in complexes with the EGCG were sustained along the simulation (Supple-
DENV2-My and DENV2-Thai E proteins was stably mentary Table S3). Asp98 and Val151 formed the

123
506 Interdiscip Sci Comput Life Sci (2017) 9:499–511

Fig. 4 Molecular dynamics

simulation trajectories for the
unbound E proteins and E
protein–flavonoid complexes
plotted as a function of
simulation time; a DENV2-Thai
E protein and b DENV2-My E
protein. The unbound DENV2-
Thai and DENV2-My E
proteins are represented as the
black line. The E protein
baicalein, quercetin and EGCG
complexes are represented in
red, green, blue and yellow lines,
respectively

hydrogen bonds after ~2000 and ~5000 ps, respectively;
however, the interactions also sustained throughout the
simulation. Only Gly100 formed up to three hydrogen
bonds with EGCG, while the other interacting residues
formed up to two hydrogen bonds except for the Ile4 that
formed only one (Fig. 7a; Table 2 and Supplementary
Table S3). For the DENV2-My E protein–EGCG simula-
tion, the carbonyl oxygen of Ile4 and Gly5 formed
persistent hydrogen bonds with the 3′-OH and 4′-OH
groups of phenyl moiety, respectively (Fig. 7b; Table 2 and
Supplementary Table S3). Similarly as in the DENV2-Thai
E protein, the hydrogen bonds between the carbonyl oxy-
gen of Val151 and the 3″-OH of 3,4,5-trihydroxybenzoate
moiety were unstable and disrupted at the end of the sim-
ulation (Fig. 7b and Supplementary Table S3), whereas the
carbonyl oxygen of Asp98 and the amino group of Gly100
continuously formed interactions with the 7-OH group and
the carbonyl oxygen of EGCG at position 1, respectively.
3.7 DENV2 E Protein–Baicalin Complex
Baicalin (7-glucuronic acid,5, 6-dihydroxyflavone) is also
one of the flavones that can be extracted from the Scutel-
laria species. Structurally, baicalin has an extra bulky
glucuronic acid moiety on its 7-OH baicalein moiety. This
compound has similar inhibitory activities as the baicalein

123
Interdiscip Sci Comput Life Sci (2017) 9:499–511 507

Table 2 Hydrogen bond analysis between the flavonoids and E protein of DENV2-Thai and DENV2-My
DENV2-Thai H-B donor/acceptor DENV2-My H-B donor/acceptor
complexes complexes

Baicalein Baicalein: 6-OH—Asp98: O Baicalein Baicalein: 1-O—Asp98: O

Baicalein: 7-OH—Gly100: NH2 Baicalein: 7-OH—Gly100: O
Baicalein: 7-OH—Ile4: O Baicalein: 6-OH—Ile4: O
Baicalein: 7-OH—Gly5: O Baicalein: 7-OH—Gly5: O
Baicalein: 6-OH—Val151: O Baicalein: 6-OH—Val151: O
Quercetin Quercetin: 3′-OH—Asp98: O Quercetin Quercetin: 3′-OH—Asp98: O
Quercetin: 3′-OH—Gly100: NH2 Quercetin: 1-O—Gly100: NH2
Quercetin: 7-OH—Ile4: NH2 Quercetin: 7-OH—Ile4: O
Quercetin: 4′-OH—Gly5: O Quercetin: 7-OH—Gly5: O
Quercetin: 7-OH—Val151: O Quercetin: 5-OH—Val151: O
EGCG EGCG: 5′-OH—Asp98: NH2 EGCG EGCG: 4″-OH—Asp98: O
EGCG: 3″-OH—Gly100: NH2 EGCG: 1 = O—Gly100: NH2
EGCG: 5″-OH—Ile4: O EGCG: 3′-OH—Ile4: O
EGCG: 5″-OH—Gly5: O EGCG: 3″-OH—Gly5: O
EGCG: 4″-OH—Val151: O EGCG: 7-OH—Val151: O
Baicalin Baicalin: carbonyl oxygen of beta-D-glucopyranose Baicalin Baicalin: 6-OH—Asp98: O
acid—Asn103: NH2 Baicalin: 2-OH of beta-D-glucopyranose
Baicalin: 4-OH of beta-D-glucopyranose acid—Gly5: O acid—Arg99: NH2
Baicalin: carbonyl oxygen of beta-D-glucopyranose Baicalin: 4 = O—Lys247: NH2
acid—Asn153: NH2 Baicalin: 6-OH—Ser7: O
Baicalin: 4-OH of beta-D-glucopyranose acid—Gly152: O Baicalin: 4-OH of beta-D-glucopyranose
Baicalin: carbonyl oxygen of beta-D-glucopyranose acid—Asn153: NH2
acid—Asp154: NH2

Fig. 5 Binding conformations of baicalein to the E protein of a —green and chain B–cyan). The snapshot is taken from the final
DENV2-Thai and b DENV2-My. Residues that formed interactions 20,000 ps MD simulations
with baicalein during the simulation are represented as sticks (chain A

123
508
Fig. 6 Binding conformations of quercetin to the E protein of a
DENV2-Thai and b DENV2-My. Residues that formed interactions
with quercetin during the simulation are represented as sticks (chain A
Fig. 7 Binding conformations of EGCG to the E protein of a
DENV2-Thai and b DENV2-My. Residues that formed interactions
with EGCG during the simulation are represented as sticks (chain A—
against influenza A virus [53]. In addition, baicalin was
also shown to inhibit HIV-1 entry [27]. Docking of baicalin
to the E proteins produced the highest binding affinity
among the nine studied flavonoids and higher in the
DENV2 E proteins compared to the other E proteins
(Table 1).
During the molecular dynamics simulations, baicalin
stably bound to the E proteins. However, compared to the
baicalein, baicalin was observed to provide different
123
Interdiscip Sci Comput Life Sci (2017) 9:499–511
—green and chain B—cyan). The snapshot is taken from the final
20,000 ps MD simulations
green and chain B–cyan). The snapshot is taken from the final
20,000 ps MD simulations
binding mode in DENV2-My and DENV2-Thai E protein.
Furthermore, residues involved in the hydrogen bonding
between baicalin and both E proteins of DENV2-My and
DENV2-Thai were not exactly the same. Hydrogen bond
interactions of the baicalin with the DENV2-Thai E protein
were formed by Asn103, Gly5, Gly152, Asn153 and
Asp154 residues. In detail, we observed the carbonyl
oxygen of both Gly5 and Gly152 consistently formed
interactions to the 4-OH. Meanwhile, the amino group of
Interdiscip Sci Comput Life Sci (2017) 9:499–511
Asn103, Asn153 and Asp154 formed hydrogen bonds to
the carbonyl oxygen of the glucuronic acid moiety (Fig. 8a;
Table 2 and Supplementary Table S4). However, during
the simulation of DENV2-My E protein–baicalin, residues
that involved in the interactions were Asp98, Arg99,
Lys247, Ser7 and Asn153 residues. The consensus residue
between both DENV2-Thai and DENV2-My E proteins
was only Asn153 (Fig. 8a, b; Table 2 and Supplementary
Table S4 and Supplementary Table S5). The amino groups
of Arg99 and Asn153 were observed to form consistent
hydrogen bond interactions to the 2-OH and 4-OH of
glucuronic acid moiety of baicalin, respectively (Fig. 8b;
Table 2 and Supplementary Table S5). Meanwhile, the
carbonyl oxygens of Asp98 and Ser7 continuously inter-
acted to the 6-OH of baicalein moiety throughout the
simulation.
4 Discussion
In this study, docking and molecular dynamic simulations
showed consistent results. Moreover, the DENV2-My E
protein structure, which is a homology model, produced
highly similar results as the DENV2-Thai E protein
structure. These data describe that the constructed E pro-
tein model is in good agreement with the template
structure. Consistently, a recent study constructed a
DENV1 E protein model based on the DENV2 E protein
structure successfully developed a lead inhibitor. This
potential DENV1 entry inhibitor was shown to potently
inhibit the viral membrane fusion [55].
Fig. 8 Binding conformations of baicalin to the E protein of a
DENV2-Thai and b DENV2-My. Residues that formed interactions
with baicalin during the simulation are represented as sticks (chain A
509
The flavonoid consensus pocket observed in this study is
proposed as an alternative binding pocket. This pocket is
present during the E protein dimeric state and consists of
residues known to form the fusion loop, which are con-
served among the flaviviruses. This conserved region could
be useful for the antiviral development targeting the four
serotype of DENV. However, several studies reported the
presence of other cavities on the surface of the E protein
that exists during different states of the virus. For example,
Yennamali et al. [18, 56] identified potential inhibitor
binding pockets using another crystal structure of DENV2
E protein. Studies using molecular dynamics simulations
also reported different pockets on the surface of the
DENV2 E protein dimeric prefusion structure that were
observed during different histidine protonation states [57,
58].
The flavonoids that were observed to dock into the
consensus pocket could be used as the potential leads to
design the DENV entry inhibitor. Residues Ile4, Gly5,
Asp98, Gly100 and Val151 located in the potential binding
pocket were consistently observed to form interactions
with the E proteins of DENV2. These residues can be used
as the references for the bioassay and mutagenesis studies
in order to validate this computational prediction. Despite
the observed binding consensus between DENV2-My and
DENV2-Thai E proteins, there are possibilities that these
flavonoids could also bind to different E protein cavities
that exist during the rearrangement process or they could
interact with other non-structural proteins at the later stage
of the viral life cycle. Such binding events could be
addressed more specifically in the future studies. We also
—green and chain B—cyan). The snapshot is taken from the final
20,000 ps MD simulations
123
510
note that some of these flavonoids are categorized as
amphiphilic phytochemical, which may affect the host
membrane lipid perturbations rather than specifically bind
to the target protein [59].
5 Conclusions
We have identified an alternative binding pocket on the
surface of the DENV2 E protein based on the consensus
binding site of the flavonoids. We describe here the
important amino acid residues of the E protein that may
interact with the flavonoids. For the DENV2-Thai and
DENV2-My E proteins, Ile4, Gly5, Asp98, Gly100 and
Val151 residues were identified to form interactions with
baicalein, quercetin and EGCG. These data are useful for
the future mutagenesis and structural studies of DENV E
protein and the development of the DENV entry inhibitor.
Acknowledgments The authors thank Dr. Ozlem Demir for the
helpful discussion and careful reading of the manuscript. This
research was supported by the Universiti Teknologi MARA (UiTM)
Dana Cluster 600-RMI/DANA 5/3/CG (2/2012). NAI was funded by
the scholarship from the Ministry of Higher Education (Malaysia)
through the MyBrain15 program. We thank Faculty of Pharmacy,
UiTM Puncak Alam Campus for providing the computing facilities in
the Bioinformatics Lab, as well as Research Management Institute
(RMI), UiTM and Ministry of Science and Technology Malaysia
(MOSTI) for the financial and administrative support.
Compliance with Ethical Standards
Conflict of interest The authors declare that they have no conflict
of interest.
Ethical Approval This study does not contain any studies with
human participants or animals performed by any of the authors.
References
1. Wang E, Ni H, Xu R, Barrett AD, Watowich SJ, Gubler DJ,
Weaver SC (2000) Evolutionary relationships of endemic/epi-
demic and sylvatic dengue viruses. J Virol 74(7):3227–3234
2. Moncayo AC, Fernandez Z, Ortiz D, Diallo M, Sall A, Hartman
S, Davis T, Coffey L, Mathiot CC, Tesh RB, Weaver SC (2004)
Dengue emergence and adaptation to peridomestic mosquitoes.
Emerg Infect Dis 10(10):1790–1796
3. Bhatt S, Gething PW, Brady OJ, Messina JP, Farlow AW, Moyes
CL, Drake JM, Brownstein JS, Hoen AG, Sankoh O, Myers MF,
George DB, Jaenisch T, Wint GR, Simmons CP, Scott TW,
Farrar JJ, Hay SI (2013) The global distribution and burden of
dengue. Nature 496(7446):504–507
4. Christian E, Kahle KM, Mattia K, Puffer BA, Pfaff JM, Miler A,
Paes C, Davidson E, Doranz BJ (2013) Atomic level functional
model of dengue virus envelope protein infectivity. PNAS 110
(46):18662–18667
5. Abu Bakar S, Shafee N (2002) Outlook of dengue in Malaysia: a
century later. Malays J Pathol 24(1):23–27
6. Rodenhuis-Zybert IA, Wilschut J, Smit JM (2010) Dengue virus
life cycle: viral and host factors modulating infectivity. Cell Mol
Life Sci 67(16):2773–2786
123
Interdiscip Sci Comput Life Sci (2017) 9:499–511
7. Mukhopadhyay S, Kuhn RJ, Rossmann MG (2005) A structural
perspective of the flavivirus life cycle. Nat Rev Microbiol 3
(1):13–22
8. R-f Qi, Zhang L, C-w Chi (2008) Biological characteristics of
dengue virus and potential targets for drug design. Acta Biochim
Biophys Sin 40(2):91–101
9. Acosta EG, Castilla V, Damonte EB (2008) Functional entry of
dengue virus into Aedes albopictus mosquito cells is dependent
on clathrin-mediated endocytosis. J Gen Virol 89(Pt 2):474–484
10. Zhang Y, Zhang W, Ogata S, Clements D, Strauss JH, Baker TS,
Kuhn RJ, Rossmann MG (2004) Conformational changes of the
flavivirus E glycoprotein. Structure 12(9):1607–1618
11. Zhang X, Ge P, Yu X, Brannan JM, Bi G, Zhang Q, Schein S,
Zhou ZH (2013) Cryo-EM structure of the mature dengue virus at
3.5-A resolution. Nat Struct Mol Biol 20(1):105–110
12. Stiasny K, Heinz FX (2006) Flavivirus membrane fusion. J Gen
Virol 87(Pt 10):2755–2766
13. Huang CY, Butrapet S, Moss KJ, Childers T, Erb SM, Calvert
AE, Silengo SJ, Kinney RM, Blair CD, Roehrig JT (2010) The
dengue virus type 2 envelope protein fusion peptide is essential
for membrane fusion. Virology 396(2):305–315
14. Klein DE, Choi JL, Harrison SC (2012) Structure of a dengue
virus envelope protein late-stage fusion intermediate. J Virol 87
(4):2287–2293
15. Modis Y, Ogata S, Clements D, Harrison SC (2003) A ligand-
binding pocket in the dengue virus envelope glycoprotein. Proc
Natl Acad Sci USA 100(12):6986–6991
16. Poh MK, Yip A, Zhang S, Priestle JP, Ma NL, Smit JM, Wilschut
J, Shi PY, Wenk MR, Schul W (2009) A small molecule fusion
inhibitor of dengue virus. Antivir Res 84(3):260–266
17. Wang QY, Patel SJ, Vangrevelinghe E, Xu HY, Rao R, Jaber D,
Schul W, Gu F, Heudi O, Ma NL, Poh MK, Phong WY, Keller TH,
Jacoby E, Vasudevan SG (2009) A small-molecule dengue virus
entry inhibitor. Antimicrob Agents Chemother 53(5):1823–1831
18. Lim SP, Wang QY, Noble CG, Chen YL, Dong H, Zou B,
Yokokawa F, Nilar S, Smith P, Beer D, Lescar J (2013) Ten years
of dengue drug discovery: progress and prospects. Antivir Res
100(2):500–519
19. Pugach P, Ketas TJ, Michael E, Moore JP (2008) Neutralizing
antibody and anti-retroviral drug sensitivities of HIV-1 isolates
resistant to small molecule CCR5 inhibitors. Virology 377
(2):401–407
20. Lalezari JP, Henry K, O’Hearn M, Montaner JSG, Piliero PJ,
Trottier B, Walmsley S, Cohen C, Kuritzkes DR, Eron JJ, Chung
J, DeMasi R, Donatacci L, Drobnes C, Delehanty J, Salgo M
(2003) Enfuvirtide, an HIV-1 fusion inhibitor, for drug-resistant
HIV infection in North and South America. N Engl J Med 348
(22):2174–2185
21. Volz T, Allweiss L, ḾBarek MB, Warlich M, Lohse AW, Pollok
JM, Alexandrov A, Urban S, Petersen J, Lütgehetmann M, Dandri
M (2013) The entry inhibitor Myrcludex-B efficiently blocks
intrahepatic virus spreading in humanized mice previously
infected with hepatitis B virus. J Hepatol 58(5):861–867
22. Murota K, Terao J (2003) Antioxidative flavonoid quercetin:
implication of its intestinal absorption and metabolism. Arch
Biochem Biophys 417(1):12–17
23. Cushnie TPT, Lamb AJ (2005) Antimicrobial activity of flavo-
noids. Int J Antimicrob Agents 26(5):343–356
24. Snijman PW, Swanevelder S, Joubert E, Green IR, Gelderblom WC
(2007) The antimutagenic activity of the major flavonoids of rooibos
(Aspalathus linearis): some dose-response effects on mutagen acti-
vation-flavonoid interactions. Mutat Res 631(2):111–123
25. Calland N, Albecka A, Belouzard S, Wychowski C, Duverlie G,
Descamps V, Hober D, Dubuisson J, Rouille Y, Seron K (2012)
(-)-Epigallocatechin-3-gallate is a new inhibitor of hepatitis C
virus entry. Hepatology 55(3):720–729
Interdiscip Sci Comput Life Sci (2017) 9:499–511
26. Haid S, Novodomska A, Gentzsch J, Grethe C, Geuenich S,
Bankwitz D, Chhatwal P, Jannack B, Hennebelle T, Bailleul F,
Keppler OT, Poenisch M, Bartenschlager R, Hernandez C,
Lemasson M, Rosenberg AR, Wong-Staal F, Davioud-Charvet E,
Pietschmann T (2012) A plant-derived flavonoid inhibits entry of
all HCV genotypes into human hepatocytes. Gastroenterology
143(1):213e5–222e5
27. Li BQ, Fu T, Dongyan Y, Mikovits JA, Ruscetti FW, Wang JM
(2000) Flavonoid baicalin inhibits HIV-1 infection at the level of
viral entry. Biochem Biophys Res Commun 276(2):534–538
28. Zandi K, Teoh B-T, Sam S-S, Wong P-F, Mustafa MR, AbuBakar
S (2011) In vitro antiviral activity of Fisetin, Rutin and Narin-
genin against Dengue virus type-2. J Med Plants Res 5(23):5534–
5539
29. Zandi K, Teoh B-T, Sam S-S, Wong P-F, Mustafa MR, Abubakar
S (2011) Antiviral activity of four types of bioflavonoid against
dengue virus type-2. Virol J 8:560
30. Zandi K, Teoh B-T, Sam S-S, Wong P-F, Mustafa MR, AbuBakar
S (2012) Novel antiviral activity of baicalein against dengue
virus. BMC Complementary and Alternative Medicine 12
(214):1–9
31. Benson DA, Cavanaugh M, Clark K, Karsch-Mizrachi I, Lipman
DJ, Ostell J, Sayers EW (2013) GenBank. Nucleic Acids Res 41
(Database issue):D36–D42
32. Rose PW, Bi C, Bluhm WF, Christie CH, Dimitropoulos D, Dutta
S, Green RK, Goodsell DS, Prlic A, Quesada M, Quinn GB,
Ramos AG, Westbrook JD, Young J, Zardecki C, Berman HM,
Bourne PE (2013) The RCSB Protein Data Bank: new resources
for research and education. Nucleic Acids Res 41(Database
issue):D475–D482
33. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990)
Basic local alignment search tool. J Mol Biol 215:403–410
34. Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W,
Lopez R, McWilliam H, Remmert M, Soding J, Thompson JD,
Higgins DG (2011) Fast, scalable generation of high-quality
protein multiple sequence alignments using Clustal Omega. Mol
Syst Biol 7:539
35. Clamp M, Cuff J, Searle SM, Barton GJ (2004) The Jalview Java
alignment editor. Bioinformatics 20(3):426–427
36. Eswar N, Webb B, Marti-Renom MA, Madhusudhan MS, Era-
mian D, Shen MY, Pieper U, Sali A (2006) Comparative protein
structure modeling using Modeller. Curr Protoc Bioinform.
doi:10.1002/0471250953.bi0506s15
37. Laskowski AR (2001) PDBsum: summaries and analyses of PDB
structures. Nucleic Acids Res 29(1):221–222
38. Wiederstein M, Sippl MJ (2007) ProSA-web: interactive web
service for the recognition of errors in three-dimensional struc-
tures of proteins. Nucleic Acids Res 35(Web Server issue):
W407–W410
39. Holm L, Park J (2000) DaliLite workbench for protein structure
comparison. Bioinformatics (Oxford, England) 16(6):566–567
40. Weininger D (1988) SMILES, a chemical language and infor-
mation system. Introduction to methodology and encoding rules.
J Chem Inf Comput Sci 28(1):31–36
41. Trott O, Olson AJ (2010) AutoDock Vina: improving the speed
and accuracy of docking with a new scoring function, efficient
optimization, and multithreading. J Comput Chem 31(2):455–461
42. Kubinyi H (2007) Hydrogen bonding: the last mystery in drug
design? Pharmacokinet Optim Drug Res 30:513–524
43. Hess B, Kutzner C, van der Spoel D, Lindahl E (2008) GRO-
MACS 4: algorithms for highly efficient, load-balanced, and
scalable molecular simulation. J Chem Theory Comput 4(3):435–
447
511
44. Pronk S, Pall S, Schulz R, Larsson P, Bjelkmar P, Apostolov R,
Shirts MR, Smith JC, Kasson PM, van der Spoel D, Hess B,
Lindahl E (2013) GROMACS 4.5: a high-throughput and highly
parallel open source molecular simulation toolkit. Bioinformatics
29:845–854
45. Oostenbrink C, Villa A, Mark AE, van Gunsteren WF (2004) A
biomolecular force field based on the free enthalpy of hydration
and solvation: the GROMOS force-field parameter sets 53A5 and
53A6. J Comput Chem 25(13):1656–1676
46. Pol-Fachin L, Fernandes CL, Verli H (2009) GROMOS96 43a1
performance on the characterization of glycoprotein conforma-
tional ensembles through molecular dynamics simulations.
Carbohydr Res 344(4):491–500
47. Schuttelkopf AW, Van Aalten DMF (2004) PRODRG: a tool for
high-throughput crystallography of protein-ligand complexes.
Acta Cryst D60:1355–1363
48. Darden T, York D, Pedersen L (1993) Particle mesh Ewald: an
N·log(N) method for Ewald sums in large systems. J Chem Phys
98(12):10089
49. Berendsen HJC, Postma JPM, van Gunsteren WF, DiNola A,
Haak JR (1984) Molecular dynamics with coupling to an external
bath. J Chem Phys 81(8):3684
50. Zandi K, Lani R, Wong P-F, Teoh B-T, Sam S-S, Johari J,
Mustafa MR, AbuBakar S (2012) Flavone enhances dengue virus
type-2 (NGC strain) infectivity and replication in Vero cells.
Molecules 17(3):2437–2445
51. Modis Y, Ogata S, Clements D, Harrison SC (2005) Variable
surface epitopes in the crystal structure of dengue virus type 3
envelope glycoprotein. J Virol 79(2):1223–1231
52. Sithisarn P, Michaelis M, Schubert-Zsilavecz M, Cinatl J Jr
(2013) Differential antiviral and anti-inflammatory mechanisms
of the flavonoids biochanin A and baicalein in H5N1 influenza A
virus-infected cells. Antivir Res 97(1):41–48
53. Hour MJ, Huang SH, Chang CY, Lin YK, Wang CY, Chang YS,
Lin CW (2013) Baicalein, Ethyl Acetate, and Chloroform
Extracts of Scutellaria baicalensis Inhibit the Neuraminidase
Activity of Pandemic 2009 H1N1 and Seasonal Influenza A
Viruses. Evid Based Complement Alternat Med 2013:750803
54. Kaul TN, Middleton E, Ogra PL (1985) Antiviral effect of fla-
vonoids on human viruses. J Med Virol 15(1):71–79
55. Abe T, Sando A, Teraoka F, Otsubo T, Morita K, Tokiwa H,
Ikeda K, Suzuki T, Hidari KI (2014) Computational design of a
sulfoglucuronide derivative fitting into a hydrophobic pocket of
dengue virus E protein. Biochem Biophys Res Commun 449
(1):32–37
56. Yennamalli R, Subbarao N, Kampmann T, McGeary RP, Young
PR, Kobe B (2009) Identification of novel target sites and an
inhibitor of the dengue virus E protein. J Comput Aided Mol Des
23(6):333–341
57. Degreve L, Fuzo CA (2013) Structure and dynamics of the
monomer of protein E of dengue virus type 2 with unprotonated
histidine residues. Genet Mol Res 12(1):348–359
58. Fuzo CA, Degrève L (2013) New pockets in dengue virus 2
surface identified by molecular dynamics simulation. J Mol
Model 19(3):1369–1377
59. Ingolfsson HI, Thakur P, Herold KF, Hobart EA, Ramsey NB,
Periole X, de Jong DH, Zwama M, Yilmaz D, Hall K, Maretzky
T, Hemmings HC Jr, Blobel C, Marrink SJ, Kocer A, Sack JT,
Andersen OS (2014) Phytochemicals perturb membranes and
promiscuously alter protein function. ACS Chem Biol 9(8):1788–
1798
123