An Insilico attempt was made to characterize a newly sequenced Interferon induced GTP-binding protein MX1protein (organism: Mus musculus (mouse)) of influenza- A virus (H1N1) to deduce its structural information and to identify the potential drug to inhibit the protein. But, due to unavailability of further sequences a ‘template’ 3LJB (Interferon induced GTP-binding protein MX1protein (organism: Homo sapiens) is selected. For that an effort was taken to deduce the 3-D structure of this template protein and to identify and bind the active site of Interferon induced GTP-binding protein MX1protein with docking technique.
3.
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.
4.
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.
5.
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.
7. 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
8.
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.
9.
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.
10.
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 threedimensional 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.
11.
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’.
12.
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.
13.
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 Unixlike environment.
14.
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
15.
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
16.
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’
17.
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
19. 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 GTPbinding protein MX1.
20.
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.
21.
Homology Modelling :-
The target protein Interferon induced GTPbinding 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:-
22.
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
24.
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.
Energy values after Docking with Ligands :Sl.No
Pdb id
Ligand
Lowest Binding
Mean Binding
Energy
Run
Energy
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)
-2.89
-2.89
7
amide
25.
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
27.
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
29. 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.
31.
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Schnell JR, Chou JJ. 2008. Structure and mechanism of the M2 proton channel of influenza A virus. Nature 451:591-6.
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34. 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.