Title

In-silico identification of potential drug targets from different Mycobacterium sp Dissertation submitted in partial fulfillment of the requirements for the degree of Master of Science In Biotechnology

By Divakaran.G.K [07MSB037]

SCHOOL OF BIOTECHNOLOGY, CHEMICAL AND BIOMEDICAL ENGINEERING VIT UNIVERSITY Vellore-632014, Tamil Nadu, India May-2009

Certificate This is to certify that dissertation entitled “In-silico identification of potential drug targets from different Mycobacterium sp” submitted by Divakaran.G.K (07MSM037) to the School of Biotechnology, Chemical and Biomedical engineering, VIT University, Vellore in partial fulfillment of the requirements for the degree of Master of Science in Biotechnology is a bonafide record of work carried out by him under my supervision. The contents of this dissertation, in full or in parts have not been submitted to any other institute or university for the award of any degree or diploma.

Project Guide

Division Leader Division of Medical Biotechnology SBCBE

Internal Examiner

External Examiner

Declaration I hereby declare that the study entitled “In-silico identification of potential drug targets from different Mycobacterium sp” is bonafide and has been prepared under the supervision and guidance of Dr.Sundandira doss, Professor, School of Biotechnology, Chemical and Biomedical Engineering, VIT University, Vellore in partial fulfillment of the requirements for the degree of Master of Science in Biotechnology.

Date:

Divakaran.G.K

Place:

07MSB037

Acknowledgement It gives me immense pleasure to acknowledge our honourable Chancellor Mr. G. Viswanathan who is a quintessence of determination and excellence. His penchant for being ahead of his contemporaries has set new benchmark for both the staff and the students of VIT. I would also like to thank Honourable Pro-Chancellors, Mr. G. V. Sampath(Administration) and

Mr. G. V. Shankar (Academic) and

Vice-Chancellor Dr. Radha Krishnan for providing amenities for my project work. I am very much grateful to Dr. Lazar Mathew, Dean of SBCBE who was always for his support during the time of my project. I extend my hearty gratitude to my Guide Dr. Sutindira doss who has been a source of inspiration for suggesting the topic, inspiring guidance, constructive criticism and invaluable advice for the successful completion of the project. I wish to thank Mr. George priya doss (research scholar), and my friends for their support and help throughout the project.

Abstract Infectious diseases remain as a serious threat to the mankind because of the emergence of multidrug resistant varieties of different Mycobacterium sp has led to a search for novel drug targets. I have performed an in-silico comparative analysis of metabolic pathways of different Mycobacterium sp with the metabolic pathway of the host (Homo sapiens). Enzymes involved in different metabolic pathways are extracted from KEGG metabolic pathway and were compared with the proteins from the host (Homo sapiens). The e-value threshold was set to 0.01 using the option available in NCBI blast search engine. The enzymes which do not have similarity were filtered out and listed as potential targets. These potential drug targets can be useful in developing broad spectrum drugs. Potential drug targets from pathways amino acid metabolism, nucleotide metabolism, energy metabolism, carbohydrate metabolism were identified and listed out. The identified drug-targets from M.leprae were subjected to choke point analysis for the further identification of best targets from its whole genome. Around 3500 genes were subjected to these analysis and about 170 genes were identified as potential drug targets. Preference can be given to these identified targets in drug-discovery process so that any chances for side effects will be very less and the chances of developing drug-resistance will be low. The study was successful in listing out the potential drugs targets from different Mycobacterium sp involved in vital aspects of the pathogen’s metabolism, persistence and virulence. This method can be adapted to other pathogens and further the identified targets can be analyzed using molecular docking techniques for screening of inhibitors.

Contents S.No. 1.

Title INTRODUCTION

Page No. 1

1.1 Impact of Leprosy on mankind

2

1.2 Global Prevalence of leprosy

3

1.3 Diagnosis and treatment methods for leprosy

5

1.4 Emergence of Drug-resistance varieties

6

1.5 Genome of Mycobacterium leprae TN

7

2.

REVIEW OF LITERATURE

10

3.

OBJECTIVES

24

4.

MATERIALS AND METHODS 4.1.1 Identification of potential drug targets from M.leprae Genome

25

4.1.2 Identification of orthologous groups

25

4.1.3 Choke point analysis of the potential targets

25

4.1.4 Isozyme analysis

26

4.2 Identification of potential drug targets from Mycobacterium Avium paratuberculosis k-10 genome.

27

4.3 Identification of potential drug targets from Mycobacterium gilvum PYR-GCK genome

27

4.4 Identification of potential drug targets from Mycobacterium avium 104 genome

28

5. RESULTS AND DISCUSSION 5.1 Potential drug-target genes from different Mycobacterium sp.

29

5.2.1 Pathways unique to M.leprae (pathogen) compared to Homo sapiens (host)

29

5.2.2 Targets from Amino acid metabolism

30

5.2.3. Targets from Carbohydrate metabolism

31

5.3 Genes involved in multiple metabolic pathways

32

6. SUMMARY

59

7. BIBLIOGRAPHY

60

Lists of tables Table no. Table 1

Particulars Identified potential gene targets from Mycobacterium leprae TN

Table 2

40

Identified potential gene targets from Mycobacterium avium 104 genome

Table 5

37

Identified potential gene targets from Mycobacterium gilvum PYR-GCK genome

Table 4

33

Identified potential gene targets from Mycobacterium Avium paratuberculosis k-10 genome

Table 3

Page no.

44

Identified potential gene targets from Mycobacterium leprae TN after choke point analysis

56

Lists of figures Fig no.

Particulars

1.1

Genome of M.leprae

9

2.1

The average length of proteins present in M.leprae

30

3.1

Nucleotide composition values of Mycobacterium leprae genome

Page no.

31

ABBREVIATIONS

BB

Mid –borderline

BL

Borderline lepromatous

BT

Borderline tuberculoid

ENL

Erythema nodosum leprosum

IFN

interferon

IL

interleukin

LL

lepromatous

MB

Multibacillary

MDT

Multidrug therapy

PB

Paucibacillary

PGL-I

Phenolic glycolipid

RR

Reversal reaction

TT

Tuberculoid

MDT

Multiple drug treatment method

ng

nanogram

ml

milliliter