A

Project Report On

““Insilico analysis towards influenza virus : A homology modeling and molecular

docking study”

at Biotech Park, Lucknow

INTRODUCTION

Influenza A virus causes influenza in birds and some mammals, and is the only species of influenza virus A. Influenza virus A is a genus of the Orthomyxoviridae family of viruses. Strains of all subtypes of influenza A virus have been isolated from wild birds, although disease is uncommon. Some isolates of influenza A virus cause severe disease both in domestic poultry and, rarely, in humans.

Types of Influenza A virus :- Influenza type A viruses are categorized into subtypes based on the type of two proteins on the surface of the viral envelope: Hemagglutinin, a protein that causes red blood cells to agglutinate. Neuraminidase, an enzyme that cleaves the glycosidic bonds of the monosaccharide, neuraminic acid Variants of Influenza A virus :-

Avian influenza or Bird flu :- The bird flu, also known as avian influenza and H5N1, is an infection caused by avian influenza A. Bird flu can infect many bird species, including domesticated birds such as chickens. Rarely, humans can be infected by these bird viruses. People who get infected with bird flu usually have direct contact with the infected birds or their waste products.

Human flu or Annual flu :- The annual flu (also called "seasonal flu" or "human flu") in the U.S. "results in approximately 36,000 deaths and more than 200,000 hospitalizations each year. The annually updated, trivalent influenza vaccine consists of hemagglutinin (HA) surface glycoprotein components from influenza H3N2, H1N1, and B influenza viruses. Swine influenza :- Swine influenza (or "pig influenza") refers to a subset of Orthomyxoviridae that create influenza and are endemic in pigs. The known subtypes of influenza A virus that create influenza and are endemic in pigs are H1N1, H1N2, H3N1 and H3N2. Causes :- The flu is a highly contagious disease. The flu virus is spread when you either inhale infected droplets in the air (spread when an infected person coughs or sneezes) or when you come in direct contact with an infected person's secretions (by kissing, touching, sharing objects such as spoons and forks). You can also transfer the flu virus to your hands by touching smooth surfaces such as doorknobs, handles, television remotes, computer keyboards, and telephones. Then when you touch your hands to your nose, eyes, or mouth, the flu virus gets absorbed.

Symptoms :- Typical clinical features of influenza may include fever (usually 100 F-103 F in adults and often even higher in children), chills, respiratory symptoms such as

cough (more often in adults), sore throat (more often in adults), runny or stuffy nose (especially in children),

headache, muscle aches, fatigue, sometimes extreme. Treatment :- Treatments for influenza include a range of medications and therapies that are used in response to disease influenza. Treatments may either directly target the influenza virus itself; or instead they may just offer relief to symptoms of the disease, while the body's own immune system works to recover from infection. The two main classes of antiviral drugs used against influenza are neuraminidase inhibitors, such as zanamivir and oseltamivir, or inhibitors of the viral M2 protein, such as amantadine and rimantadine. These drugs can reduce the severity of symptoms if taken soon after infection and can also be taken to decrease the risk of infection. However, viral strains have emerged that show drug resistance to both classes of drug.

MATERIAL AND METHODS

For the pharmaceutical industry, the discovery of a new drug presents an enormous scientific challenge, and consists essentially in the identification of new molecules or compounds. Ideally, the latter will become drugs that act in new ways upon biological targets specific to the diseases requiring new therapeutic approaches. The identification of therapeutic targets requires knowledge of a disease’s etiology and the biological systems associated with it. Molecular biology has revolutionized the process of drug discovery. Not too long ago, scientists searched for new targets employing a long and costly process of trial and error. Today, the collective contribution of genomics, proteomics and bioinformatics allows for the much more rapid and precise discovery of those genes and/or proteins involved in the etiology of certain diseases. Databases used:- NCBI Protein DataBank (PDB) Swissprot/ Uniprot DrugBank Pubchem compound

Sequence identification :- Sequence for Influenza A virus has been identified from UniProtKB/Swiss-Prot. UniProtKB/Swiss-Prot is a high-quality, manually annotated, non-redundant protein sequence database that provide all known relevant information about a particular protein. Annotation is regularly reviewed to keep up with current scientific findings. Template selection :- Once the target was identified, template identification relies on serial pairwise sequence alignments aided by database search techniques such as FASTA and BLAST. Ligand identification :- Once the therapeutic target has been identified, we must then find one or more leads (e.g., chemical compounds or molecules) that interact with the therapeutic target so as to induce the desired therapeutic effect, e.g., through antiviral or antibacterial activity. In order to discover the compounds whose pharmacological properties are likely to have the required therapeutic effects, researchers must test a large variety of them on one or more targets.

Ligand identification is done :- Using Literature Survey :- After a thorough literature survey, it was found that Amantadine is one of the potential drugs used against InfluenzaA virus. So a DrugBank database search was done and Amantadine was selected as one of the leads. Softwares used :- Chem Sketch Open babel Chem Sketch :- ACD/Chem Sketch is a chemically intelligent drawing interface that allows you to draw almost any chemical structure including organics and it can be used to produce structures of organic molecules, names of organic molecules as well as Lewis structures, 3-D structures or ball stick model, space filling models etc. Various selected ligands were drawn using Chem Sketch. The files drawn were saved in (.mol) format. Open babel :- It is software, mainly used for converting chemical file formats. Here we convert downloaded .mol files into (.pdb) files format.

Homology Modelling :- Homology Modelling or Protein Modelling, also known as comparative modeling of protein, refers to constructing an atomic-resolution model of the "target" protein from its amino acid sequence and an experimental three-dimensional structure of a related homologous protein (the "template"). Homology Modelling using Swiss-PdbViewer (SPDBV):- Swiss-PdbViewer (or SPDBV), is an interactive molecular graphics program for viewing and analyzing protein and nucleic acid structures. In combination with Swiss-Model(a server for automated comparative protein modeling maintained at http://www.expasy.org/swissmod) new protein structures can also be modeled. Model Analyzation using Structure Analysis and Validation Server (SAVS):- Structure Analysis and Validation Server greatly simplifies computational analysis of the molecular structure and sequence of proteins. Ramachandran plot is a way to visualize dihedral angles φ against ψ of amino acid residues in protein structure. It shows the possible conformations of φ and ψ angles for a polypeptide. The Ramachandran plot displays the psi and phi backbone conformational angles for each residue in a protein.

Steps involved in Modelling:- The template structure from file (.pdb file) is opened. ‘Inverse selection’ is done. The 'selected residues' are removed. The file is saved in (.pdb) format. The ‘raw target sequence’ is then loaded. Then 'magic fit' is done. The file is then saved in (.pdb) format. The model is then submitted by 'submit modeling request'(A new browser will be opened loading the pdb file and

give the Email ID for receiving the modeled structure). Upload the model according to the path given. Download the new model from Email in (.pdb) format. The new model is then saved as ‘Model-1.pdb’. Analyse the model using SAVS. Save the Ramachandran plot as ‘Ramachandran.pdf’.

Loop modeling :- Loop Modelling is a problem in protein structure prediction requiring the prediction of the conformations of loop regions in proteins with or without the use of a structural template. Steps involved in Loop Modelling :- In SPDBV the template structure is opened in pdb format. From Windows open the Ramachandran plot. The residue in disallowed region is coloured. ‘Build Loop’ is done. Save in (.pdb) format.

Docking :- Docking is a method which predicts the preferred orientation of one molecule to a second when bound to each other to form a stable complex Applications of Docking :- Docking is frequently used to predict the binding orientation of small molecule drug candidates to their protein targets in order to in turn predict the affinity and activity of the small molecule. Hence docking plays an important role in the rational design of drugs. Softwares used in Docking :- Autodock 4 Cygwin terminal

Autodock :- Auto Dock is a suite of automated docking tools. It is designed to predict how small molecules, such as substrates or drug candidates, bind to a receptor of known 3D structure. Cygwin terminal :- Cygwin is a Unix-like environment and command-line interface for Microsoft Windows. Cygwin provides native integration of Windows-based applications, data, and other system resources with applications, software tools, and data of the Unix-like environment.

Steps involved in Docking :- Initially save the protein and the ligand molecule as protein.pdb and ligand.pdb in a new folder in the desktop. Here we do AutoDock4 in Windows. Preparation of Protein : Open the Protein file(.pdb format) Edit the Hydrogen, Charges and Atoms of the protein Save the protein in (.pdbqt) format Preparation Of Ligand : Open the Ligand file(.pdb format) From ‘Torsion tree’ choose and detect the root Set the no. of torsions Save the ligand in (.pdbqt) format

Grid Preparation : From the Grid select ‘Macromolecule’ to open the protein Then choose the Ligand From Grid Box select ‘centre’ to ‘pick an atom’ Save the protein in (.gpf) format

Dock Preparation : From Docking select ‘Macromolecule’ to open the protein in

(.pdbqt format) Then open the ligand file (.pdbqt format) From Docking select the ‘Genetic Algorithm’ Save the protein in (.dpf) format

Open Cygwin : Open the Folder where the Proteins and ligands are present with the use of ‘cd’ command Command for autogrid is given : ./autogrid4.exe –p protein.gpf –l protein.glg& (command to run grid file) Command for autodock is given : ./autodock4.exe –p protein.dpf –l protein.dlg&(command to run dock file) Again Open Cygwin : Open the Folder where the Proteins and ligands are present with the use of ‘cd’ command Commands given : grep ‘^DOCKED’ protein.dlg | cut –c9- > protein1.pdbqt cut –c-66 protein1.pdbqt > prt.pdb Open protein.dlg file and search for the minimum energy run number from the RMSD table.(say8th ) Now open ‘prt.pdb file’ and select the atoms of the above selected run (here 8th ) and copy it. Now paste these atoms at the last of the original protein file in (.pdb) format and save it as ‘Docked_protein.pdb’

Post Docking Analysis :- Understanding protein-ligand interactions is a critical step in rational drug design/virtual ligand screening. Using Chimera software we identified the ligand-receptor interactions in the optimized complexes at different levels of protein flexibility. Chimera :- The selected ligands were optimized to their minimum energy configuration using Chimera Steps involved in Post Docking Analysis :- Open Chimera : Open the ‘Docked_protein.pdb’ file Select the ‘Ligand’ from ‘Residues’ Select Zone=6.5 Then do ‘invert’ and hide the atoms and bonds Again select the ‘Ligand’ from ‘Residues’ and specify the ‘Zone’ Now specify the name and residue number from ‘Action’ Again select the ‘Ligand’ From ‘Tools’ do structure analysis and find the Hydrogen bond

RESULT AND DISCUSSION

Prediction of interaction energies between ligand and receptor has been a major challenge for drug docking. Interferon induced GTP-binding protein MX1 is an antiviral protein of Influenza A virus and its structure has been deduced by homology modeling and further Docking has been performed for further study on protein orientation & to predict the affinity, activity, binding orientation of ligand and the target protein Interferon induced GTP-binding protein MX1. Sequence identification :- Sequence for Influenza A virus that has been identified from UniProtKB/Swiss-Prot is Interferon induced GTP-binding protein MX1.

Template selection :- BLAST OUTPUT :-

By analyzing the BLAST result it was found that Chain A, Structural Basis Of Oligomerisation In The Mxa Stalk is having :- Score = 279 bits (714), Expect = 3e-88, Method: Compositional matrix adjust. Identities = 145/274 (53%), Positives = 199/274 (72%), Gaps = 3/274 (1%) Was selected as template. This template is further used in modeling the target.

Homology Modelling :- The target protein Interferon induced GTP-binding protein MX1 structure deduced by homology modeling shows that there are 35 possible templates for protein modeling out of which we have chosen 3LJB having 94.7% residues in most favoured regions (i.e 94.7%identity) to Interferon induced GTP-binding protein MX1protein structure (viewed through Ramachandran Plot)of modeled protein by Swiss-PdbViewer (SPDBV). 3LJB:-

Loop Modelling :- 2IF5 having 88.6% identity (Ramachandran Plot) has been chosen for loop modeling and is further modeled in order to increase its percentage of identity. After loop modeling the percentage of 2FI5 has increased to 91.4% (Ramachandran Plot) 2IF5 :- Before loop modeling :- 2IF5 having 88.6% identity

After loop modelling:- The percentage of 2FI5 has increased to 91.4%

Docking:- For further study on protein orientation & to predict the affinity, activity, binding orientation of ligand and the target protein Interferon induced GTP-binding protein MX1 docking has been performed between already available 10 market ligands:-Amantadine, AmantadineHCL, Rimantadine, RimantadineHCL, AmphotericinB, Flumadine, Famciclovir, Zanamivir, Oseltamivir and Hemagglutinin precursor(114-122) amide and the target protein. These ligands were selected because it would bind to the enzyme as substrate molecule. Docking has been performed by using the software Autodock4. The lowest binding energy (in Kcal/ mol) between the ligand AmphotericinB and the target protein Interferon induced GTP-binding protein MX1 is-8.04 Kcal/mol out of all the 10 ligands.

Sl.No Pdb id Ligand Lowest Binding

Energy

Mean Binding

Energy

Run

1 3LJB Amantadine -4.04 -4.02 8

2 3LJB AmantadineHCL -3.92 -3.91 5

3 3LJB RimantadineHCL -3.84 -3.84 4

4 3LJB Rimantadine -4.09 -4.09 6

5 3LJB AmphotericinB -8.04 -7.97 5

6 3LJB Famciclovir -4.18 -4.18 6

7 3LJB Flumadine -3.41 -3.41 3

8 3LJB Oseltamivir -3.64 -3.64 10

9 3LJB Zanamivir -5.26 -5.26 1

10 3LJB Hemagglutininprecursor (114-122)

amide

-2.89 -2.89 7

Energy values after Docking with Ligands :-

Post docking analysis :- Docking is followed by post docking analysis which helps in understanding the protein-ligand interactions. Post-docking analysis is performed using Chimera software. Docking between protein and ligand- Zanamivir

Docking between protein and ligand- Zanamivir having 6 Hydrogen bonds

Table for Hydrogen Bonds :- Sl.No Pdb id Ligand Active Site Residue Number of

Hydrogen Bonds

1 3LJB Amantadine GLU 516 1 2 3LJB AmantadineHCL GLU 516 1 3 3LJB RimantadineHCL - 0 4 3LJB Rimantadine - 0 5 3LJB AmphotericinB GLN 513

LYS 519 2

6 3LJB Famciclovir ` - 0 7 3LJB Flumadine GLU 516 1 8 3LJB Oseltamivir GLU 516

GLU516 2

9 3LJB Zanamivir TYR 532 TYR 532 GLU 587 ARG 591 SER 594 SER 594

6

10 3LJB Hemagglutininprecursor (114-122) amide

- 0

Docking between protein and ligand- Rimantadine having No Hydrogen bonds

After Post-docking analysis is performed, we can see that :- The number of Hydrogen bonds between the ligand Zanamivir and the target protein Interferon induced GTP-binding protein MX1is maximum, that is 6 in number out of all the 10 ligands

CONCLUSION

Influenza A virus causes influenza in birds and some mammals, and is the only species of influenza virus A. Strains of all subtypes of influenza A virus have been isolated from wild birds, although disease is uncommon. Some isolates of influenza A virus cause severe disease both in domestic poultry and, rarely, in humans. Occasionally, viruses are transmitted from wild aquatic birds to domestic poultry, and this may cause an outbreak or give rise to human influenza pandemics. Influenza A virus is the most virulent human pathogens among the three influenza types. It is capable of infecting human as well as animals (ducks, chickens, pigs, whales, horses and seals). Wild aquatic birds are the natural hosts for a large variety of influenza A. All type A influenza viruses, including those that regularly cause seasonal epidemics of influenza in humans, are genetically labile and well adapted to elude host defenses. Influenza A virus is the main cause of worldwide pandemics. Interferon induced GTP-binding protein MX1was selected as target protein. Its 3D structure was developed using Spdbv. Compound known to inhibits MX1 were taken as inhibitors. There was 10 ligand. Docking was performed to check the binding conformation of each protein ligand complex . The compound AmphotericinB showed least binding energy among all selected compound. And the compound Zanamivir showed maximum number of Hydrogen bonds among all selected compound. The study end up with the selection of compounds AmphotericinB and Zanamivir as the potential compound which can be developed as a drug against Influenza.

REFERENCES

Homology Modeling and Docking Studies Of Hemagglutinin Protein Of Influenza A (H1N1) Virus With Selected Ligands - A Computer Aided Structure Based Drug Design Approach To Find A Suitable Inhibitor - A.Bhattacharjee , P.Yadav, B.J. Mylliemngap, E.Annipindi

Schnell JR, Chou JJ. 2008. Structure and mechanism of the M2 proton channel of influenza A virus. Nature 451:591-6. Nature 2009, 459:1122-1125. PubMed Abstract | Publisher Full Text OpenURL A. Bairoch, R. Apweiler, C. H. Wu, W. C. Barker, B. Boeckmann, S. Ferro, E. Gasteiger,H. Huang, R. Lopez, M. Magrane, M. J. Martin,

D. A. Natale, C. O’Donovan,N. Redaschi, and L.-S. L. Yeh. The Universal Protein Resource (UniProt). Nucleic Acids Res, 33(Database issue):D154–159, 2005.

A. Bairoch. The enzyme database in 2000. Nucleic Acids Res, 28:304–305, 2000. Structural basis and sequence co-evolution analysis of the hemagglutinin protein of pandemic influenza A H1N1 (2009) virus.htm The CPSF30 Binding Site on the NS1A Protein of Influenza A Virus Is a Potential Antiviral Target.htm The CPSF30 binding site on the NS1A protein of influ... [J Virol. 2006] - PubMed - NCBI.htm Influenza Book Virology of Human Influenza.htm Avian Influenza - Symptoms, Diagnosis, Treatment of Avian Influenza - NY Times Health Information.htm CDC - Seasonal Influenza (Flu) - ACIP Recommendations Introduction and Biology of Influenza.htm CDC - Seasonal Influenza (Flu) - Flu Symptoms & Severity.htm CDC - Seasonal Influenza (Flu) - Types of Influenza Viruses.htm

Web links :- https://en.wikipedia.org/wiki/Influenza_A_virus http://en.wikipedia.org/wiki/Influenza_treatment http://hten.wikipedia.org/wiki/Homology_modeling en.wikipedia.org/wiki/Docking_(molecular) http://swissmodel.expasy.org/ http://blast.ncbi.nlm.nih.gov/Blast.cgi http://www.pdb.org/pdb/home http://pubchem.ncbi.nlm.nih.gov/ http://www.rcsb.org/pdb/home/home. http://www.cgl.ucsf.edu/chimera/ http://nihserver.mbi.ucla.edu/SAVES/

ACKNOWLEDGEMENT

I am thankful to many people because the success of my project is a joint effort of many people and by the grace of god I gathered the strength to do the project and a great support of him to make me accomplish my project in Biotech Park. Thankful to my college Bengal College of Engineering and Technology to permit me the permission to work on my project here and gave me letter of recommendation. I am grateful and show my gratitude to CEO P.K. Seth of biotech who provide me the opportunity to work here as a trainee and perform my project. I would like to thank to my teacher or guide Mr. Siddharath Sinha as I know without his full support and guidance I would have not been able to complete my project successfully and he is the only one who cleared doubts. I also express my sincere thanks to Mr. Sunil Kumar Gupta and Dr. Sarita Singh who also helped me in the way to clear my doubts. I am also thankful to my friends who directly or indirectly helped me in my project. Last but not the least I owe my regards to my parents Dr. Asit Kumar Pal and Mrs. Bharati Pal and my family who supported me mentally. My studies would not be without their love, support, and blessings.

THANK YOU