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New Drug Targets from Mycobacterium tuberculosis : Strategies, Progress and Pitfalls from a Structural Genomics Enterprise. Ted Baker School of Biological Sciences University of Auckland New Zealand. On behalf of TB Structural Genomics Consortium.

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Ted baker school of biological sciences university of auckland new zealand

New Drug Targets from Mycobacterium tuberculosis:Strategies, Progress and Pitfalls from a Structural Genomics Enterprise

Ted Baker

School of Biological Sciences

University of Auckland

New Zealand

On behalf of TB Structural

Genomics Consortium


The challenge posed by complete genome sequences

The challenge posed by complete genome sequences


The mycobacterium tuberculosis genome

The Mycobacterium tuberculosis genome

  • Approx. 3900 open reading frames (ORFs)

  • ~60% of gene products have an inferred

    function (mostly by homology)

  • ~25% are “conserved hypotheticals”

  • ~15% are “unknowns”

  • ~30% can be related to proteins of known 3D

    structure - but only ~25 TB protein structures

  • Many metabolic pathways appear incomplete


Function from structure

Function from structure?

Relationships that are hidden at the sequence level

SpeB – virulence factor from S. pyogenes

Actinidin – plant cysteine protease

- < 10% sequence identity


Structural genomics

Structural Genomics

  • The use of genomic information to guide protein structure discovery

    - and its inverse

  • The use of protein structure analysis to add value to genomic sequence data – to deduce function

    -

  • Reversal of the ‘traditional’ direction of structural analysis

  • Many targets – whole genomes, pathways,

    functional classes, folds


Beginnings 1998 a pilot pilot programme pyrobaculum aerophilum

Beginnings…~1998A pilot pilot programme – Pyrobaculum aerophilum

  • Using laboratory-scale approaches

    - PCR cloning

    - Expression in E. coli, cleavable affinity tags

    - Variation of expression temperature

    - Purification by affinity chromatography and

    gel filtration

  • Genomic approach – most tractable first


Results p aerophilum

Results – P. aerophilum

  • Cloned 25 (274)

  • Expressed 20 (168)

  • Soluble 12 (80)

  • Purified 12 (43)

  • Crystallized 6 (24)

  • Structures 4 (11)

Main bottlenecks - solubility

- crystallization


Pa 989 tb homologue

HisF (imidazoleglycerol phosphate synthase)

Banfield et al.Acta Cryst. D (2001)

Pa_989 (TB homologue)


Pa 2307 unknown

Pa_2307 (unknown)

  • ‘Ancient conserved domain’ found in bacteria and archaea. No functional annotation

  • Reproducible crystals with Li2SO4

    - but twinned

  • Two crystals grown from PEG/phosphate

  • 1.5 A native data from one, SAD data from Pt(NO2)4 deriv of the other (used gel shift)

  • Structure solved: SAD/Solve/Resolve/ARP


Pa 2307

Pa_2307


The next phase larger enterprises

The next phase – larger enterprises

  • Publicly funded

    - NIH Protein Structure Initiative (USA)

    - Initiatives in Japan, Germany, UK,

    France, Canada

  • Biotech companies

    - Structural Genomix, Syrrx


Nih protein structure initiative

NIH Protein Structure Initiative

  • 10 groups (consortia) funded

  • Aim to develop methods and tools for “high throughput” structure determination

  • Goals primarily structural

    - representative structures for

    all protein sequence families

    - discover novel folds (cover

    “fold space”)

    - estimate 10,000 structures

    needed

    But evolving


Ted baker school of biological sciences university of auckland new zealand

Mycobacterium tuberculosis

Causative agent of TB

One-third of world’s population affected

- approximately 3 million deaths annually

Five front-line drugs (isoniazid, pyrazinamide, ethambutol, rifampin, streptomycin) but…

- effective only against actively-growing

bacteria

- very long treatment regime (6-9 months)

- resistance rising

- need for new drugs


Peculiarities of the organism

Peculiarities of the organism

  • Very slow-growing Gram-positive organism

  • Complex waxy cell wall – outer layer rich

    in unusual lipids, glycolipids, polysaccharides

  • Novel biosynthetic pathways

  • Complex lifestyle - persistence

    - enters dormant state within

    active macrophages

    - survives through switches

    in metabolism

    - can be reactivated years later


Ted baker school of biological sciences university of auckland new zealand

  • Led in United States by:

    - Tom Terwilliger (Los Alamos NL)

    - David Eisenberg (UCLA)

    - Jim Sacchettini (Texas A&M)

    - Bill Jacobs (Albert Einstein Coll. of Med.)

    - Tom Alber (UC Berkeley)….. and many others

  • Aims are focused on function:

    - understanding TB biology

    - discovery and structural analysis of

    novel drug targets

http://www.doe-mbi.ucla.edu/TB/


Philosophy and policies

Philosophy and policies

  • Open participation - to all with an interest in TB

  • Operates as a wider consortium of >30

    participating labs in 13 countries worldwide

  • Collaboration between structural biologists

    TB biologists, chemists….

  • Commitment to common policies

    - collaboration and cooperation

    - shared database for logging progress

    - sharing of data and materials

    - structures to be placed in public domain


Operational aspects

Operational aspects

  • Central facilities for

    - bioinformatic analysis and data storage

    - protein expression and evolution

    - crystallization

    - synchrotron data collection

    - gene knockouts

  • Technologies and facilities available to all

  • Individuals choose their own targets according to

    their own interests – and assign priorities

  • Targeting scores determine priorities of facilities

  • Parallel efforts in individual labs


Progress to date

Progress to date

  • Most of structural results to date come as a result

    of efforts in individual labs

  • But - availability of high-throughput facilities gives

    flexible options for individual labs

    and for efforts in the facilities

  • Within facilities – 688 genes cloned (out of 720

    targeted to date)

  • First phase – concentrate on soluble proteins

  • Next phase – the insoluble proteins


Ted baker school of biological sciences university of auckland new zealand

Folding Reporter - GFP

  • Function of R (GFP) depends on solubility of X-L-R.

  • Solubility of X-L-R depends on X.

Express fusion protein X-L-R

L

N

C

X

R

Non-functional R

Detect function R

Insoluble

Soluble

Dealing with insoluble proteins

GFP fusions as reporter of solubility– G. Waldo


Ted baker school of biological sciences university of auckland new zealand

Cell Colonies

X-L-GFP FUSION FLUORESCENCE

In Vitro

Transcription

+

Translation

Soluble Fraction

SDS-PAGE

X (Non-Fusion)

Pellet Fraction


Using gfp fusions to engineer proteins for solubility

Insoluble Protein

Mutate Gene

FORWARD

EVOLUTION

Recombine

Optima

Clone

Select

Recombine

Optima &

Wild type

Clone

BACKCROSSING

Select

Soluble Protein

Using GFP-fusions to engineer proteins for solubility

G.Waldo


Solubilisation by evolution rv2002 se won suh

Solubilisation by evolutionRv2002 – Se Won Suh

  • Putative ketoacyl ACP

    reductase

  • Rendered soluble by

    3 random mutations

  • I6T and T69K

    mutations are on

    the molecular surface

  • V47M mutation

    enhances a semi-

    exposed hydrophobic

    contact


Potential new tb drug targets

Potential new TB drug targets

Early results from the TB

Structural Genomics Consortium


Target orf selection in mycobacterium tuberculosis

Target ORF Selection in Mycobacterium tuberculosis

  • Selection of ORFs: (a) potential drug targets

    and (b) to understand TB biology

    • Biosynthetic enzymes for essential amino

      acids, cofactors, lipids, polysaccharides

    • Secreted proteins

    • Proteins implicated in antibiotic resistance

      or response

    • Proteins implicated in persistence


Cell wall biosynthesis mycolic acids sacchettini lab

Cell wall biosynthesis- mycolic acids (Sacchettini lab)

  • Long chain branched lipids - form dense waxy

    outer layer of the mycobacterial cell wall

  • Contribute to its impenetrability

  • Implicated in both virulence and persistence

  • Either covalently attached to cell wall

    or released as trehalose dimycolate

    (“cord factor”)

  • Modification of mycolic acids, eg. cyclopropanation

    – varies between pathogenic and non-pathogenic

    species


Cyclopropanation of mycolic acid chains

Cyclopropanation of mycolic acid chains

  • Cyclopropane groups introduced by methylation


Three cyclopropane synthases c smith j sacchettini texas a m

Three cyclopropane synthases(C. Smith, J. Sacchettini – Texas A&M)

CmaA1

CmaA2

PcaA


Secreted proteins eisenberg lab

Secreted proteins(Eisenberg lab)

Secreted proteins attractive drug targets

for M. tuberculosis because:

  • Often determinants of virulence or persistence

    - involved in cell wall modification

    - role in survival in macrophages

  • M. tuberculosis secretes large number of proteins

  • Cell wall is impermeable to many anti-

    bacterial agents


Secreted proteins c goulding d anderson h gill d eisenberg ucla

C

N

Secreted proteins(C. Goulding, D. Anderson, H. Gill, D. Eisenberg – UCLA)

Rv1886c

Antigen 85B

Mycolyl transferase

Rv2220

Glutamine synthetase

- Synthesis of

poly-(L-Glu-L-Gln)

for cell wall

Rv1926c

Unknown, resembles cell surface binding proteins (invasin, adaptin, arrestin)


3 targets against persistence sacchettini lab

3. Targets against persistence(Sacchettini lab)

  • Persistence within activated macrophages

    facilitated by switch in metabolism

  • Glycolysis downregulated – instead

    glyoxalate shunt allows use of C2 substrates

    generated by b-oxidation of fatty acids

  • Enzymes isocitrate lyase and malate synthase

    are drug targets for persistent bacteria


Glyoxalate shunt enzymes v sharma j sacchettini texas a m

Glyoxalate shunt enzymes(V. Sharma, J. Sacchettini - Texas A&M)

Rv0867

Isocitrate lyase

Rv1837c

Malate synthase


4 antibiotic resistance isoniazid response genes

DNA microarray analysis of TB ORFs upregulated by exposure to isoniazid

Some code for proteins ofknown function – cell wall

biosynthesis

Others represent ‘unknowns’

The proteins encoded bythese ORFs may represent the bacterial response to thetoxic effects of the antibiotic

4. Antibiotic resistance- Isoniazid response genes

Wilson et al.,PNAS96:12833-12838 (1999)


Putative inh response operon

Rv0340

Rv0341

Rv0342

Rv0343

Putative INH response operon

  • Four ORFs appear to make up part of a putative operon in the TB genome: Rv0340, Rv0341, Rv0342, Rv0343.

  • None of the four ORFs have detectable sequence homologues in other organisms.

  • Rv0340 and Rv0341 are paralogues, as are Rv0342 and Rv0343

  • Same genes also upregulated by ethambutol.


Isoniazid response rv0340 moyra komen vic arcus shaun lott

Isoniazid response – Rv0340Moyra Komen, Vic Arcus, Shaun Lott

  • Crystallization attempts

  • NMR – shows only partially folded

  • Limited proteolysis – gives N-terminal fragment with excellent NMR spectrum

Oil

Spherulites


Nmr spectrum rv0340 residues 1 131

NMR spectrum – Rv0340(residues 1-131)

  • Indicates helical

    bundle with flexible tail

  • Possible homology

    with acyl carrier

    protein

  • Gives putative

    role in cell wall

    biosynthesis


Problems of partial or incorrect functional annotation

Problems of partial or incorrect functional annotation

  • Widespread in bacteria, but

    not eukaryotes

  • No clearly indicated function

    - closest sequence homologs:

    malonyl CoA decarboxylase

    siderophore biosynthesis

    aminoglycoside acetyltransferase

  • No structure prediction

Rv1347c


Rv1347c structure graeme card

Rv1347c structure - Graeme Card

Rv1347cAcetyl-CoA dependent aminoglycoside

acetyltransferase (11% identity)

Aminoglycoside N-acetyl transferase (GCN5 family)

~ 11% sequence identity

Rv1347c


Problem of partial or incorrect functional annotations

Problem of partial or incorrect functional annotations

  • Putative SAM-dependent methyltransferase

    catalysing final step in menaquinone biosynthesis

  • Potential drug target – menaquinone pathway is

    essential and is not present in humans

  • Genome also includes ubiE (Rv0558) - catalyses this step in both menaquinone and ubiquinone biosynthesis (menG is specific for menaquinone)

  • Expressed, refolded, crystallized, solved to 1.9Å by SIRAS

Rv3853 - “menG”


Common methyltransferase fold

Common methyltransferase fold


Meng structure jodie johnston

MenG structure – Jodie Johnston

  • Structure does not look like a methyltransferase

  • Resembles a phosphate transfer domain?

  • Incorrect annotation


Challenges for the future

Challenges for the future

  • Membrane proteins

  • Solubility of expressed proteins

  • Hetero-oligomeric proteins

  • Protein-protein interactions

  • Assignment of function to “unknowns”

  • Cellular pathways - metabolic pathways

    - signalling pathways


Conclusions

Conclusions

  • Structural biology is being transformed by

    new technologies – some driven by genomics

  • Less effort in solving initial structures – more

    emphasis on “downstream” studies

  • TB structural genomics consortium – a different

    model for large scale structure determination

    - access to centralised facilities

    - international effort on a common goal

    - collaboration rather than competition

    - opportunities for smaller labs


Thanks

Thanks

  • Mycobacterium tuberculosis structural genomics consortium

  • Members of Auckland Structural Biology Laboratory – Vic Arcus, Kristina Backbro, Mark Banfield, Heather Baker, Graeme Card, Jodie Johnston, Rainer Knijff, Moyra Komen, Shaun Lott, Andrew McCarthy, Clyde Smith

  • Marsden Fund

    Health Research Council

    New Economy Research Fund


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