Potential Drug Targets Mycobacterium tuberculosis

Dr. P. Gautam
CHEMICAL BIOLOGY GROUP
Dr.P.Gautam, Professor, Centre for Biotechnology, Anna University, Chennai -600 025. pgautam@annauniv.edu gpennathur@gmail.com www.annauniv.edu/biotech/boc/

RESEARCH INTERESTS Isolation and Characterization of Microorganisms for Lipases and Biosurfactants . Development of HTS Assays for Lipases Bioinformatics and Molecular Dynamics of Lipases. Protein structure prediction using knowledge based methods.

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www.sanger.ac.uk

Growing Incidence of M. tuberculosis

www.genome.gov

www.genome.jp/kegg

Thomas R Frieden, Timothy R Sterling, Sonal S Munsiff, Catherine J Watt and Christopher Dye The Lancet, 362, 887-899 (2003 )

The Mycobacterium tuberculosis Genome

Burden of M. tuberculosis & First Line Drugs

The Global TB Alliance for Drug Development www.tballiance.org The Stop TB Partnership www.stoptb.org The World Health Organisation TB Resource www.who.in/gtb The TubercuList World-Wide Web Server (Pasteur Institute) http://genolist.pasteur.fr/TubercuList/ NIAID Pathogen Functional Genomics Resource Centre http://www.niaid.nih.gov

S. T. Cole et al Nature 393, 537-544 (1998)

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Drug Targeting Against Bacteria

The Mycobacterium tuberculosis genome

Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence S.T. Cole etal (1998) Nature 393: 537-544.
M.Tb genome approx 4000 genes Completion of the H37Rv M.Tb genome has led to An improved understanding of genes expressed Metabolic pathways Virulence factors Mycobacterial persistence

Mass Spectrometry Reviews 2005

Functional Genomics of M. tuberculosis

The need for the discovery of new drug targets in Mycobacterium tuberculosis
Though front line TB drugs are available, prolonged treatment and noncompliance are major obstacles in effective treatment

global threat due to multidrug resistance strains (MDR strains) Synergy between HIV and TB

Re-emergence TBaas a drug target Two essentials of for good

S. T. Cole et al , FEBS Lett, 452 , 7 ( 1999)

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Identification of Unique pathways in the pathogen Unique pathways identified in Mycobacterium tuberculosis
Peptidoglycan biosynthesis Mycobactin biosynthesis

Potential drug targets identified in Mycobacterium tuberculosis

Comparison of host and pathogen pathways yields unique pathway list.

Cell wall biosynthesis Lipid metabolism Carbohydrate metabolism Energy metabolism Amino acid metabolism Vitamin and cofactor biosynthesis

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Enzymes from these pathways are bacteria specific and hence represent attractive potential drug targets.

C5 branched dibasic acid metabolism D-alanine metabolism Thiamine metabolism Polyketide sugar unit biosynthesis

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Two essentials for a good drug target

Should be important for the viability of the pathogen Should not bear similarity to any host protein

Informatics approach for identifying drug targets

Metabolic pathway information of host and pathogen are available in pathway databases like KEGG Comparative metabolic pathway analysis of host and the pathogen Identification of enzymes unique to the pathogen

Elimination of pseudo drug targets

Potential targets are screened for homologues with host. Bioinformatics tools like BLAST help in comparing targets with all the proteins from the host. Targets with no similarity to the host Homo sapiens proteins are the final candidates

These represent potential drug targets

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Membrane Structure in Eubacteria

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Case study 1: The mycobacterial cell wall

The three components of the cell wall Plasma membrane Mycolic acid, arabi nogalactan and peptidoglycan complex (MAPc) Polysaccharide rich capsule like material. Existing drugs isoniazid & ethambutol target cell wall biosynthesis.

Isoniazid inhibits mycolic acid biosynthesis Ethambutol inhibits arabinan biosynthesis

Because of its complex structure, cell wall biosynthesis still remains a favourite for the discovery of new drugs Our targets from cell wall biosynthesis: 10 Prime candidate: MurD ligase

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Case study 2: Lipid metabolism

The bacterium depends on the host cell lipids Degradation of host cell lipids provides precursors to many of the bacterial cell processes. Our targets from the lipid metabolism : 20
Lipolysis might provide carbon sources that the micro-organism could use for growth; adhesion:

A large part of the coding region of M.Tb is devoted to the production of enzymes involved in Lipogenesis Lipolysis

Includes virulence factors like phospholipases and lipases

released FFA due to lipolytic activity could support cell-to-cell and/or cell-to-host tissue adhesion; synergism: a lipase might work hand in hand with another enzyme or it might optimize conditions for other enzymes; unspecific hydrolysis: lipases might possess additional phospholipolytic activity; immune system: lipases and their catalytical end products may have an effect on different immune cells and might initiate inflammatory processes; defense: micro-organisms that secrete lipolytic enzymes might have a selection advantage by lysing competing microflora.

Journal of Molecular Catalysis B ,347 , 2003

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Case study 3 : Iron acquisition

Mycobacterium tuberculosis inhabits one M.Tb produces the mycobactin of the most hostile environments class of siderophores. the alveolar macrophage One of the host defensive mechanisms Dramatic reduction of iron availability to support bacterial growth.
Mutant studies have shown i mpaired growth of the bacterium in macrophage like THP1 like cells under iron limiting conditions

Iron Uptake & Synthesis of Mycobactin

Tuberculosi s

Mycobactin biosynthesis is important for virulence

One of our targets is from the Bacteria have evolved sophisticated iron mycobactin biosynthetic pathway. acquisition systems in the form of iron sequestering molecules : siderophores

Iron, mycobacteria and tuberculosis Colin Ratledge, Tuberculosis, 81, 110 (2004)

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Case study 4: Mycobacterial persistence : Latent TB

The pathogen can survive for prolonged periods in infected individuals in a dormant form : Latent TB
Under the microaerophilic conditions of the macrophage the bacterium survives the low oxygen conditions by a metabolic downshift. It also Switches over to anaerobic nitrate respiration Need for new drugs to combat latent forms of TB Genes expressed during mycobacterial persistence have been identified. Uses glyoxylate bypass to produce carbohydrates from fatty acid s Targets from these pathways could prove useful for treatment of latent forms of TB

Structure of the mycobactin siderophores of M. tuberculosis. For mycobactin T, which occurs wholly intracellularly, n=19 (also 17) and R=---CH3; for carboxymycobactin T, which occurs extracellularly, n=29 and R=---COOH. The three pairs of chelating groups responsible for the binding of Fe(III) are indicated by asterisks (*).

Once targets are discovered structure based drug design follows.....

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Structure of isocitrate from M.tuberculosis

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Ernesto J Muoz-Elas & John D McKinney , Nature Medicine 11, 638 - 644 (2005)

Vivek Sharma, Sujata Sharma, Kerstin Hoener zu Bentrup, John D. McKinney, David G. Russell, William R. Jacobs Jr. & James C. Sacchettini Nature Structural Biology 7, 663 - 668 (2000)

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Various Enzymes in the Pathway

Structure of Mur Enzymes

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Mechanism of Action of Mur D Ligase

Structure of Mur D Ligase from E.coli

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Sequence Alignment of MurD Ligase of E. coli & M. tuberculosis

Homology modeling of one of s the targets MurD ligase : A prime candidate for broad spectrum drug discovery
Why target MurD ?
Essential for cell wall biosynthesis viability of the bacterium Present across all eubacteria
Homology modeling
An alternative when structures are not available

Substrate binding in E. coli MurD, Leu 15, Thr 16, Gly 73, Asn 138 and His 183 (Jay et al., 1997).The corresponding residues in MurD of M. tuberculosis are Val 18, Thr 19, Gly 75, Asn 147 and His 192. Similarly, the residues for the ATP binding in E. coli MurD are Gly 114, Lys 115, Ser 116, Thr 117, Arg 302 and Asp 317 (Jay et al., 1997).The equivalent residues are MurD of M. tuberculosis, at positions Gly 123, Lys 124, Thr 125, Arg 314 and Asp 329. The residue involved in Mg2+ fixation, Glu 157 of E. coli (Jay et al., 1997) is also conserved as Glu 166 of M. tuberculosis MurD. In addition, all these residues are in absolutely conserved regions.

M.Tb MurD modelled using E.coli MurD as a template, with WHATIF Molecular modeling and drug design program

What next ??
Structure based drug design Screening of chemical libraries for ligand/drug docking Chemoinformatics

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Potential Drug Candidate for Mycobacteium Tuberculosis An Insilico Approach

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Acknowledgement Department of Science and Technology (DST) Govt. of India. Council of scientific and industrial research (CSIR) Govt. of India Department of Biotechnology- Biotechnology Information Services (DBT-BTIS) Govt. of India

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Margaret Fonteyn in Memoirs Famous Ballerina

I Cannot Imagine Feeling Lackadaisical about a Performance. I Treat Each Encounter as a Matter of Life and Death. The Important thing I have learnt over the Years is the Difference Between Taking One's Work Seriously and Oneself Seriously, the First is Imperative and the Second Disastrous

Thank You

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