8/7/2019 Swine flue drug

1/5

Abstract-- Swine flu now becomes a deadest diseasethroughout the world and the mortality rate is a greatinternational concern now. Influenza A viruses carry twosurface glycoprotein, the hemagglutinin (HA) and theneuraminidase (NA) which recognize the same host cellmolecule, sialic acid. Here in this work an attempt has beentaken to improve the binding efficiency of the most commondrugs like Oseltamivir and Zanamivir.A homology basedmodeling has been performed by MODELLER tool to get the3D structure. From docking studies the functional groups of themolecule responsible for binding to the modeled neuraminidaseenzyme was identified and for that several modification in thefunctional groups has been done accordingly. Further dockingwith analogs shows that modified functional group with CH 2COOH in case of Oseltamivir and -CH 2OCH 2CH 2OH inZanamivir having least energy value with high binding affinity.

I. INTRODUCTION

Human cases of new influenza A (H1N1) virus infectionhave been identified recently in many countries [1, 2]. After the detection of the first cases in Mexico and in the UnitedStates and the spread of infection to further countries, theWorld Health Organization (WHO) declared the outbreak of a new influenza A (H1N1) swl (swine-like) virus infection to

be a public health emergency. The recent outbreak of thenovel strain of influenza A (H1N1) virus has raised a globalconcern of the future risk of a pandemic. A recent report saysthat currently isolates of H1N1 virus are resistant to presentlyused neuraminidase inhibitor drugs like Zanamivir andOseltamivir [3]. To understand at the molecular level howthis new H1N1 virus can be inhibited by the current anti-influenza drugs and which of these drugs it is likely toalready be resistant to homology modeling and MDsimulations have been applied on the H1N1neuraminidase[4].Methods developed to facilitate andspeedup the drug designing process are Rational Drug Design(RDD). These processes are used in biopharmaceuticalindustry to discover and develop new drugs. RDD uses avariety of computational methods to identify novelcompounds. One of those methods is docking of drug

molecules with receptors. The site of drug action, which isultimately responsible for the pharmaceutical affect, is areceptor [5].

In the present study a computational approach is used to predict the three dimensional structure of neuraminidaseenzyme sequence of a novel H1N1 virus in China byhomology modeling method [6]. The approach produces thevalid structural model with the available template whichhaving 88% amino acid identity. After validation of themodel the in-silico docking process with different analogs of drug Oseltamivir and Zanamivir is performed this shows thatthe analog having significant binding energy can as be usedas the probable lead molecule for the H1N1 virus. So by therational drug design approach, an effort has been made todesign novel modified drugs by using the conventional drugslike Oseltamivir and Zanamivir and docking against the

predicted structure of H1N1 virus [6].

II. MATERIALS AND METHODS.A . Sequence retrieval and 3D model building

The sequence for the neuraminidase enzyme was retrievedfrom NCBI having ID GQ232095 [7]. Then with this querysequence a BLAST [8] search was performed against PDB(Protein Databank) to retrieve the corresponding template for the neuraminidase. Searching for the suitable template for theneuraminidase in the PDB (protein data bank) was performed

basing on criteria viz, the native protein should have E value85% with the target sequence. The modelwas built by homology modeling and for this MODELLER [9] program was used. The MODELLER program uses anautomated approach to comparative protein structuremodeling by satisfaction of spatial restraints. Briefly, thecore modeling procedure begins with an alignment of thesequence to be modeled (target) with related known 3Dstructures (templates). This alignment is usually the input tothe program. The output is a 3D model for the targetsequence containing all main chain and side chain non-hydrogen atoms [10].

In-silico modeling and docking Studies of Antiviral Drugs for Swine flu

Raghunath Satpathy*, Rashmiranjan Behera and Rajesh Kumar Guru

Department of Biotechnology, MIRC Laboratory,Majhighariani Institute Of Technology & Science, Rayagada, Odisha, India-765017

E-mail: rnsatpathy@gmail.com

1National Conference On Computational Biology (NCCB-09)December 28 th -29,2009Sikhya O Anusandhan University (SOA University),Bhubaneswar

8/7/2019 Swine flue drug

2/5

Figure1 Showing the working principle of MODELLER program (Sali and Blundell 1993)

B. Model ValidationThe MODELLER generated structure was further verified

by PROCHECK [11].The PROCHECK program provides theinformation about the stereo chemical quality of a given protein structure. The PROCHECK was used to generateRamachandran plot and the quality of the structure wascomputed in terms of % of residues in favorable regions, %of non Proline Glycine residues etc.The quality of structurewas also further accessed by using ERRAT [12]. ERRAT is a

protein structure verification algorithm that is especiallywell-suited for evaluating the progress of crystallographicmodel building and refinement. Along with ERRAT data theDOPE (discrete optimized potential energy) score [13] per residues was observed. DOPE score is calculated by Modeler

program which is the distance dependent statistical potential based on probabilistic theory. This is extremely useful in

making decisions about reliability. Then the backbonealignment and RMSD (root mean square deviation) study was

performed by using PYMOL tool [14]. PyMOL is an usefulmolecular modeling software allowing the visualization of three dimensional molecular structures as well as for the

backbone alignment between the model and template.

C. Preparation of drug analogsFrom the PDB the drug molecular structure of Oseltamivir

and Zanamivir were retrieved. Protein Data Bank (PDB) is arepository for the storing of X-ray and NMR retrievedstructural data of molecules for proteins and nucleic acids.The structural analog for the above drugs were prepared byMARVIN SKETCH [15]. Marvin sketch is a Java basedchemical drawing tool which allows creating and editing of molecules in various file formats.

D. Docking studyDocking was performed between the validated structure of

neuraminidase and the structural analogs by using HEX 5.0tool [16]. HEX is an interactive molecular graphics programfor protein-ligand docking, assuming the ligand is rigid, andit can superpose pairs of molecules using only knowledge of their 3D shapes. It is still the only docking and superposition

program to use spherical polar Fourier (SPF) correlations toaccelerate the calculations [17]. The molecules binding to a

receptor, inhibit its function, and thus act as drug. Thecollection of Oseltamivir, Zanamivir and neuraminidasecomplexes was identified via docking. The binding energywas computed and the ligand binding pattern was observed.

III. RESULTS

A . Homology modeling of h1n1 neuraminidaseThe sequence for the neuraminidase protein having 468

amino acid was obtained from NCBI having accessionnumber gq 232095.The sequence was directly submitted to

NCBI from Chinese national influenza centre on 4 th June2009. The template structure 3CYE [18] is considered as thesuitable template by considering the E-value (0.0), identity(88%) for the homology modeling by Modeller program.

Fig.2 . Showing Rasmol [18] view of final 3D structure of theh1n1 neuraminidase. Yellow color shows beta sheets and Redone is the alpha helixes.

Table 1 The structural information of the modeledneuraminidase protein

Structural features Information No. of residues 468 No. of atoms 3616 No. of Hydrogen bonds 252 No. of helices 3 No. of sheets 42 No. of turns 60

IV . VALIDATON OF THE MODEL

A. Structure validation by ERRAT and DOPE score.To verify further the predicted structures, the coordinates

of the modeled protein was fed into the ERRAT ProteinVerification Server. The overall quality factor was obtainedas 64. 978 which is satisfactory. The result obtained is shownin Figure 3. (B).The DOPE scores of both template andmodel obtained from Modeller output has been shown inFigure 3(A). From the comparative chart the peak is showingthat there is no defect in the loop regions in the residues. Soin the present case the loop refinement method is notrequired for the model.along with the original molecule by the HEX tool. Theenergy after each docking was computed given in table 3(A)and 3(B).

2

8/7/2019 Swine flue drug

3/5

(A)

(B) Fig.3 (A), (B) .showing the DOPE score comparison betweenthe model and template and the ERRAT validation server output respectively.

B. Structure validation by PROCHECK The validation of the model was carried out using

Ramachandran plot calculations computed with thePROCHECK program Figure 4. The and distributions of the Ramachandran plots of non-Glycine, non-Proline residues

are summarized in Figure 8 and table 2. Altogether 100% of the residues were in favored and allowed regions. The overallG-factor used was computed as -0.46.

Fig.4 .

Showing the Ramachandran plot view from PROCHECK output.

Table2 Ramachandran plot calculations on 3D model of Neuraminidase protein computed with the PROCHECK program

% residues in favorable regions 81.7% residues in additional residue regions 15.5% residues in generously regions 1.3% residues in disallowed regions 1.5% of non Proline and non Glycine residues 100

B. Docking study

The docking study was performed between the modeledstructure and molecular analogs of Zanaminvir andOseltamivir

Table 3 (A) Docking results of modeled neuraminidase byZanmaivir

Drugs docked E -valuesZamanivir -207.88Zamanivir analog 1 -205.81Zamanivir analog 2 -202.92Zamanivir analog 3 -215.49Zamanivir analog 4 -204.48Zamanivir analog 5 -203.74Zamanivir analog 6 -206.54Zamanivir analog 7 -217.52Zamanivir analog 8 -212.19Zamanivir analog 9 -222.02Zamanivir analog 10 -228.44

Table 3 (B) Docking results of modeled neuraminidase byOseltamivir

Drugs docked E -valuesOseltamivir -193.53Oseltamivir analog 1 -195.58Oseltamivir analog 2 -201.36

Oseltamivir analog 3 -198.22Oseltamivir analog 4 -198.83Oseltamivir analog 5 -207.61Oseltamivir analog 6 -208.03Oseltamivir analog 7 -229.53Oseltamivir analog 8 -231.21Oseltamivir analog 9 -209.74Oseltamivir analog 10 -224.83

3

8/7/2019 Swine flue drug

4/5

(A) Zanamivir (original molecule)

(B) Oseltamivir (original molecule)

(C) Zanamivir (Analog 10)

(D) Oseltamivir (analog 8)

Fig.5 . Showing the original drug molecule of Zanamivir and

Oseltamivir (A, B) and analogs of original drug molecule (C,

D).

V. C ONCLUSION

The current drugs like Oseltamivir and Zamanivir are potential Neuraminidase inhibitor hence used against swineflu virus H1N1. In this work, we have constructed ahomology based 3D model of Neuraminidase of China H1N1virus, using the MODELLER software. The final refined

model was further assessed by ERRAT and PROCHECK program, and the results obtained that this model is reliable.The stable model is further subjected for docking with thedifferent drug analogs of Oseltamivir and Zanamivir.Docking results indicate that the analog 8 in case of Oseltamivir and analog 10 in case of Zamanivir show more

potent binding activity than the original drugs, thiscorresponds to inhibition activity against the viralneuraminidase. More over this type of analysis show that thesome of the modified drugs having more potential activitythan the original one. So in our study the drug analogsshowing higher binding activities can be used as the probablelead molecules than the rest of drugs for Swine flu.

ACKNOWLEDGMENT

We are thankful to CEO, Director and Dean of Majhighariani Institute Of Technology & Science ,Ryagadato provide us the MIRC lab for computing facility.

R EFERENCES[1] Salomon R, Webster RG, The influenza virus enigma, Cell, vol 136,

pp.402-410, 2009.[2] Butler D, Swine flu goes global, Nature, vol 458, pp.1082-1083, 2009.[3] Sheri mossad,The resurgence of swine-origin influenza A (H1N1)Cleveland clinical journal of medicine,vol 76, pp.337-343, 2009.[4] Rungrotmongkol T, etal,Susceptibility of antiviral drugs against 2009influenza A (H1N1) virus, Biochem Biophys Res Commun. May 2009.

[5] Virupakshaiah DBM, Chandrakanth Kelmani, Rachanagouda Patil, andPrasad Hegade,

Computer Aided Docking Studies on Antiviral Drugs for SARS PWASET,vol 24, pp. 297-299, 2008.

[6] Huang IC, Li W, Sui J, Marasco W, Choe H, Farzan M , "Influenza A virusneuraminidase limits viral super infection", J. Virol. vol 10, pp. 48344843,2007.[7] http://www.ncbi.nlm.nih.gov/nuccore/GQ232095.[8] Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman D.J Basic

local alignment search tool. J. Mol. Biol. vol 215, pp.403-410, 1990[9] Eswar N, Eramian D, Webb B, Shen M, Sali A Protein structure modelingwithMODELLER Methods, Mol. Biol, vol 426 pp.145-159, 2008.[10] A. Sali & T.L. Blundell. Comparative protein modeling by satisfactionof spatial restraints J. Mol. Biol., vol 234 pp.779-815, 1993.

[11]Laskowski R A, MacArthur M W, Moss D S & Thornton J M:PROCHECK: a program to check the stereo chemical quality of proteinstructures J. Appl. Cryst., vol 26 pp.283-291,1993[12] V C. Colovos and T. O. Yeates Verification of protein structures:

patterns of non bonded atomic interactions Protein Sci vol 2, pp. 15111519,1993[13] Min-Yi Shen, Andrej Sali Statistical potential for assessment and

prediction of Protein structures Protein Science vol 15 pp.2507-2524, 2006[14] Delano, W.L The PyMol Molecular Graphics System (2002) on WorldWide Web www.pymol.org.

4

8/7/2019 Swine flue drug

5/5