Bangladesh Journal of Pharmacology Research Article In silico antigenic site evaluation and antiviral therapy against dengue serotypes BJP Introduction Dengue disease, caused by dengue virus infection which is found to be an endemic in over 100 countries (Brinkworth et al., 1999). It was found that 100 million cases of dengue fever occur annually. Of which, 500,000 cases require hospitalization, and 25,000 are fatal (Gubler et al., 1998; Ligon et al., 2005; Gratz et al., 1999; Halstead, 2007), due to limited healthcare facilities in developing and underdeveloped countries. DENV (Dengue virus) is an arthropod-borne flavivirus that comprises four distinct serotypes (DENV I, DENV II, DENV III and DENV IV) that constitute an antigenic complex of the genus flavivirus, family Flaviviridae (Mason et al., 1990; Henchal et al., 1982; Gentry et al., 1982; Monath et al., 1986; Russell et al., 1967). Every step in the life cycle of the dengue virus is a potential target for inhibiting viral replication (Qi et al., 2008). NS3 protein constitutes protease, helicase and polymer- ase that are essential for dengue virus replication (Bera et al., 2008). NS3 is responsible for proteolysis of den [gue viral RNA polyprotein as well as carrying out various enzymatic reactions that are mandatory for replication of dengue virus (Luo et al., 2008). Currently there is no antiviral therapy available for Dengue (Muhamad et al., 2010). We assessed the ability of myco -phenolic acid (MPA) and ribavirin (RBV), drug currently used as an immunosuppressive agent, to inhibit dengue virus antigen expression, RNA replica- tion, and virus production (Diamond et al., 2012; Allison et al., 1993; Koff et al., 1983; Conner et al., 1984). The aim of this study was to examine the mutation in antigenic site of dengue virus and the antiviral action of mycophenolic acid (MPA) and ribavirin (RBV) on NS3 proteins DENV I-IV determining the best drug that can be most active against the virus from the binding ener- gy and the pocket which fits the drug. Materials and Methods Sequence retrieval The nonstructural protein 3 sequence of DENV I, DENV II, DENV III and DENV IV were obtained from the A Journal of the Bangladesh Pharmacological Society (BDPS) Bangladesh J Pharmacol 2014; 9: 83-95 Journal homepage: www.banglajol.info Abstracted/indexed in Academic Search Complete, Agroforestry Abstracts, Asia Journals Online, Bangladesh Journals Online, Biological Abstracts, BIOSIS Previews, CAB Abstracts, Current Abstracts, Directory of Open Access Journals, EMBASE/Excerpta Medica, Global Health, Google Scholar, HINARI (WHO), International Pharmaceutical Abstracts, Open J-gate, Science Citation Index Expanded, SCOPUS and Social Sciences Citation Index ISSN: 1991-0088 Abstract Nonstructural protein 3 (NS3) constitute protease, helicase and polymerase that are essential for dengue virus replication. The aim of the present study is to block the replication of the virus by targeting the NS3 Protein. The retrieved sequences of NS3 protein from National Centre for Biotechnology information shows that the antigenic sites of the protein are highly variable in all the four serotypes of dengue virus (DENV) i.e. DENV I, DENV II, DENV III and DENV IV. DENV III found to be most distantly related serotype among all the serotypes studied using UPGMA method. The 3D structure of NS3 protein was modeled using homology modeling by MODELLER 9v8. Evaluation of the constructed NS3 protein models were done by PROCHECK, WhatIf using Exome Horizon. The derived compounds of mycophenolic acid and ribavirin were docked as ligands to the constructed models of NS3 protein using AutoDock 4.2 for Protein-ligand interaction study. Article Info Received: 7 January 2014 Accepted: 19 February 2014 Available Online: 21 February 2014 DOI: 10.3329/bjp.v9i1.17583 Cite this article: Parida P, Deka P, Shankar B, Yadav RNS. In silico antigenic site evaluation and antiviral therapy against dengue serotypes. Bangladesh J Pharmacol. 2014; 9: 83-95. This work is licensed under a Creative Commons Attribution 3.0 License. You are free to copy, distribute and perform the work. You must attribute the work in the manner specified by the author or licensor. In silico antigenic site evaluation and antiviral therapy against dengue serotypes Pratap Parida, Priyadarshini Deka, Brajesh Shankar and RNS Yadav Bioinformatics Centre, Centre for Studies in Biotechnology, Dibrugarh University, Assam 786 004, India. National Centre for Studies in Biotechnology (http:// www.ncbi.nlm.nih.gov). The numbers of sequences were found to be 25, 17, 103 and 25 for DENV I, DENV II, DENV III and DENV IV respectively. Antigenic site finding The Exome Horizon antigenic site finder tool was used to find the antigenic sites of protein. The accession No., sequence length, hits, positions, antigenic sites, anti- genic site length and the highest score were given in the Table I-IV. Phylogenetics analysis The phylogenetic trees were also built for analyzing the highly variable sequences and to find the conserved domains of the non structural protein sequences by using UPGMA method using Exome Horizon. Sequence alignment and homology modeling The amino acid sequences of NS3 of DENV were taken from the National Center for Biotechnology Information (NCBI) Database (Wheeler et al., 2008). A modified Needleman and Wunsch (1970) method implemented in Exome Horizon and the BLOSUM62 substitution matrix (Henikoff et al., 1992) was applied for aligning the sequences. The homology models of DENV NS3 were constructed applying the MODELER 9v8 program using same tem- plate structures for each domain. All the models (M1, M2, M3, and M4 for DENV I, DENV II, DENV III and DENV IV respectively) were generated using the Crystal Structure of the NS3 Protease-Helicase from Dengue virus as template structures (2VBC) (Bhattacharya et al., 2008). Ligand preparation The Molecules (In this study referred as ligands) were prepared using moldraw tool of Exome Horizon. The different parameters of the ligands were studied given in the Table V and VI. Molecular docking study The docking of ligands to the catalytic triad of NS3- NS2B protease was performed using AutoDock 4.0 software (http://autodock. scripps.edu/). AutoDock is reported to be a very common docking program (Sousa et al., 2006) and is reliable (Hetényi et al., 2002). Using the software, polar hydrogen atoms were added to the enzyme and its nonpolar hydrogen atoms were merged, whereas for the ligand, nonpolar hydrogen atoms were merged and Gasteiger charges were added. All rota- table bonds of ligands were set to be rotatable. All calculation for protein-fixed ligand-flexible docking was done using the Lamarckian Genetic Algorithm (LGA) method. A population size of 150 and 10 millions energy evaluations were used for 100 search runs. The grid box with a dimension of 60 × 60 × 60 points and 0.375 Å grid spacing was used around the catalytic triad to cover the entire enzyme binding site and allow accommodated ligands to move freely. After the docking searches were completed, clustering histogram analysis was performed based on an RMSD (root mean square deviation) of not more than 1.5 Å. The best conformation was chosen from the most populated cluster with the lowest docked energy. The interactions of complex enzyme-ligand conformations, including hydrogen bond and other interactions, were analyzed using Viewerlite (www.accelrys.com) and UCSF Chimera software (www.cgl.ucsf.edu/chimera/) (Figure 1-4). Results The antigenic sites analyzed from all the serotypes were found to be highly variable. In DENV-I the sites were found to be IVGLYGNGVVTTSGTYVSPIAQAK, MRLLSPVRVP, EVQVIAVE, MAVGIVSILLSSLLKND VPLAGPLIAGGMLIACYVISG, FTVVVGDVVGILAQ and HGTVLVQVKY. It is observed that the amino acids present at the site producing highest scores were found to be A, P, V, C, G and Q for DENV I. For DENV-II, the antigenic sites were found to be EVQVLALE, MRLLSPVRVPNYNLI, QLGQVMLLVLCVTQVLM, RYLPAIVREA, TSLSVSLVLVGIVTLYLGVMVQAD, and QLGQVMLLVLCVTQVLM. The amino acids producing highest scores for DENV-II were found to be V, P and L. However DENV-III showed much conserved antigenic sequences and was found to be KYLPAIVREA, DRVIDPRRCLKPVILT, and ADRVI DPRRCLKPVILTD. Proline was found to be the most conserved antigenic site for DENV-III. The DENV-IV found to be having the most similar antigenic sites as of DENV-III. The sites were found to be IVDLMCHAT, GRVIDPRRCLKPVILT, MADLSLEKAANVQ, QLGQVMLLVLCAGQLLLM, KHMILVVVITLCAIIL GG, and GRVIDPRRCLKPVILT. The high scoring regions were found to be L, P, E and I. Hence, the anti- genic site P is found to be the most conserved region among all the four serotypes, suggesting as the potent inhibition point of the ligands. The geometry and stereochemistry of the models were evaluated by using the program PROCHECK (Sousa et al., 2006). The stereochemical quality of the generated homology models as well as the crystal structure was evaluated using Ramachandran plots. Results revealed that 79.7 and 2.4% of the residues of the models are located in the most favored regions and the additional allowed regions for Model1. Similarly the residues present in favored region and disallowed regions were found to be 88.6 and 0.2, 87.1 and 11.2, 89.3 and 0.4% for Model2, Model3 and Model4 respectively. The obtained results indicate that all models possess sufficient stereo- 84 Bangladesh J Pharmacol 2014; 9: 83-95 Bangladesh J Pharmacol 2014; 9: 83-95 85 Table I Antigenic site of nonstructural proteins (DENV I) Accession No. Seqeunce length Hits Positions Antigenic sites Antigenic site length Maximum score pose at Score ACJ05959.1 58 1 14->37 IVGLYGNGVVTTSGTYVSPI- AQAK 24 A 1.122 ACJ05958.1 55 1 14->37 IVGLYGNGVVTTSGTYVSAI- AQAK 24 A 1.122 ACJ05957.1 55 1 14->37 IVGLYGNGVVTTSGTYVSAI- AQAK 24 A 1.122 ACJ05956.1 56 1 14->37 IVGLYGNGVVTTSGTYVSAI- AQAK 24 A 1.122 ACJ05955.1 55 1 14->37 IVGLYGNGVVTTSGTYVSAI- AQAK 24 A 1.122 ACJ05954.1 56 1 14->37 IVGLYGNGVVTTSGTYVSAI- AQAK 24 A 1.122 ACJ05953.1 57 1 14->37 IVGLYGNGVVTTSGTYVSPI- AQAK 24 P 1.122 ACJ05952.1 56 1 14->37 IVGLYGKGVVTTSGTYVSAI- AQAK 24 K 1.122 ACJ05951.1 57 1 14->37 IVGLYGNGVVTTSGTYVSPI- AQAK 24 P 1.122 ACJ05950.1 57 1 14->37 IVGLYGNGVVTTSGTYVSPI- AQAK 24 P 1.122 ACJ05949.1 56 1 14->37 IVGLYGKGVVTTSGTYVSAI- AQAK 24 K 1.122 ACJ05948.1 56 1 14->37 IVGLYGNGVVTTSGTYVSAI- AQAK 24 A 1.122 ACJ05947.1 55 1 14->37 IVGLYGNGVVTTSGTYVSAI- AQAK 24 A 1.122 ACJ05946.1 56 1 14->37 IVGLYGKGVVTTSGTYVSAI- AQAK 24 K 1.122 AAA18245.1 142 6 129->138 MRLLSPVRVP 10 P 1.174 AAB03618.1 143 6 129->138 MRLLSPVRVP 10 P 1.174 AAB03617.1 143 6 129->138 MRLLSPVRVP 10 P 1.174 AAB03616.1 143 6 129->138 MRLLSPVRVP 10 P 1.174 3LKWA 236 9 144->151 EVQVIAVE 8 V 1.176 3L6PA 236 9 144->151 EVQVIAVE 8 V 1.176 POLG_DENV IW 3392 13 4 1354- >1391 MAVGIVSILLSSLLK- NDVPLAGPLIAGGMLIACYV ISG 38 C 1.225 POLG_DENV IS 3396 13 7 860->873 FTVVVGDVVGILAQ 14 G 1.225 POLG_DENV IC 791 39 597->606 HGTVLVQVKY 10 Q 1.236 POLG_DENV IA 792 38 597->606 HGTVLVQVKY 10 Q 1.215 86 Bangladesh J Pharmacol 2014; 9: 83-95 Table II Antigenic site of nonstructural proteins (DENV II) Accession No. Seqeunce length Hit Position Antigenic sites Antigenic site length Score CAA40704.1 618 26 94->101 EVQVLALE 8 1.171 NP_739587.2 618 25 267->281 MRLLSPVRVPNYNLI 15 1.174 AAA73185.1 3391 135 2410->2426 QLGQVMLLVLCVTQVLM 17 1.262 AAA73186.1 3391 134 2410->2426 QLGQVMLLVLCVTQVLM 17 1.262 AAB03619.1 108 5 64->73 RYLPAIVREA 10 1.135 AAA66406.1 886 38 331->348 TGPLVAGGLLTVCYVLTG 18 1.25 POLG_DENV IIT 1683 69 473->503 TSLSVSLVLVGIVTLYL- GVMVQADSGCVVSW 31 1.239 POLG_DENV IIJ 3391 135 753->783 TSLSVSLVLVGVVTLYL- GAMVQADSGCVVSW 31 1.272 POLG_DENV IID 1127 49 753->783 TSLSVSLVLVGVITLYL- GAMVQADSGCVVSW 31 1.239 POLG_DENV IIU 679 34 653->676 TSLSVSLVLVGIVTLYLGVMVQAD 24 1.239 POLG_DENV II6 3391 135 2410->2426 QLGQVMLLVLCVTQVLM 17 1.262 POLG_DENV IIN 3391 131 753->783 TSLSVSLVLVGVVTLYL- GVMVQADSGCVVSW 31 1.272 POLG_DENV II8 3391 133 2410->2426 QLGQVMLLVLCVTQVLM 17 1.262 POLG_DENV IIQ 3391 134 2410->2426 QLGQVMLLVLCVTQVLM 17 1.262 POLG_DENV II7 3391 134 2410->2426 QLGQVMLLVLCVTQVLM 17 1.262 POLG_DENV IIP 3388 132 2407->2423 QLGQVMLLVLCVTQVLM 17 1.262 Table III Antigenic site of nonstructural proteins (DENV III) Accession No. Sequence length Hits Positions Antigenic sites Antigenic site length Score ABU88348.1 102 3 P KYLPAIVREA 10 1.143 ABU88347.1 102 3 P KYLPAIVREA 10 1.143 YP_001531172.2 619 24 P DRVIDPRRCLKPVILT 16 1.206 ACJ06087.1 86 3 P KYLPAIVREA 10 1.143 ACJ06086.1 88 3 P KYLPAIVREA 10 1.143 ACJ06085.1 18 1 P PAIVREA 7 1.143 ACJ06081.1 87 2 P KYLPAIVREA 10 1.143 ACJ06082.1 88 2 P KYLPAIVREA 10 1.143 ACJ06083.1 88 2 P KYLPAIVREA 10 1.143 ACJ06080.1 87 2 P KYLPAIVREA 10 1.143 ACJ06079.1 89 4 P KYLPAIVREA 10 1.143 ACJ06078.1 89 2 P KYLPAIVREA 10 1.143 ACJ06077.1 88 2 P KYLPAIVREA 10 1.143 ACJ06076.1 88 2 P KYLPAIVREA 10 1.143 ACJ06074.1 88 2 P KYLPAIVREA 10 1.143 ACJ06075.1 88 2 P KYLPAIVREA 10 1.143 Bangladesh J Pharmacol 2014; 9: 83-95 87 Table IV Antigenic site of nonstructural proteins (DENV IV) Accession No. Sequence length Hits Positions Antigenic sites Antigenic site length Score AAA18247.1 140 7 115->123 IVDLMCHAT 9 1.142 2VBCA 618 28 420->435 GRVIDPRRCLKPVILT 16 1.206 2VBCB 31 1 4->16 MADLSLEKAANVQ 13 1.06 2WHXA 618 28 420->435 GRVIDPRRCLKPVILT 16 1.206 POLG_DENV IVT 3387 150 2406->2423 QLGQVMLLVLCAGQLLLM 18 1.251 POLG_DENV IVH 3387 151 2406->2423 QLGQVMLLVLCAGQLLLM 18 1.251 POLG_DENV IVS 3387 147 1156->1173 KHMILVVVITLCAIILGG 18 1.267 POLG_DENV IVP 3387 151 2406->2423 QLGQVMLLVLCAGQLLLM 18 1.251 POLG_DENV IVD 3387 148 1156->1173 KHMILVVVITLCAIILGG 18 1.267 2JLXB 451 20 253->268 GRVIDPRRCLKPVILT 16 1.206 2JLXA 451 20 253->268 GRVIDPRRCLKPVILT 16 1.206 2JLZB 451 20 253->268 GRVIDPRRCLKPVILT 16 1.206 2JLZA 451 20 253->268 GRVIDPRRCLKPVILT 16 1.206 2JLYB 451 19 253->268 GRVIDPRRCLKPVILT 16 1.206 2LYBA 451 19 253->268 GRVIDPRRCLKPVILT 16 1.206 2JLWB 451 20 253->268 GRVIDPRRCLKPVILT 16 1.206 2JLWA 451 20 253->268 GRVIDPRRCLKPVILT 16 1.206 2JLVB 451 20 253->268 GRVIDPRRCLKPVILT 16 1.206 2JLVA 451 20 253->268 GRVIDPRRCLKPVILT 16 1.206 2JLUB 451 20 253->268 GRVIDPRRCLKPVILT 16 1.206 2JLUA 451 20 253->268 GRVIDPRRCLKPVILT 16 1.206 2JLSA 451 20 253->268 GRVIDPRRCLKPVILT 16 1.206 2JLRA 451 20 253->268 GRVIDPRRCLKPVILT 16 1.206 2JLQA 451 19 253->268 GRVIDPRRCLKPVILT 16 1.206 NP_740321.1 618 28 420->436 GRVIDPRRCLKPVILPD 17 1.206 Table V Molecular properties of mycophenolic acid derivatives Sl. No IUPAC Name Chemical formula Molecular weight Log P Structure 1 6-(4-hydroxy-6-methoxy-7- methyl-3-oxo-1,3-dihydro- isobenzofuran-5-yl)-4- methyl-hex-4-enoic acicd C17H20O6 320.34 2.55 O O OH O HO O 2 7-hydroxy-6(6- hydroxyamino-3-methyl- hepta-2,6-dienyl)-5- methoxy-4-methyl-3H- isobenzofuran-1one C18H23NO5 333.38 2.63 OH O O O H N OH 3 ACMPHA C20H26N2O5 374.43 1.91 NHOH O O O H N O 4 LMPHA C31H46N2O4 510.71 NHOH C10H19 O O O H N chemical quality. The Phylogenetic analysis showed the high intraspecies variation in NS3 protein of all the serotypes (Figure 5). The distances within group was found to be 0.05, 1.27, 0.70 and 0.20 for DENV-I, DENV- II, DENV-III and DENV-IV respec-tively. The mean distances within groups showed that DENV-I has high distance related from DENV-II, DENV-III and DENV- IV. The homology models of the NS3 protein of four serotypes also showed very good variation in Rama- chandran plot. The docking study of the derived compound of mycophenolic acid (4 nos.) and ribavirin (5 nos.) was done with AutoDock4.2. The binding energy obtained in the range of -0.97 to +190.03. The ligand 3 of mycophenolic acid was found to be the best drug in Model1 having the minimum binding energy - 9.2 kcal/mol. The ligand 4 of the ribavirin revealed as the best inhibitor for model2 and showed the minimum binding energy of -16.5 kcal/mol and was found to be the best ligand among all the ligands studied. The number 4 ligand of the mycophenolic acid found to be second best inhibitor for model3 among all the selected ligands after the ligand4 of mycophenolic acid with binding energy -15.9 kcal/mol. The ligand1 of ribavirin was found to be the best inhibitor for model4 having minimum binding energy -10.7 kcal/mol (Figure 6). Discussion Dengue fever epidemics has increased numerously over the last few decades (Ligon et al., 2005) therefore Developing antiviral drug and vaccine is becoming very important due to the global threat of viral disease pandemics (Noble et al., 2010; Wang et al., 2009). The functional similarity between the NS2B/NS3 proteases from the four genetically and antigenically distinct serotypes was identified by the differences in their substrate specificity using tetrapeptide and oc- 88 Bangladesh J Pharmacol 2014; 9: 83-95 Table VI Molecular properties of ribavirin derivatives Sl. No. IUPAC Chemical formula Mol. Wt. Log P Structure 5 5-amino-1-(3,4-dihydroxy-5- hydroxymethyl-tetrahydro- furan-2-yl)-1H-imidazole-4- carboxylic acid amide C9H14N4O5 258.23 -3.28 O N OH OH HO H2N N NH2 O 6 4-hydroxy-5-(3,4,5-trihydroxy -tetrahydro-furan-2-yl)-4,5- dihydro-1H-pyrazole-3- carboxylic acid amide C8H13N3O6 247.21 -3.73 O HO HO OH HN N OH O NH2 7 1-(3,4-dihyroxy-5- hydroxymethyl-tetrahydro- furan-2-yl)-1H-[1,2,4]triazole- 3-carbozamidine C8H13N5O4 243.22 -1.61 O HO OH HO N N N NH2 HN 8 Ribavirin 5 triphosphate C8H12N4O14P33- 481.12 O HO OH N O P O - O O P - O O O P - O HO O N N NH2 O 9 1-(3,,4,5-trihydroxy- tetrahydro-furan-2-yl)-1H- [1,2,4]triazole-3carboxylic acid amide C7H10N4O5 230.18 -1.38 O HO N HO OH N N NH2 O Bangladesh J Pharmacol 2014; 9: 83-95 89 Figure 1: Docking Interaction of Models with ligand1 and 2. The interaction energy was calculated and the hydrogen bonds were observed 90 Bangladesh J Pharmacol 2014; 9: 83-95 Figure 2: Docking interaction of Models with ligand 5 and 6. The interaction energy was calculated and the hydrogen bonds were observed Bangladesh J Pharmacol 2014; 9: 83-95 91 Figure 3: Docking interaction of Models with ligand 7 and 8. The interaction energy was calculated and the hydrogen bonds were observed 92 Bangladesh J Pharmacol 2014; 9: 83-95 B in di ng e ne rg y 200 150 100 50 0 -50 Serial number 1 2 3 4 5 6 7 8 9 M1 M2 M3 M4 Figure 4: Docking interaction of Models with ligand 9. The interaction energy was calculated and the hydrogen bonds were ob- served Figure 5: Binding enegry analysis from the interaction study of the ligands it was observed that the ligands 3, 8, 4 and 5 are the best inhibitors for DEN-I, DEN-II, DEN-III and DEN-IV respectively. These ligands showed the binding energy of -9.18, -16.52, - 15.87 and -10.66 Kcal/mol respectively for the corresponding serotypes tapeptide libraries in a positional scanning format, each containing 130,321 substrates (Guzman et al., 2010). Development of new genomic and proteomic studies coupled with computational sciences could provide the discovery of various target proteins and poten- tial inhibitor to be developed as drugs (Li et al., 2005; Tambunan et al., 2011). The NS3 enzyme of dengue is responsible for replication of the virus. The replication complex include the NS3 nucleotide the NS3 protease and with its NS2B cofactor, the NS3 nucleotide triphosphatase. This protein serves as the potential inhi- bitory targets for antiviral agents since they are required for virus replication. The multifunctional C- terminal domain of NS3 encodes NTPase, helicase and RTPase activities. NTP hydrolysis is thought to provide the chemical energy required for helicase activity. There is currently no antiviral therapy available against dengue virus. In this study we generated some antiviral ligands of mycophenolic acid and ribavirin. We predic- ted the binding activity of the ligands against NS3 protein of Dengue virus. From the interaction study of the ligands it was observed that the ligands 3, 8, 4 and 5 are the best inhibitors for DEN-1, DEN-2, DEN-3 and DEN-4 respectively. These ligands showed the binding energy of -9.2, -16.5, -15.9 and -10.7 Kcal/mol respec- tively for the corresponding serotypes. To study the interaction of ligands at the inhibition point, it is necessary to know the antigenic sites of the disease causing protein. It is observed that, there is great variation in the antigenic site of the proteins. However the most conserved antigenic site found to be the aminoacid, Proline almost in every serotype. The ligands were docked into each antigenic site of the protein and the binding energy was reported. Phylo- genetic analysis also suggested the typical variations in all the NS3 sequences of NCBI. Finding antigenic site of a mutated protein is the primary aim of any computer aided drug design. The current research discovered several variations in the original sequences as well as antigenic site in the NS3 protein of the 4 serotypes. The antigenic sites were targeted to block the replication activity of the virus. The antigenic sites were targeted by the derivative compounds of mycophenolic acid and ribavirin to block the replication process of the virus. Hence the current study may be useful in designing the drugs that may be synthesized in wet lab and can be used as antiviral drugs against dengue serotypes. The docking of two groups of inhibitors from mycophenolic acid and ribavirin again NS3 were carried out. In this work, the complexation energy of the docking was used as the descriptors for selecting new candidates for competitive dengue inhibitors. The antigenic sites were highly variable. The phylogenetics studies were also evaluated for finding the interspecies variation in NS3. Homologies of protein were constructed for all the serotypes. AutoDock4.2 helped in carrying out the Bangladesh J Pharmacol 2014; 9: 83-95 93 Figure 6: Phylogenetic analysis of DENV I interaction of the drugs with the protein models. Complexation energies for all the new ligand-enzyme complexes were evaluated. Detailed structural information is becoming increasingly available for the dengue NS3 proteins. Since these proteins are requested for virus infectivity and replication. Structure based computational approaches offer an attractive strategy for the discovery and optimization of dengue antiviral drugs. Moreover, these computational approaches promise and improve the effectiveness of current structure based calculations. Conclusion Based on the complexation energies calculated, the 3rd compound of the mycophenolic was found to be the best inhibitor for DENV I. Similarly drug4 of ribavirin, drug4 of mycophenolic acid and drug1 of ribavirin were found to be best drugs against DENV II, DENV III and DENV IV respectively the lowest and closest energies to the reference compounds. Acknowledgement The authors gratefully acknowledge Bioinformatics Infrastructure Facility (BIF) funded by Department of Biotechnology, Govt. of India, at Centre for Studies in Biotechnology, Dibrugarh University. References Allison AC, Eugui EM. Immunosuppressive and other effects of mycophenolic acid and an ester prodrug, mycophenolate mofetil. Immunol Rev. 1993; 136: 5-28. Bera AK, Kuhn RJ, Smith JL. Functional characterization of cis and trans activity of the flavivirus NS2B-NS3 protease. J Biol Chem. 2007; 282: 12883-92. Bhattacharya A, Wunderlich Z, Monleon D, Tejero R, Montelione GT. Assessing model accuracy using the homology modeling automatically software. Proteins 2008; 70: 105-18. Brinkworth RI, Fairlie DP, Leung D, Young PR. Homology model of the dengue 2 virus NS3 protease: Putative interactions with both substrate and NS2B cofactor. J Gen Vir. 1999; 80: 1167-77. Conner CS. Ribavirin. Drug Intell Clin Pharm. 1984; 18: 137-38. Diamond MS, Zachariah M, Harris E. Mycophenolic acid inhibits dengue virus infection by preventing replication of viral RNA. Virology 2012; 304: 211-21. Gentry MK, Henchal EA, McCOWN LM, Brandt WE, Dalrymple JM. Identification of distinct antigenic determi- nants on dengue-2 virus by using monoclonal antibodies. Am J Trop Med Hyg. 1982; 31: 548-55. Gratz NG. Emerging and resurging vector-borne diseases. Annu Rev Entomol. 1999; 44: 51-75. Gubler DJ. Dengue and dengue hemorrhagic fever. Clin Microbiol Rev. 1998; 11: 480-96. Guzman MG, Halstead sb, Artsob H, Buchy P, Farrar J, Gubler DJ, Hunsperger E, Kroeger A, Margolis HS, Martínez E, Nathan MB, Pelegrino JS, Simmons C, Yoksan S, Peeling RW. Dengue: A continuing global threat. Nature Rev Micro. 2010; S7-S16. Halstead SB. Dengue. Lancet 2007; 370: 1644-52. Henchal EA, Gentry MK, McCowN JM, Brandt WE. Dengue virus-specific and flavivirus group determinants identified with monoclonal antibodies by indirect immunofluo- rescence. Am J Trop Med Hyg. 1982; 31: 830-36. Henikoff S, Henikoff JG. Amino acid substitution matrices from protein blocks. Proc Natl Acad Sci. 1992; 15: 10915-19. Hetényi C, van der Spoel D. Efficient docking of peptides to proteins without prior knowledge of the binding site. Protein Sci. 2002; 11: 1729-37. Koff WC, Elm JL Jr, Halstead SB. Antiviral effects of ribavirin and 6-mercapto-9-tetrahydro-2-furylpurine against dengue viruses in vitro. Antivir Res. 1982; 2: 69-79. Li J, Lim SP, Beer D, Patel V, Wen D, Tumanut C, Tully DC, Williams JA, Jiricek J, Priestle JP, Harris JL, Vasudevan SG. Functional profiling of recombinant NS3 proteases from all four serotypes of dengue virus using tetrapeptide and octapeptide substrate libraries. J Bio Chem. 2005; 280: 28766- 74. Ligon BL. Dengue fever and dengue hemorrhagic fever: A review of the history, transmission, treatment, and prevention. Semin Pediatr Infect Dis. 2005; 16: 60-65. Luo D, Xu T, Hunke C, Grüber G, Vasudevan SG, Lescar J. Crystal Structure of the NS3. J Virol. 2008; 82: 173-83. Malachowska-Ugarte M, Cholewinski G, Dzierzbicka K, Trzonkowski P. Synthesis and biological activity of novel mycophenolic acid conjugates containing nitroacridine/ acridone derivatives. Euro J Med Chem. 2012; 54: 197-201. Mason PW, Zfigel MU, Semproni AR, Fournier MJ, Mason L T. The antigenic structure of dengue type 1 virus envelope and NS1 proteins expressed in Escherichia coil. J Gen Vir. 1990; 71: 2107-14. Monath TP. Pathobiology of the flaviviruses. In: The Togaviridae and Flaviviridae. Schlesinger S, Schlesinger MJ (eds). New York, Plenum Press, 1986, 275-440. Muhamad M, Kee LY, Rahman NA, Yusof R. Antiviral actions of flavanoid-derived compounds on dengue virus type-2. Int J Biol Sci. 2010; 6: 294-302. Noble CG, Chen YL, Dong H, Gu F, Lim SP, Schul W, Wang QY, Shi PY. Strategies for development of dengue virus inhibitors. Antiviral Res. 2010; 85: 450-62. Qi RF, Zhang L, Chi CW. Biological characteristics of dengue virus and potential targets for drug design. Acta Biochim Biophys Sin. 2008; 40: 91-101. Russell PK, Nisalak A. Dengue virus identification by the plaque reduction neutralization test. J Immun. 1967; 99: 291- 96. Sousa SF, Fernandes PA, Ramos MJ. Protein-ligand docking: 94 Bangladesh J Pharmacol 2014; 9: 83-95 Current status and future challenges. Proteins 2006; 65: 15- 26. Tambunan USF, Apriyanti N, Parikesit AA, Chua W, Wuryani K. Computational design of disulfide cyclic peptide as potential inhibitor of complex NS2B-NS3 dengue virus protease. Afr J Biotechnol. 2011; 10: 12281-90. 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. A small-molecule dengue virus entry inhibitor. Antimicrob Agents Chemother. 2009; 53: 1823-31. Wheeler DL, Barrett T, Benson DA, Bryant SH, Canese K, Chetvernin V, Church DM, Dicuccio M, Edgar R, Federhen S, Feolo M, Geer LY, Helmberg W, Kapustin Y, Khovayko O, Landsman D, Lipman DJ, Madden TL, Maglott DR, Miller V, Ostell J, Pruitt KD, Schuler GD, Shumway M, Sequeira E, Sherry ST, Sirotkin K, Souvorov A, Starchenko G, Tatusov RL, Tatusova TA, Wagner L, Yaschenko E. Database resources of the National Center for Biotechnology Information. Nucleic Acids Res. 2008; 36: D13-21. Author Info Pratap Parida (Principal contact) e-mail: pratap_parida2007@yahoo.com Bangladesh J Pharmacol 2014; 9: 83-95 95 DatePrinted: This article was downloaded by you on: Sep 28, 2018