Interdisciplinary Sciences: Computational Life Sciences

pp 1–13

Molecular Docking and Molecular Dynamics Simulation Studies to Predict Flavonoid Binding on the Surface of DENV2 E Protein

Authors

  • Nurul Azira Ismail
    • Department of Pharmaceutical Life Sciences, Faculty of PharmacyUniversiti Teknologi MARA
    • Department of Pharmaceutical Life Sciences, Faculty of PharmacyUniversiti Teknologi MARA
Original Research Article

DOI: 10.1007/s12539-016-0157-8

Cite this article as:
Ismail, N.A. & Jusoh, S.A. Interdiscip Sci Comput Life Sci (2016). doi:10.1007/s12539-016-0157-8

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 flavonoids (baicalin, baicalein, EGCG, fisetin, glabranine, hyperoside, ladanein, quercetin and flavone) to the soluble ectodomain of DENV type 2 (DENV2) E protein. Interestingly, 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, quercetin and EGCG during the simulations. This study demonstrates flavonoids potentially form interactions with the E protein of DENV2.

Keywords

Dengue virus MalaysiaDENV2FlavonoidsBaicalinBaicaleinEGCGFisetinGlabranineHyperosideLadaneinQuercetinDockingMolecular dynamics simulationsEnvelope 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 countries [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 categorized 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 anchored onto the viral lipid bilayer. The RNA genome encodes a single polyprotein that is co-translationally and posttranslationally processed into three structural (capsid, 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 membrane fusion process [1214]. 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 discovered [1618]; 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 compounds that possess various pharmacological activities, such as antioxidative [22], antibacterial [23] and antimutagenic 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 compound 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 baicalein are among the flavonoids that were reported to inhibit the DENV2 replication by targeting the viral RNA [2830].

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 flavonoids were selected for this study based on their known 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. Additionally, 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 evaluated 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 to study their potential interactions with the flavonoids. The docking results were compared among the studied structures 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]. Flavonoids 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 flavonoids. 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 consensus 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 constrained using LINCS algorithm, and van der Waals interactions were evaluated with a cutoff distance of 1.0 nm. Particle mesh Ewald (PME) was employed to treat long-range electrostatics interactions with a Coulomb cutoff 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 analysis 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 flaviviruses. Residues located in the flavonoid consensus binding pocket are highlighted and discussed in the molecular docking section.
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Fig. 1

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

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 conformational space of proteins determined by X-ray crystallography method (Supplementary Figure S1).
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Fig. 2

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

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 glycosylation 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.
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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

In the second docking stage, the docking area was targeted 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 antagonistically 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].
Table 1

Docking binding affinity of the nine flavonoids to the E proteins of flaviviruses

Flavonoids

E proteins

DENV2-Thai

DENV2-My

DENV3

TBEV

JEV

Binding affinity (kcal/mol)

Baicalein

−8.7

−8.1

−6.7

−7.6

−6.6

Flavone

−6.8

−6.7

−6.5

−7.6

−5.9

Fisetin

−8.5

−8.1

−7.4

−8.0

−7.3

Quercetin

−8.4

−8.6

−7.5

−8.0

−6.5

Glabranine

−8.9

−9.0

−7.0

−7.7

−6.7

Hyperoside

−8.1

−7.7

−7.4

−7.5

−6.8

Baicalin

−9.2

−9.6

−8.7

−8.5

−7.6

EGCG

−8.2

−8.4

−6.7

−7.0

−7.3

Ladanein

−8.9

−8.2

−6.7

−7.3

−6.4

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 hyperoside 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 consensus pocket (Supplementary Figure S2). The explanation to this behavior is highly likely due to the different orientations 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 complexes 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 simulations 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.
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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 yellowlines, respectively

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 complex 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 bond interactions is explained for each of the complexes below.
Table 2

Hydrogen bond analysis between the flavonoids and E protein of DENV2-Thai and DENV2-My

DENV2-Thai complexes

H-B donor/acceptor

DENV2-My complexes

H-B donor/acceptor

Baicalein

Baicalein: 6-OH—Asp98: O

Baicalein: 7-OH—Gly100: NH2

Baicalein: 7-OH—Ile4: O

Baicalein: 7-OH—Gly5: O

Baicalein: 6-OH—Val151: O

Baicalein

Baicalein: 1-O—Asp98: O

Baicalein: 7-OH—Gly100: O

Baicalein: 6-OH—Ile4: O

Baicalein: 7-OH—Gly5: O

Baicalein: 6-OH—Val151: O

Quercetin

Quercetin: 3′-OH—Asp98: O

Quercetin: 3′-OH—Gly100: NH2

Quercetin: 7-OH—Ile4: NH2

Quercetin: 4′-OH—Gly5: O

Quercetin: 7-OH—Val151: O

Quercetin

Quercetin: 3′-OH—Asp98: O

Quercetin: 1-O—Gly100: NH2

Quercetin: 7-OH—Ile4: O

Quercetin: 7-OH—Gly5: O

Quercetin: 5-OH—Val151: O

EGCG

EGCG: 5′-OH—Asp98: NH2

EGCG: 3″-OH—Gly100: NH2

EGCG: 5″-OH—Ile4: O

EGCG: 5″-OH—Gly5: O

EGCG: 4″-OH—Val151: O

EGCG

EGCG: 4″-OH—Asp98: O

EGCG: 1 = O—Gly100: NH2

EGCG: 3′-OH—Ile4: O

EGCG: 3″-OH—Gly5: O

EGCG: 7-OH—Val151: O

Baicalin

Baicalin: carbonyl oxygen of beta-D-glucopyranose acid—Asn103: NH2

Baicalin: 4-OH of beta-D-glucopyranose acid—Gly5: O

Baicalin: carbonyl oxygen of beta-D-glucopyranose acid—Asn153: NH2

Baicalin: 4-OH of beta-D-glucopyranose acid—Gly152: O

Baicalin: carbonyl oxygen of beta-D-glucopyranose acid—Asp154: NH2

Baicalin

Baicalin: 6-OH—Asp98: O

Baicalin: 2-OH of beta-D-glucopyranose acid—Arg99: NH2

Baicalin: 4 = O—Lys247: NH2

Baicalin: 6-OH—Ser7: O

Baicalin: 4-OH of beta-D-glucopyranose acid—Asn153: NH2

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 DENV2-Thai, the carbonyl oxygen of Asp98 and Val151 formed interactions with the 6-OH of the baicalein. However, hydrogen bond interactions between the baicalein and Asp98 disrupted after 13,000 ps of the simulation (Supplementary Table S1). Meanwhile, the amino group of Gly100, carbonyl oxygen of Gly5 and Ile4 formed hydrogen bonds with to the 7-OH of the baicalein throughout the simulation time. For the E protein of DENV2-My, the carbonyl oxygen of Val151 and Ile4 formed hydrogen bonds with the 6-OH of baicalein. Meanwhile, Gly100 and Gly5 formed hydrogen bond with 7-OH of baicalein. Similar as in the E protein of DENV2-Thai, hydrogen bond interactions formed by Asp98 with the baicalein were not retained after 15,000 ps of simulation time (Fig. 5b; Table 2 and Supplementary Table S1). Final conformations of E protein–baicalein complexes from the both DENV2-Thai and DENV2-My obtained at 20,000 ps molecular dynamics simulations are shown in Fig. 5a, b.
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Fig. 5

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

3.5 DENV2 E Protein–Quercetin Complex

Quercetin is a phenolic type of flavonoids commonly found in many fruits and vegetables. Although no specific study relates this compound with DENV E protein, however, there were reports showed that quercetin caused concentration-dependent reduction in viral infectivity in other type of viruses [54]. This suggests potential activity of quercetin during the viral entry. In the molecular docking section, the result showed quercetin consistently docked to the consensus binding pocket of E proteins of DENV2, JE and TBEV except for the DENV3. Therefore, the E protein–quercetin complexes of DENV2-My and DENV2-Thai were further investigated using the molecular dynamics simulations.

During the simulations, quercetin in complexes with DENV2-My and DENV2-Thai E proteins was stably interacting throughout the 20,000 ps simulation (Fig. 6). The carbonyl oxygen of Asp98 and amino group of Gly100 of DENV2-Thai E protein formed hydrogen bonds to the 3′-OH group of quercetin (Fig. 6a; Table 2). Meanwhile, the carbonyl oxygen of Gly5 formed hydrogen bonds with the 4′-OH hydroxyl group; the 7-OH group interacted with both the amino group of Ile4 and the carbonyl oxygen of Val151. The hydrogen bonds between both the Gly5 and Val151 and quercetin were consistently formed along the simulation time (Supplementary Table S2). The hydrogen bonds between Asp98 and Gly100 and the quercetin occurred only after 2000 ps simulation and consistent till the end of the simulation. The interaction between quercetin 7-OH group and Ile4 occurred at the beginning of the 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 oxygen of Ile4 and Gly5 formed a hydrogen bond to the 7-OH group of quercetin (Fig. 6b; Table 2). At the same time, the carbonyl oxygens of Asp98 and Val151 formed hydrogen bonds to the 3′-OH and 5-OH groups of quercetin, respectively. The amino group of Gly100 formed an interaction to the oxygen atom of quercetin at position 1. The hydrogen bond interactions of Asp98, Gly100 and Ile4 of the E protein DENV2-My and quercetin were consistent during the simulation (Supplementary Table S2); however, the interactions of Gly5 and Val151 with quercetin were disrupted before the end of the simulation.
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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—green and chain B—cyan). The snapshot is taken from the final 20,000 ps MD simulations

3.6 DENV2 E Protein–EGCG Complex

EGCG (also known as epigallocatechin-3-gallate), a compound present in green tea extract, was shown to be a potent HCV entry inhibitor that blocked the virus attachment to the host cells [25]. Similar to other selected complexes, EGCG docked to the same flavonoid consensus pocket; therefore, we selected the E protein–EGCG complexes for further analysis using the molecular dynamics simulations.

The E protein–EGCG complexes were stable in all simulations. Hydrogen bonds between the DENV2-Thai E protein and EGCG were observed between the 3,4,5-trihydroxybenzoate moiety of EGCG and four residues of DENV2-Thai E protein Gly100, Ile4, Gly5 and Val151 as shown in Fig. 7a and Table 2. We observed the carbonyl oxygen of Ile4 formed hydrogen bonds with 5″-OH, and both Gly5 and Val151 formed hydrogen bonds with the 4″-OH of the 3,4,5-trihydroxybenzoate moiety of EGCG, respectively. The amino group of Gly100 formed interactions to the 3″-OH of the same moiety. The hydrogen bond interactions between Gly100, Ile4 and Gly5 residues and the EGCG were sustained along the simulation (Supplementary Table S3). Asp98 and Val151 formed the 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 simulation, 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 oxygen of Val151 and the 3″-OH of 3,4,5-trihydroxybenzoate moiety were unstable and disrupted at the end of the simulation (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.
https://static-content.springer.com/image/art%3A10.1007%2Fs12539-016-0157-8/MediaObjects/12539_2016_157_Fig7_HTML.gif
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—green and chain B–cyan). The snapshot is taken from the final 20,000 ps MD simulations

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 Scutellaria 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 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 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 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 interacted to the 6-OH of baicalein moiety throughout the simulation.
https://static-content.springer.com/image/art%3A10.1007%2Fs12539-016-0157-8/MediaObjects/12539_2016_157_Fig8_HTML.gif
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—green and chain B—cyan). The snapshot is taken from the final 20,000 ps MD simulations

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 protein 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].

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 conserved 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 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.

Supplementary material

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Supplementary material 1 (PDF 1303 kb)

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© International Association of Scientists in the Interdisciplinary Areas and Springer-Verlag Berlin Heidelberg 2016