Mycobacterum Drug Targets

8/13/2019 Mycobacterum Drug Targets

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Dr. P. Gautam

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CHEMICAL BIOLOGY GROUP

RESEARCH INTERESTSIsolation 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.

Dr.P.Gautam,

Professor,

Centre for Biotechnology,

Anna U niversity, Chennai -600 025.

[email protected]

[email protected]

www.annauniv.edu/biotech/boc/

www.sanger.ac.uk

www.genome.gov

www.genome.jp/kegg

Thomas R Frieden, Timothy R Sterling, Sonal S Munsiff, Catherine J Watt andChristopher Dye The Lancet, 362, 8 87-899 (2003 )

Growing Incidence of M. tuberculosis

Burden ofM. tuberculosis & First Line Drugs

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

The Mycobacterium tuberculosis Genome

The Global TB Alliance for Drug Developme nt

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


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Mass Spectrometry Reviews 2005

Drug Targeting Against BacteriaThe Mycobacterium tuberculosis genome

Deciphering the biology of Mycobacterium tuberculosis from the completegenome sequence S.T. Cole etal (1998)

Nature 393: 537-544.

Completion of the H37Rv M.Tb genome has ledto

An improved understanding of genes e xpressed

Metabolic pathways

Virulence factors

Mycobacterial persistence

M.Tb genome approx 4000 genes

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

unctional Genomics of M. tuberculosis

Two essentia ls for a good drug target

The need for the discovery of new drug targets in

Mycobacterium tuberculosis

Though front line TB drugs are available, prolonged treatment and non-

compliance are major obstacles in effective treatment

Re-emergence of TB as a

global threat due

to multidrug resistance strains (MDR strains)

Synergy between HIV and TB

Identification of Unique pathways in the pathogen

Comparison of host andpathogen pathways yields

unique pathway list.

Enzymes from these pathwaysare bacteria specific and hence

represent attractive potential

drug targets.

Peptidoglycan biosynthesis

Mycobactin biosynthesis

C5 branched dibasic acidmetabolism

D-alanine metabolism

Thiamine metabolism

Polyketide sugar unitbiosynthesis

Unique pathways identified inMycobacterium tuberculosis

Potential drug targets identified in Mycobacterium tuberculosis

Cell wall biosynthesis 10

Lipid metabolism 20

Carbohydrate metabolism 40

Energy metabolism 30

Amino acid metabolism 80

Vitamin and cofactor 35biosynthesis


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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

Elimination of pseudo drug targets

Potential targets are screened for homologues w ith 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

Informatics approach for identifying drug targets

Metabolic pathway information of host and p athogen are available in

pathway databases like KEGG

Comparative metabolic pathway analysis of host and the pathogen

Identification of enzymes unique to the pathogen

These represent potential drug targets

Case study 1: The mycobacterial cell wall

Isoniazid inhibits mycolic acidbiosynthesis

Ethambutol inhibits arabinanbiosynthesis

The three components of the

cell wall

Plasma membrane

Mycolic acid,

arabinogalactan

and peptidoglycan complex(MAPc)

Polysaccharide rich capsulelike material.

Existing drugs isoniazid &ethambutoltarget cell wall biosynthesis.

Because of its complex structure, cell wall

biosynthesis still remains a favourite for thediscovery of new drugs

Our targets from cell wall biosynthesis: 10

Prime candidate: MurD ligase

Membrane Structure in Eubacteria

Case study 2: Lipid metabolism

The bacterium depends on the hostcell lipids

Degradation of host cell lipids

provides precursors to many of thebacterial cell processes.

A large part of the codingregion of M.Tb is devoted tothe production of enzymesinvolved in

Lipogenesis

LipolysisOur targets from the lipid metabolism

: 20

Includes virulence factors likephospholipases and lipases

Lipolysis might provide carbon sources that the micro-organism could use for growth; adhesion:

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 conditionsfor other enzymes; unspecific hydrolysis: lipases might possess additional phospholipolytic

activity; immune system: lipases and their catalytical end products may have an effect on differentimmune cells and might initiate inflammatory processes; defense: micro-organisms that secrete

lipolytic enzymes might have a selection advantage by lysing competing microflora.

Jour nal of Molecul ar Catalysis B ,347 , 2003


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Case study 3 : Iron acquisition

One of the host defensivemechanisms

Dramatic reduction of ironavailability to support bacterialgrowth.

Mycobacterium tuberculosis inhabits oneof the most hostile environments

the alveolar macrophage

Bacteria have evolved sophisticated ironacquisition systems in the form of ironsequestering molecules : siderophores

M.Tb produces the mycobactinclass of siderophores.

Mutant studies have shown impaired growth of the bacteriumin macrophage li ke THP1 like

cells under iron limiting conditions

Mycobactin biosynthesisis important for vir ulence

One of our targets is from themycobactin biosynthetic pathway.

Iron Uptake & Synthesis of Mycobactin

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

Tuberculosis

Structure of the mycobactin sideropho res of M. tuberculosis. For mycobactin T, whichoccurs wholly intracellularly, n=19 (also 17 ) and R=---CH3; for ca rboxymycobactin T,which occurs extracellularly, n=29 and R=---COOH. The three pairs of chelating group s

responsible for the binding o f Fe(III) are indicated by asterisks (*).

Case study 4: Mycobacterial persistence : Latent TB

Need for new drugs to combat

latent forms of TB

Genes expressed duringmycobacterial persistence havebeen identified.

The pathogen can survive for prolongedperiods in infected individuals in a

dormant form : Latent TB

Under the microaerophilic

conditionsof the macrophage the

bacteriumsurvives the low oxygen conditionsby a metabolic downshift. It also

Switches over to anaerobic nitraterespiration

Uses glyoxylate bypass to producecarbohydrates from fatty acids

Targets from these pathwayscould prove useful for t reatment

of latent forms of TB

Once targets are discovered structurebased drug design follows.....

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

Structure of isocitrate from M.tuberculosis

Vivek Sharma, Sujata Sharma, Kersti n

H oe ner zu B en tru p, Jo hn D . M cK in ney,Dav id G. Rus se ll, W il li am R. J acobs J r. &James C. Sac chett in i Na tu re Struc tu ra lBiology 7, 663 - 668 (2000)


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Various Enzymes in the PathwayStructure of Mur Enzymes

Mechanism of Action of Mur D Ligase Structure of Mur D Ligase fromE.coli

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

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, Asn147 and His 192.

Similar ly, the residues for the ATP binding in E. coliMurD are Gly 114, Lys 115, Ser 116, Thr117, 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 asGlu 166 of M. tuberculosis MurD. In addition, all these residues are in absolutely conservedregions.

Homology modeling of one of the targets MurD ligase :A prime candidate for broad spectrum drug discovery

Why target MurD ?

Essential for cell wall

biosynthesis

viability of thebacterium

Present across all

eubacteria

Homology modelingAn alternative when structures are not available

M.Tb MurD modelled

using E.coliMurD as atemplate, with

WHATIF

Molecular modelingand drug design program

What next ??

Structure based drug design

Screening of c hemical libraries for l igand/drug dockingChemoinformatics

s


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Potential Drug Candidate for

Mycobacteium Tuberculosis An Insilico

Approach

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

I Cannot Imagine Feel ing 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 TakingOne's Work Seriously and Oneself Seriously, the First is Imperative

and the Second Disastrous

Famous Ballerina

Margaret Fonteyn in Memoirs

Thank You

Mycobacterum Drug Targets

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