INTRODUCTION:

Bird flu" is a phrase similar to swine flu, dog flu, horse flu or human flu in that it refers to an illness caused by any of many different strains of influenza viruses that have adapted to a specific host. All known viruses that cause influenza in birds belong to the species influenza A virus. Avian influenza is flu infection in birds. The disease is of concern to humans, who have no immunity against it. The virus that causes this infection in birds can mutate (change) to easily infect humans. Such mutation can start a deadly worldwide epidemic.

The highly pathogenic influenza-A virus subtype H5N1 virus is an emerging avian influenza virus that has been causing global concern as a potential pandemic threat. It is often referred to simply as "bird flu" or "avian influenza" even though it is only one subtype of avian influenza causing virus. Symptoms of avian flu infection in humans depend on the particular strain of virus. In case of the H5N1 virus, infection in humans causes more classic flu-like symptoms, which might include cough, sore throat, fever, difficulty breathing, diarrhea, runny nose, head ache and muscle aches¹.

Different types of avian flu virus may cause different symptoms. Therefore, treatment may vary. In general, treatment with the antiviral medication oseltamivir (Tamiflu) or zanamivir (Relenza) may make the disease less severe if start taking the medicine within 48 hours after symptoms start. The virus that causes human avian flu appears to be resistant to the antiviral medicines amantadine and rimantadine. Therefore these medications cannot be used if an H5N1 outbreak occurs. Studies done in laboratories suggest that the prescription medicines approved for human influenza viruses should work in treating avian influenza infection in humans. However, influenza viruses can become resistant to these drugs, so these medications may not always work. Additional studies are needed to determine the effectiveness of these medicines.

Computational Biology and bioinformatics have the potential not only of speeding up the drug discovery process thus reducing the costs, but also of changing the way drugs are designed. Rational Drug Design (RDD) helps to facilitate and speedup the drug designing process, which involves variety of methods to identify novel compounds. One such method is the docking of the drug molecule with the receptor (target). The site of drug action, which is ultimately responsible for the pharmaceutical effect, is a receptor². Docking is the process by which two molecules fit together in 3D space.

TOOLS AND MATERIALS USED:

For our present study we used bioinformatics tools, biological databases like PubMed, Drug Bank, PDB (Protein Data Bank) and software’s like Hex, ACD ChemSketch.. Hex is an Interactive Molecular Graphics Program for calculating and displaying feasible docking modes of pairs of protein and DNA molecules. Hex can also calculate Protein-Ligand Docking, assuming the ligand is rigid, and it can superpose pairs of molecules using only knowledge of their 3D shapes³. It uses Spherical Polar Fourier (SPF) correlations to accelerate the calculations and its one of the few docking programs which has built in graphics to view the result⁴.

Drug Bank is a unique Bioinformatics/Cheminformatics resource that combines detailed drug (i.e. chemical) data with comprehensive drug target (i.e. protein). Each Drug Card entry contains greater than 80 data fields with half of the information being devoted to drug/chemical data and the other half devoted to drug target or protein data⁵. The PDB (Protein Data Bank) is the single worldwide archive of Structural data of Biological macromolecules, established in Brookhaven National Laboratories (BNL)⁶. It contains Structural information of the macromolecules determined by X-ray crystallographic, NMR methods etc. PubMed is a free digital archive of biomedical and life sciences journal literature at the U.S. National Institutes of Health (NIH) developed and managed by NIH's National Center for Biotechnology Information (NCBI) in the National Library of Medicine (NLM). PubMed is a free search engine for accessing the MEDLINE database of citations and abstracts of biomedical research articles⁷. RASMOL [Raster Display of Molecules] is a molecular graphics program intended for the structural visualization of proteins, nucleic acids and small biomolecules. The program reads in molecular coordinate files and interactively displays the molecule on the screen in variety of representations and color schemes.

METHODOLGY:

Bioinformatics is seen as an emerging field with the potential to significantly improve how drugs are found, brought to the clinical trials and eventually released to the marketplace. Computer – Aided Drug Design (CADD) is a specialized discipline that uses computational methods to simulate drug – receptor interactions. CADD methods are heavily dependent on bioinformatics tools, applications and databases². The structure of virus H5N1 receptor was retrieved from PDB (2Q06). Using Chemsketch the structures of the drugs were generated by their SMILES notation obtained from Drug Bank and the structural analogues of these drugs were sketched. The docking analysis of zanamivir and oseltamivir with influenza A virus H5N1 receptor was carried by HEX docking software.

Docking allows the scientist to virtually screen a database of compounds and predict the strongest binders based on various scoring functions. It explores ways in which two molecules, such as drugs and an virus H5N1 receptor fit together and dock to each other well, like pieces of a three-dimensional jigsaw puzzle. The molecules binding to a receptor, inhibit its function, and thus act as drug. The collection of zanamivir and receptor complex was identified via docking and their relative stabilities were evaluated using molecular dynamics and their binding affinities, using free energy simulations.

The parameters used for the docking process were

• Correlation type – Shape only

• FFT Mode – 3D fast lite

• Grid Dimension – 0.6

• Receptor range – 180

• Ligand Range – 180

• Twist range – 360

• Distance Range – 40

The drug and its analogues were docked with the receptor using the above parameters.

RESULTS AND DISCUSSION:

Docking results tabulated between influenza A virus H5N1 receptor and the conventional drug zanamivir (Table 1) as well as with the modified drugs are shown below along with the changes or modification within them. It was observed using RasMol that ester group present in the drug zanamivir was the site of binding to the receptor. Several modifications were made to these probable functional groups, which resulted in a decrease in the energy value. The energy values were calculated using Hex. This was the pharmacophoric part of the drug was partially identified.

Fig.1 Structure of influenza A virus H5N1 receptor

Fig.2. Interaction and binding energy of Zanamivir with influenza A virus H5N1 receptor

TABLE 1: DOCKING RESULTS OF H5N1 RECEPTOR WITH ZANAMIVIR ANALOGS

Drug docked	R	X	E-value
Zanamivir	-OH		-231.74
Zanamivir analog 1	-OCH₃		-244.89
Zanamivir analog 2	-OC₂H₅		-265.01
Zanamivir analog 3	-OC₆H₅		-292.46
Zanamivir analog 4			-269.96
Zanamivir analog 5	-C₆H₄(p-Cl)		-274.42
Zanamivir analog 6	-C₆H₄(o-NO₂)		-263.12
Zanamivir analog 7	-C₆H₄(p-NH₂)		-273.31
Zanamivir analog 8	-COOH		-234.92
Zanamivir analog 9	-COOH		-231.14
Zanamivir analog 10	-COOH		-237.32

Fig.3. Interaction and the binding energy of Zanamivir analog 3 with influenza A virus H5N1 receptor

TABLE 2: DOCKING RESULTS OF H5N1 RECEPTOR WITH OSELTAMIVIR ANALOGS

Drug docked	R	X	E-value
Oseltamivir	H	-CH₂-	-243.74
Oseltamivir analog 1		-CH₂-	-258.17
Oseltamivir analog 2		-CH₂-	-257.36
Oseltamivir analog 3		-CH₂-	-256.18
Oseltamivir analog 4	-OH	-CH₂-	-247.70
Oseltamivir analog 5		-CH₂-	-332.02
Oseltamivir analog 6	-CH₂-CH₂-O-CH₂-CH₃	-CH₂-	-293.01
Oseltamivir analog 7		-CH₂-	-263.06
Oseltamivir analog 8		-CH₂-	-262.07
Oseltamivir analog 9		-O-	-256.18
Oseltamivir analog 10		-S-	-262.12

Fig.4. Interaction and the binding energy of Oseltamivir with influenza A virus H5N1 receptor

Docking results tabulated between human H5N1 influenza viral receptor and the conventional drug oseltamivir (Table 2) as well as with the modified drugs are shown below along with the changes or modification within them. It was observed using RasMol that ester group present in the drug oseltamivir was the site of binding to the receptor. Several modifications were made to these probable functional groups, which resulted in a decrease in the energy value. The energy values were calculated using Hex. This was the pharmacophoric part of the drug was partially identified

Based on the literature it has been shown clearly that the drugs zanamivir and oseltamivir have been used to target receptor H5N1. Zanamivir and oseltamivir on docking with H5N1 produce an energy value of -231.74 and -243.74 respectively. An analog with phenyl ester (zanamivir analog 3) was prepared virtually using chemsketch. This particular analog showed an increase in the energy values (-292.46) and an analog with additional functional group (oseltamivir analog 5) was prepared virtually using chemsketch. This particular analog of oseltamivir showed an increased in energy values (-332.02) which means the zanamivir analog 3 and oseltamivir analog 5 was more compatible with the receptor than its predecessor. However, the binding site of the analog was similar to that of its predecessor, which means that functional groups involved were the same and by preparing the analog only the steric compatibility was increased.

Fig.5. Interaction and the binding energy of Oseltamivir analog 5 with influenza A virus H5N1 receptor

CONCLUSION:

The Protein-Ligand interaction plays a significant role in structural based drug designing. In the present work we have taken influenza A virus H5N1 receptor and identified the drugs that were used against bird flu. When the receptor (2Q06) was docked with standard drugs Zanamivir and oseltamivir and the energy values obtained were -231.74 and -243.74 respectively. When the modified drugs were docked against the same receptor the energy values obtained were Zanamivir Analog 3 (-292.46) and oseltamivir analog 5 (-332.02) from this we can conclude that some of the modified drugs are better than the commercial drugs available in the market. In future research work the ADME/T (Absorption, Distribution, Metabolism, Excretion / Toxicity) properties of these compounds can be calculated using the commercial ADME/T tools available thus reducing the time and cost in drug discovery process.

REFERENCES:

1. http://en.wikipedia.org/wiki/Avian_influenza.

2. Computational Biology and Drug Discovery: From single – network Drugs”, Current Bioinformatics, 2006; 1: 3-13.

3. David. W. Rithcie, Evaluation of Protein Docking Predictions using Hex 3.1 in CAPRI rounds 1-2, Proteins, Structure, Fucntion and Genetics, Wiley-liss Inc.

4. D.W. Ritchie and G.J.L. Kemp, Protein Docking Using Spherical Polar Fourier Correlations, PROTEINS: Struct. Funct. Genet. 2000; 39: 178-194.

5. Drug Bank; a comprehensive resource for in silico drug discovery and exploration”, Nucleic Acids Research, 2006, Vol.34, Database Issue.

6. The Protein Data Bank”, Nucleic Acids Research 2000; 28. Oxfords University Press.

7. PubMed: Online Search Engine for science and biomedical articles, www.pubmedcentral.nih.gov

Received on 25.12.2009 Modified on 16.02.2010

Asian J. Research Chem. 3(2): April- June 2010; Page 370-373