WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

Sathyamurthy et al. World Journal of Pharmacy and Pharmaceutical Sciences

SJIF Impact Factor 7.421

Volume 7, Issue 9, 1188-1197 Research Article ISSN 2278 – 4357

IN SILICO DRUG DESIGNING STUDIES ON DENGUE NS5 PROTEIN

1
Sushmitha H. S. and 2*Balasubramanian Sathyamurthy

1
Department of Biochemistry, Ramaiah College of Arts, Science and Commerce, Bangalore.
2
Professor, Department of Biochemistry, Ramaiah College of Arts, Science and Commerce,
Bangalore.

Article Received on
15 July 2018,
Revised on 05 August 2018,
Accepted on 25 August 2018
DOI: 10.20959/wjpps20189-12355
ABSTRACT
The key proteins involved in causing dengue are the three structural
and five non-structural proteins, which can be considered as major
therapeutic targets for dengue drug development. Recent studies have
reported positively for NS5 protein in dysregulation of causing dengue

*Corresponding Author process in humans. Dragon fruit seed phytochemicals are reported to
Dr. Balasubramanian have antioxidant and antiviral properties. In the present study we
Sathyamurthy studied binding efficiency of 11 compounds that are present in the
Professor, Department of
dragon fruit seeds with NS5 protein through Insilico methods. By our
Biochemistry, Ramaiah
virtual screening and docking result, we found that the Compound C
College of Arts, Science and
Commerce, Bangalore. and Compound B have highest binding affinity with the NS5 protein
and also we predicted the binding site amino acid residues and the
nature of hydrogen bonding. However more invivo experimental validation of our results
with animal models will enlighten the development of more potent drugs from these
compounds for treatment of dengue.

KEYWORDS: NS5 protein, Binding interaction, molecular docking, dengue.

INTRODUCTION
Pitaya, a tropical fruit, belongs to the family of Cactaceae[1] and has the generic name
Hylocereus.[2] The fruit is scaly in appearance[3] and is oval to oblong in shape. Pitaya is a
delicate fruit with juicy pulp which contain numerous interspersed small, black seed which
are edible.[4] The three most commonly used pitaya are Hylocereus undatus, Hylocereus
polyrhizus and Hylocereus megalanthus.[2] The most common and widely used fruit is
Hylocereus undatus. The pulp of this fruit contains high amount of vitamins and water
soluble fibres. Pitaya can either be consumed directly or processed into juice and consumed

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as a drink.[5] The pitaya peel was reported to contain hylocerenin, pectin, triterpenoid,
steroids, phyllocactin, betanin and betacyanin.[6] The antioxidant properties of pitaya have
been advantageous as a source of natural food additive and ingredient.[1] Based on previous
study it was reported that betalain containing non-phenolic and phenolic structure was
responsible for the antioxidant properties of pitaya. It is also reported that the peel contains
the antioxidant property due to the colour of it. This antioxidant property of both the pulp and
peel has made it important for both the pharmaceutical and the food industry.[7] Although the
pitaya peel is acidic in nature, acidity decreases upon ripening of the fruit due to respiration
process.[8] Reports on the studies of the pitaya extract on rats were shown to increase HDL
cholesterol in plasma.[9] The edible seeds of pitaya are rich in polyunsaturated fatty acids
(PUFA).[3] The methanolic extract of pitaya seeds were subjected to GC/MS to identify the
possible compounds present in the seeds. Some of the compounds reported were
tetradecanoic acis, S-(-)-butanetriol, octadecanoic acid, 9, 17-octadecadienal, 9,12,15-
octadecatrienoic acid, phytol, nonanoic acid, n-hexadecanoic acid, 8,11,14-
[10]
heptadecatrienoate.

Dengue has been a major infectious disease which has been reported at high percentage in
India. Dengue is a viral disease which is caused by any of the four serotypes which are
similar genetically but differ antigenically.[11] DENV-1 and DENV-2 are the two prevalent
serotypes. DENV-3 is the most virulent of all the serotypes and is involved in the recent
dengue outbreaks. It has been reported that dengue caused by DENV-3 and DENV-4
serotypes are involved with the liver infections at high rates.[12] Dengue virus contains seven
main proteins.[13] The protein used for this study is the non-structural 5 (NS5) protein from
the dengue virus type 3 (strain Sri Lanka/1266/2000). This protein is classified under the
transferases. The RNA-dependent-RNA-polymerase (RdRp) domain of the NS5 protein is
involved in the replication of the viral genome. RNA is synthesized via “de novo” by NS5
protein.[14]

Bioinformatics is a trending branch of science which is used for various research aspects. It is
an interdisciplinary branch of science which is used to analyse large amounts of data with
computational methods.[15] Bioinformatics has various tools out of which one of the tools is
PDB. Protein Data Bank (PDB) is a database which the 3-D structures of the proteins, ligands
or macromolecules can be viewed. PDB has over 9000 macromolecules and proteins stored in
it.[16] Docking analysis was done in our study to analyse the binding affinities of the ligands

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with the proteins. The binding affinities of the proteins with the ligands are given in the form
of energy.[17]

MATERIALS AND METHODOLOGIES

Preparation of Macromolecule NS5 Protein
The protein data bank (PDB) was used to obtain the three-dimensional structure of the
macromolecule. PDB contains large number of proteins which are experimentally determined
and stored in this site. The structures are downloaded and saved either in mm CIF or pdb
format. NS5 protein of dengue virus was used for this study. The 3D structure of this protein
was downloaded from PDB and saved in pdb format. The downloaded protein was viewed in
Py-Mol viewer.

Preparation of Ligands
Ligands selected were from the previous studies on this fruit seeds. 11 ligands were used for
the study. Ligands were constructed using ChemSketch.[16] The constructed ligands were
optimized to add the hydrogen bonds and the obtained structures were saved in mol for
docking analysis.

Docking Study
Docking studies were conducting using iGEMDOCK software. IGEMDOCK (Generic
Evolutionary Method for molecular DOCKing) is a graphical-automatic drug design system
for docking, screening and post-analysis.[16] The protein and the ligands were loaded and the
out path was set. Standard docking parameters were used for docking (population size=200,
generations=70 and no. of solutions=2). The docking process was initiated. After the docking
process, the best docking pose for the individual ligands can be obtained. The best binding
pose, the binding affinity and the total binding energy values were saved in the output folder.
The saved files were visualized in Py-Mol viewer.

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RESULTS AND DISCUSSION

Table 1: The fitness and interaction profile for NS5 with the ligands.
V-S V-S V-M
E(pharma) GLU HIS ASP
Total Vander
40 53 254 H -Bond Electrostatic AverCon
Binding Waal’s
Ligand Compound name Z-score=> Energy Force Pair
Energy Force 3.68 1.94 2.12
-1. #J (kcal/mol) (kcal/mol) (kcal/mol)
(kcal/mol) (kcal/mol)
W(pharma)
1.00 0.53 0.58
=> 0.000
A 7,10,13-hexadecatrienoic acid -92.07 -85.66 -97.3 -6.2 -0.1 -7.2 -7.89 1.48 25.04
B 9,12,15-octadecatrienoic acid -100.51 -91.85 -102 -1.9 -1.9 0 -8.66 0 21.59
C 9,12-octadecadienoic acid -104.95 -87.22 -115 -14.6 -4.8 -5.3 -17.34 -0.39 22.25
D 9,17-octadecadienal -88.73 -79.85 -88.7 0 0 0 -8.88 0 24.81
E methyl-8,11,14-heptadecatrienoate -84.02 -77.02 88.6 -4.8 -8.2 0 -7 0 25.95
F n-hexadecanoic acid -83.99 -83.85 -92.7 -10.4 -7.2 -5.5 -1.67 1.52 32.39
G Nonanoic acid -72.18 -60.39 -72.7 0 0 0 -10.37 -1.42 30.91
H Octadecanoic acid -94.95 -86.36 -104.9 -14.7 -3.6 -5.6 -8.59 0 25.1
I Phytol -90.27 -81.05 -90.3 0 0 0 -9.22 0 24.52
J S-(-)-1,2,4-Butanetriol -54.53 -29.37 -54.4 0 0 0 -25.15 0 26.86
K Tetradecanoic acid -95.29 -81.86 -101.9 -8.7 -4.4 -4 -11.9 -1.53 29.88

Table 2: The cluster interaction table for NS5 protein with the ligands.
E (pharma)
H - Bond Energy
Ligand Compound name Z=>-1. #J H – Bond Amino acid position
(kcal/mol)
W (pharma)=>0
A 7,10,13-hexadecatrienoic acid -113.3 H–S Lys (253) - 6.7
B 9,12,15-octadecatrienoic acid -103.9 H–M Thr (50) - 3.5
C 9,12-octadecadienoic acid -135.7 H–S Lys (623) -6.0
D 9,17-octadecadienal -90.5 H–S Asp (663) - 5.0
E methyl-8,11,14-heptadecatrienoate -91.1 H–S Arg (48) - 7.0

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F n-hexadecanoic acid -94.7 - - -

H–M Val (36) - 6.9
G Nonanoic acid -80.1
H–S Arg (57) - 3.5
H–M Met (336) - 3.0
H Octadecanoic acid -106.4
H–S Gln (339) - 5.6
I Phytol -90.3 H–M Pro (363)/ Asp (683) - 2.5
H–M Arg (57) - 3.5
J S-(-)-1,2,4-Butanetriol -67.3
H–S Arg (38) - 11.5
H–M Asp (690) - 3.5
K Tetradecanoic acid -114.4
H–S His (52) -5.9

(A) 7,10,13-hexadecatrienoic acid (B) 9,12,15 octadecatrienoic acid (C) 9,12-octadecadienoic acid ((D) 9,17-octadecadienal

(E) methyl-8,11,14- heptadecatrienoate (F) n-hexadecanoic acid (G) Nonanoic acid (H) Octadecanoic acid

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(I) Phytol (J) S-(-)-1,2,4-Butanetriol (K) Tetradecanoic acid All compounds

Fig. 1: Interaction of compounds with NS5 protein.

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From the Table – 1, the 3D structure coordinates of NS5 protein is optimized and 11
compounds from dragon fruits seeds are identified. Their total binding energy were
calculated using iGEMDOCK. Evaluation of binding conformation of 11 compounds with
NS5 protein is performed using iGEMDOCK. From docking study, we listed binding affinity
of 11 compounds based on ligand binding energy (Table.1).

The binding pose for each ligand molecule into the NS5 protein is analyzed and the one
having lowest ligand binding energy with the target protein among the different poses are
generated. The lower energy scores represent better target protein-ligand binding affinity
compared to higher energy score. Among the 11 analogs, compound C and B are found to
have lower ligand binding energy value than other analogs. Compound “C” has least binding
energy score with NS5 protein (binding energy value = - 104.95 kcal/mol) and compound
“B” has ligand binding energy value of - 100.51 kcal/mol. We further analyzed the docked
pose for finding the binding mode of compound “C” and compound “B” in to the target
protein to validate the reasonable binding conformations.

Docking of compound – C into NS5 protein

From Table – 2 and Figure – 1, the docking simulation of compound - C is performed for
NS5 protein. From the docking study, we observed that compound – C has best binding
affinity with the target protein. Interaction analysis of binding mode of compound –C in the
target protein reveals that it forms one strong hydrogen bond with the branch residue of Lys
(223) having - 6.0 kcal/mol as the bond energy. A close-up view of binding mode of
compound – C with NS5 protein is shown in Fig.2.

Fig. 2: A close-up view of binding mode of compound – C with NS5 protein.

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Docking of compound - B into NS5 protein

From Table – 2 and Figure – 1, the docking studies of 11 compounds are performed for the
target protein. In our results on the binding conformation modes of compounds with NS5
protein, compound - B shows higher affinity with the target protein. In examining the binding
interaction and position of the compound B with NS5 protein ligand binding site predicted by
your docking procedure, it is found that one strong hydrogen bond is formed with backbone
of residue Thr 50 having - 3.5 kcal/mol as its bond energy. A close-up view of binding mode
of compound – B with NS5 protein is shown in Fig.3.

Fig. 3: A close-up view of binding mode of compound – B with NS5protein.

CONCLUSION
Our molecular docking studies explored the possible binding modes of 11 compounds that
are present in dragon fruit seed with NS5 protein. It revealed that all the 11 compounds show
minimum affinity with the target protein. Especially the compound compound C (9, 12-
octadecadienoic acid) and compound B (9, 12, 5-octadecatrienoic acid) shows best result
when compared with other compounds. On comparing the binding energy and the binding
site residues, we found that all compounds differ either in their binding modes or with the
binding site residues for hydrogen bond formation. The conclusion drawn from our virtual
screening and docking result was that the Compound C and Compound B have highest
binding affinity with the NS5 Protein. Though, there are many reports on the in vitro analysis
of these compounds and its antioxidant properties, but there are no in silico studies that
predict the binding and active regions especially with NS5 protein. Our study is probably the

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first such attempt to predict the binding site. However, validation of our results through
invivo and invitro experiments and also with animal models will enlighten hope for the future
development of more potent drugs for the treating Dengue.

REFERENCES
1. Nurliyana R, Zahie ZI, Suleiman MK, Aisyah MR, Rahim KK. Antioxidant Study of Pulp
and Peels of Dragon Fruits: A Comparative Study. International Food Research Journal,
2010; 17: 367-375.
2. Luo H, Cai Y, Liu Tao and Yang S; Chemical Composition and in-vitro Evaluation of the
Cytotoxic And Antioxidant Activities of Supercritical Carbon dioxide Extracts of Pitaya
(Dragon Fruit) Peel; Chemistry Central Journal, 2014; 8: 1.
3. Mello F R, Bernardo C, Dias C O, Züge L C B, Silveira J L M, Amante E R, Candido L
M B, Evaluation of the Chemical Characteristics and Rheological Behaviour of Pitaya
(Hylocereus undatus) Peel; Fruits, 2014; 69: 381-390.
4. Mejía HA, Ruiz SM, Montoya A, Sequeda CR. In situ Morphological Characterization of
Hylocereus spp. (Fam.: Cactaceae) Genotypes from Antioquia and Córdoba (Colombia).
Rev. Fac. Nal. Agr. Medellín, 2013; 66(1): 6845-6854.
5. Phongtongpasuk S, Poadang S, Yongvanich N. Environmental-friendly Method for
Synthesis of Silver Nanoparticles from Dragon Fruit Peel Extract and Their Antibacterial
Activities. Energy Procedia, 2016; 89: 239-247.
6. Herbacha KM, Stintzinga FC, Elssb S, Prestonb C, Schreierb P, Carlea R. Isotope ratio
mass spectrometrical analysis of betanin and isobetanin isolates for authenticity
evaluation of purple pitaya-based products. Food Chem., 2006; 99: 204–209.
7. Montoya-Arroyo A, Schweiggert RM, Pineda-Castro M, Sramek m, Kohlus R, Carle R.
Characterization of Cell Wall Polysaccharides of Purple Pitaya (Hylocereus sp.) Pericarp.
Food Hydrocoll., 2014; 35: 557-564.
8. Brunini MA, Cardoso SS. Quality of the White Pulp Pitayas Stored at Different
Temperatures. Caatinga Mag., 2011; 24: 78-84.
9. Ariffin AA, Bakar J, ChinPing T, Rahman RA, Karim R, Chiachun L. Essential Fatty
Acids of Pitaya (Dragon Fruit) Seed Oil. Food Chem., 2009; 114: 561-564.
10. Sushmitha HS, Roy CL, Gogoi D, Velagala RD, Nagarathna A, Balasubramanian S,
Rajadurai M. Phytochemical and Pharmacological Studies on Hylocereus undatus Seeds:
An In Vitro Approach. World Journal of Pharmacological Research, 2018; 7(14):
986-1006.

www.wjpps.com Vol 7, Issue 9, 2018. 1196

Sathyamurthy et al. World Journal of Pharmacy and Pharmaceutical Sciences

11. Yung CF, Lee KS, Thein TL, Tan LK, Gan VC, Wong JGX, Lye DC, Ng LC, Leo YS.
Dengue Serotype-Specific Differences in Clinical Manifestation, Laboratory Parameters
and Risk of Severe Disease in Adults, Singapore. Am. J. Trop. Med. Hyg., 2015; 92(5):
999-1005.
12. Malavige GN, Fernando S, Fernando DJ and Seneviratne SL. Dengue Viral Infections.
Postgrad Med J., 2004; 80: 588-601.
13. Perera R, Kuhn RJ. Structural Proteomics of Dengue Virus. Curr Opin Microbiol, 2008;
11(4): 369-377.
14. Lim SP, Noble CG, Seh CC, Soh TS, El Sahili A, Chan GK, Lescar J, Arora R, Benson
T, Nilar S, Manjunatha U, Wan KF, Dong H, Xie X, Shi PY, Yokokawa F. Potent
Allosteric Dengue Virus NS5 Polymerase Inhibitors: Mechanism of Action and
Resistance Profiling. PLoS Pathog., 2016; 12(8): e1005737.
15. Mehmood MA, Sehar U, Ahmad N, Use of Bioinformatic Tools in Different Spheres of
Life Sciences. Journal of Data Mining in Genomics & Proteomics, 2014; 5(2): 1000158.
16. Al-Khaboori SA, Balasubramanian S, Kumar KM. In Silico Drug Designing on Human
Protein Kinases Inhibitors. Int. J. Rec. Biotech., 2015; 3(4): 10-17.
17. Ferreira LG, Ricardo N, Oliva G, Andricopulo AD. Molecular Docking and Structure-
Based Drug Design Strategies. Molecules, 2015; 20: 13384-13421.

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