New Drug Targets from Mycobacterium tuberculosis: Strategies, Progress and

Pitfalls from a Structural Genomics Enterprise

New Drug Targets from Mycobacterium tuberculosis: Strategies, Progress and

Pitfalls from a Structural Genomics Enterprise

Ted BakerTed Baker

School of Biological SciencesSchool of Biological Sciences

University of AucklandUniversity of Auckland

New ZealandNew Zealand

On behalf of TB StructuralOn behalf of TB Structural

Genomics Consortium 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)Approx. 3900 open reading frames (ORFs) ~60% of gene products have an inferred~60% of gene products have an inferred

function (mostly by homology)function (mostly by homology) ~25% are “conserved hypotheticals”~25% are “conserved hypotheticals” ~15% are “unknowns”~15% are “unknowns” ~30% can be related to proteins of known 3D~30% can be related to proteins of known 3D

structure - structure - butbut only ~25 TB protein structures only ~25 TB protein structures Many metabolic pathways appear incompleteMany metabolic pathways appear incomplete

Function from structure? Function from structure?

Relationships that are hidden at the sequence levelRelationships that are hidden at the sequence level

SpeB – virulence factor SpeB – virulence factor from from S. pyogenesS. pyogenes

Actinidin – plant cysteine proteaseActinidin – plant cysteine protease- < 10% sequence identity- < 10% sequence identity

Structural GenomicsStructural Genomics

The use of genomic information to guide protein The use of genomic information to guide protein structure discoverystructure discovery

- - and its inverseand its inverse The use of protein structure analysis to add value The use of protein structure analysis to add value

to genomic sequence data – to deduce functionto genomic sequence data – to deduce function--

Reversal of the ‘traditional’ direction of structural Reversal of the ‘traditional’ direction of structural analysisanalysis

Many targets – whole genomes, pathways,Many targets – whole genomes, pathways,functional classes, foldsfunctional classes, folds

Beginnings…~1998A pilot pilot programme – Pyrobaculum aerophilum

Beginnings…~1998A pilot pilot programme – Pyrobaculum aerophilum

Using laboratory-scale approachesUsing laboratory-scale approaches

- PCR cloning- PCR cloning

- Expression in E. coli, cleavable affinity - Expression in E. coli, cleavable affinity tagstags

- Variation of expression temperature- Variation of expression temperature

- Purification by affinity chromatography and- Purification by affinity chromatography andgel filtrationgel filtration

Genomic approach – most tractable firstGenomic approach – most tractable first

Results – P. aerophilumResults – P. aerophilum

ClonedCloned 25 (274) 25 (274) ExpressedExpressed 20 (168) 20 (168) SolubleSoluble 12 (80) 12 (80) PurifiedPurified 12 (43) 12 (43) CrystallizedCrystallized 6 (24) 6 (24) StructuresStructures 4 (11) 4 (11)

Main bottlenecks Main bottlenecks - solubility - solubility - crystallization- crystallization

Pa_989 (TB homologue)Pa_989 (TB homologue)

HisF (imidazoleglycerol phosphate synthase)HisF (imidazoleglycerol phosphate synthase) Banfield Banfield et al.et al. Acta Cryst. DActa Cryst. D (2001) (2001)

Pa_2307 (unknown)Pa_2307 (unknown)

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

Reproducible crystals with LiReproducible crystals with Li22SOSO44

- but twinned- but twinned Two crystals grown from PEG/phosphateTwo crystals grown from PEG/phosphate

1.5 A native data from one, SAD data from 1.5 A native data from one, SAD data from Pt(NOPt(NO22))44 deriv of the other (used gel shift) deriv of the other (used gel shift)

Structure solved: SAD/Solve/Resolve/ARPStructure solved: SAD/Solve/Resolve/ARP

Pa_2307Pa_2307

The next phase – larger enterprises

The next phase – larger enterprises

Publicly fundedPublicly funded

- NIH Protein Structure Initiative (USA)- NIH Protein Structure Initiative (USA)

- Initiatives in Japan, Germany, UK,- Initiatives in Japan, Germany, UK, France, CanadaFrance, Canada

Biotech companiesBiotech companies

- Structural Genomix, Syrrx- Structural Genomix, Syrrx

NIH Protein Structure InitiativeNIH Protein Structure Initiative

10 groups (consortia) funded10 groups (consortia) funded Aim to develop methods and Aim to develop methods and

tools for “high throughput” tools for “high throughput” structure determinationstructure determination

Goals primarily structuralGoals primarily structural - representative structures for- representative structures for all protein sequence familiesall protein sequence families - discover novel folds (cover - discover novel folds (cover

“ “fold space”)fold space”) - estimate 10,000 structures- estimate 10,000 structures

neededneeded

But evolvingBut evolving

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 organismPeculiarities of the organism

Very slow-growing Gram-positive organismVery slow-growing Gram-positive organism

Complex waxy cell wall – outer layer richComplex waxy cell wall – outer layer rich in unusual lipids, glycolipids, polysaccharidesin unusual lipids, glycolipids, polysaccharides

Novel biosynthetic pathwaysNovel biosynthetic pathways

Complex lifestyle - Complex lifestyle - persistencepersistence - enters dormant state within- enters dormant state within active macrophagesactive macrophages - survives through switches- survives through switches in metabolism in metabolism - can be reactivated years later- can be reactivated years later

Led in United States by:Led in United States by:- - Tom Terwilliger (Los Alamos NL)Tom Terwilliger (Los Alamos NL)- David Eisenberg (UCLA)- David Eisenberg (UCLA)- Jim Sacchettini (Texas A&M)- Jim Sacchettini (Texas A&M)- Bill Jacobs (Albert Einstein Coll. of Med.)- Bill Jacobs (Albert Einstein Coll. of Med.)- Tom Alber (UC Berkeley)….. - Tom Alber (UC Berkeley)….. and many othersand many others

Aims are focused on function:Aims are focused on function:- understanding TB biology- understanding TB biology- discovery and structural analysis of- discovery and structural analysis of novel drug targetsnovel drug targets

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

Philosophy and policies Philosophy and policies

Open participationOpen participation - to all with an interest in TB - to all with an interest in TB Operates as a wider consortium of >30Operates as a wider consortium of >30

participating labs in 13 countries worldwide participating labs in 13 countries worldwide Collaboration between structural biologistsCollaboration between structural biologists

TB biologists, chemists….TB biologists, chemists…. Commitment to common policiesCommitment to common policies

- - collaboration and cooperationcollaboration and cooperation- - shared database for logging progressshared database for logging progress- sharing of data and materials- sharing of data and materials- structures to be placed in public domain- structures to be placed in public domain

Operational aspectsOperational aspects

Central facilities forCentral facilities for- bioinformatic analysis and data storage- bioinformatic analysis and data storage- protein expression and evolution- protein expression and evolution- crystallization- crystallization- synchrotron data collection- synchrotron data collection- gene knockouts- gene knockouts

Technologies and facilities available to allTechnologies and facilities available to all Individuals choose their own targets according toIndividuals choose their own targets according to

their own interests – and assign prioritiestheir own interests – and assign priorities Targeting scores determine priorities of facilitiesTargeting scores determine priorities of facilities Parallel efforts in individual labsParallel efforts in individual labs

Progress to dateProgress to date

Most of structural results to date come as a resultMost of structural results to date come as a result of efforts in individual labs of efforts in individual labs

ButBut - availability of high-throughput facilities gives - availability of high-throughput facilities gives

flexible options for individual labsflexible options for individual labs and for efforts in the facilitiesand for efforts in the facilities

Within facilities – 688 genes cloned (out of 720Within facilities – 688 genes cloned (out of 720 targeted to date)targeted to date)

First phaseFirst phase – concentrate on soluble proteins – concentrate on soluble proteins Next phaseNext phase – the insoluble proteins – the insoluble proteins

Dealing with insoluble proteins GFP fusions as reporter of solubility – G. Waldo

XR

CN L

Non-functional R

Insoluble

Detect function R

Soluble

Express fusion protein X-L-R

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

Folding Reporter - GFP

Cell Colonies

In VitroTranscription

+Translation

Soluble Fraction

Pellet Fraction

SDS-PAGEX (Non-Fusion)

X-L-GFP FUSION FLUORESCENCE

Using GFP-fusions to engineer proteins for solubility

Using GFP-fusions to engineer proteins for solubility

Insoluble Protein

FORWARDEVOLUTION

Clone

Select

Mutate Gene

RecombineOptima

Soluble Protein

BACKCROSSING Clone

Select

RecombineOptima &Wild type

G.Waldo

Solubilisation by evolutionRv2002 – Se Won Suh

Solubilisation by evolutionRv2002 – Se Won Suh

Putative ketoacyl ACPPutative ketoacyl ACP reductasereductase

Rendered soluble byRendered soluble by 3 random mutations3 random mutations

I6T and T69KI6T and T69K mutations are onmutations are on the molecular surfacethe molecular surface

V47M mutationV47M mutation enhances a semi-enhances a semi- exposed hydrophobicexposed hydrophobic contactcontact

Potential new TB drug targetsPotential new

TB drug targets

Early results from the TBEarly results from the TB

Structural Genomics ConsortiumStructural Genomics Consortium

Target ORF Selection in Mycobacterium tuberculosis

Target ORF Selection in Mycobacterium tuberculosis

Selection of ORFs: (a) potential drug targetsSelection of ORFs: (a) potential drug targets and (b) to understand TB biologyand (b) to understand TB biology Biosynthetic enzymes for essential aminoBiosynthetic enzymes for essential amino acids, cofactors, lipids, polysaccharidesacids, cofactors, lipids, polysaccharides Secreted proteinsSecreted proteins

Proteins implicated in antibiotic resistanceProteins implicated in antibiotic resistance or responseor response

Proteins implicated in persistenceProteins implicated in persistence

1. Cell wall biosynthesis- mycolic acids (Sacchettini lab)

1. Cell wall biosynthesis- mycolic acids (Sacchettini lab)

Long chain branched lipids - form dense waxy Long chain branched lipids - form dense waxy outer layer of the mycobacterial cell wallouter layer of the mycobacterial cell wall

Contribute to its impenetrabilityContribute to its impenetrability Implicated in both virulence and persistenceImplicated in both virulence and persistence Either covalently attached to cell wallEither covalently attached to cell wall

or released as trehalose dimycolateor released as trehalose dimycolate(“cord factor”)(“cord factor”)

Modification of mycolic acids, eg. cyclopropanationModification of mycolic acids, eg. cyclopropanation – – varies between pathogenic and non-pathogenicvaries between pathogenic and non-pathogenic speciesspecies

Cyclopropanation of mycolic acid chains

Cyclopropanation of mycolic acid chains

Cyclopropane groups introduced by methylation 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

2. Secreted proteins(Eisenberg lab)

2. Secreted proteins(Eisenberg lab)

Secreted proteins attractive drug targetsSecreted proteins attractive drug targets for for M. tuberculosisM. tuberculosis because: because:

Often determinants of virulence or persistenceOften determinants of virulence or persistence- involved in cell wall modification- involved in cell wall modification- role in survival in macrophages- role in survival in macrophages

M. tuberculosisM. tuberculosis secretes large number of proteins secretes large number of proteins

Cell wall is impermeable to many anti-Cell wall is impermeable to many anti- bacterial agentsbacterial agents

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

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

Rv1886cAntigen 85BMycolyl transferase

NC

Rv2220Glutamine synthetase

- Synthesis ofpoly-(L-Glu-L-Gln)for cell wall

Rv1926cUnknown, resembles cell surface binding proteins (invasin, adaptin, arrestin)

3. Targets against persistence(Sacchettini lab)

3. Targets against persistence(Sacchettini lab)

Persistence within activated macrophagesPersistence within activated macrophages facilitated by switch in metabolismfacilitated by switch in metabolism

Glycolysis downregulated – insteadGlycolysis downregulated – instead glyoxalate shuntglyoxalate shunt allows use of C2 substrates allows use of C2 substrates generated by generated by -oxidation of fatty acids-oxidation of fatty acids

Enzymes isocitrate lyase and malate synthaseEnzymes isocitrate lyase and malate synthase are drug targets for persistent bacteriaare 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 Rv0867 Isocitrate lyaseIsocitrate lyase

Rv1837cRv1837cMalate synthaseMalate synthase

4. Antibiotic resistance- Isoniazid response genes

4. Antibiotic resistance- Isoniazid response genes

DNA microarray analysis DNA microarray analysis of TB ORFs upregulated by of TB ORFs upregulated by exposure to isoniazidexposure to isoniazid

Some code for proteins ofSome code for proteins ofknown function – cell wallknown function – cell wall

biosynthesisbiosynthesis Others represent ‘unknowns’Others represent ‘unknowns’ The proteins encoded byThe proteins encoded by

these ORFs may represent these ORFs may represent the bacterial response to thethe bacterial response to thetoxic effects of the antibiotictoxic effects of the antibiotic

Wilson et al., PNAS 96:12833-12838 (1999)

Putative INH response operonPutative INH response operon

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

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

Rv0340 and Rv0341 are paralogues, as are Rv0340 and Rv0341 are paralogues, as are Rv0342 and Rv0343Rv0342 and Rv0343

Same genes also upregulated by ethambutol.Same genes also upregulated by ethambutol.

Rv0340 Rv0341 Rv0342 Rv0343

Isoniazid response – Rv0340Moyra Komen, Vic Arcus, Shaun Lott

Isoniazid response – Rv0340Moyra Komen, Vic Arcus, Shaun Lott

Crystallization attemptsCrystallization attempts

NMR – shows only partially foldedNMR – shows only partially folded

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

Oil Spherulites

NMR spectrum – Rv0340(residues 1-131)

NMR spectrum – Rv0340(residues 1-131)

Indicates helicalIndicates helicalbundle with flexible bundle with flexible tailtail

Possible homologyPossible homologywith acyl carrierwith acyl carrierproteinprotein

Gives putativeGives putativerole in cell wallrole in cell wallbiosynthesisbiosynthesis

Problems of partial or incorrect functional annotation

Problems of partial or incorrect functional annotation

Widespread in bacteria, butWidespread in bacteria, but not eukaryotesnot eukaryotes

No clearly indicated functionNo clearly indicated function- closest sequence homologs:- closest sequence homologs:

malonyl CoA decarboxylasemalonyl CoA decarboxylase

siderophore biosynthesissiderophore biosynthesis aminoglycoside acetyltransferaseaminoglycoside acetyltransferase

No structure predictionNo structure prediction

Rv1347cRv1347c

Rv1347c structure - Graeme Card

Rv1347c structure - Graeme Card

Rv1347cRv1347c Acetyl-CoA dependent Acetyl-CoA dependent aminoglycosideaminoglycoside

acetyltransferase acetyltransferase (11% identity)(11% identity)

Rv1347cRv1347cAminoglycoside N-acetyl Aminoglycoside N-acetyl transferase (GCN5 family)transferase (GCN5 family)~ 11% sequence identity~ 11% sequence identity

Problem of partial or incorrect functional annotations

Problem of partial or incorrect functional annotations

Putative SAM-dependent methyltransferasePutative SAM-dependent methyltransferasecatalysing final step in menaquinone biosynthesiscatalysing final step in menaquinone biosynthesis

Potential drug target – menaquinone pathway isPotential drug target – menaquinone pathway isessential and is not present in humansessential and is not present in humans

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

Expressed, refolded, crystallized, solved to 1.9Expressed, refolded, crystallized, solved to 1.9ÅÅ by by SIRASSIRAS

Rv3853 - “menG”Rv3853 - “menG”

Common methyltransferase foldCommon methyltransferase fold

MenG structure – Jodie JohnstonMenG structure – Jodie Johnston

Structure does not Structure does not look like a look like a methyltransferasemethyltransferase

Resembles a Resembles a phosphate transfer phosphate transfer domain?domain?

Incorrect annotationIncorrect annotation

Challenges for the futureChallenges for the future

Membrane proteinsMembrane proteins Solubility of expressed proteinsSolubility of expressed proteins Hetero-oligomeric proteinsHetero-oligomeric proteins Protein-protein interactionsProtein-protein interactions Assignment of function to “unknowns”Assignment of function to “unknowns” Cellular pathways - metabolic pathwaysCellular pathways - metabolic pathways

- signalling pathways- signalling pathways

ConclusionsConclusions

Structural biology is being transformed byStructural biology is being transformed by new technologies – some driven by genomicsnew technologies – some driven by genomics

Less effort in solving initial structures – more Less effort in solving initial structures – more emphasis on “downstream” studiesemphasis on “downstream” studies

TB structural genomics consortium – a differentTB structural genomics consortium – a different model for large scale structure determinationmodel for large scale structure determination - access to centralised facilities- access to centralised facilities - international effort on a common goal- international effort on a common goal - collaboration rather than competition- collaboration rather than competition - opportunities for smaller labs- opportunities for smaller labs

ThanksThanks

Mycobacterium tuberculosisMycobacterium tuberculosis structural genomics structural genomics consortiumconsortium

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

Marsden FundMarsden Fund Health Research CouncilHealth Research Council New Economy Research FundNew Economy Research Fund