Meeting Highlights

2004 © Ashley Publications Ltd ISSN 1472-8222 165

Ashley Publicationswww.ashley-pub.com

1. Introduction

2. G-protein-coupled receptors

and their interacting proteins

3. An integrated approach to

drug discovery

4. G-protein-coupled receptor

dimerisation

5. A selection of agonists

and antagonists

6. Single nucleotide

polymorphisms in G proteins

as determinants of drug

responsiveness

7. Molecular pathological

screening

8. Target validation through

gene disruption

9. Selection, validation and

prosecution of

inflammatory targets

10. Structural genomics on

G-protein-coupled receptors

11. The analysis of human

G-protein-coupled receptors in

fission yeast

12. Screening technologies and

library design

13. Conclusion

G-Protein-Coupled Receptors: New Approaches to Maximise the Impact of GPCRs in Drug Discovery19 – 20 January 2004, London, UK

John DaveyDepartment of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK

IBC’s Drug Discovery Technology Series is a group of conferences highlightingtechnological advances and applications in niche areas of the drug discoverypipeline. This 2-day meeting focused on G-protein-coupled receptors (GPCRs),probably the most important and certainly the most valuable class of targetsfor drug discovery. The meeting was chaired by J Beesley (Vice President, Euro-pean Business Development for LifeSpan Biosciences, Seattle, USA) andincluded 17 presentations on various aspects of GPCR activity, drug screensand therapeutic analyses. Keynote Addresses covered two of the emergingareas in GPCR regulation; receptor dimerisation (G Milligan, Professor ofMolecular Pharmacology and Biochemistry, University of Glasgow, UK) andproteins that interact with GPCRs (J Bockaert, Laboratory of FunctionalGenomics, CNRS Montpellier, France). A third Keynote Address from W Thom-sen (Director of GPCR Drug Screening, Arena Pharmaceuticals, USA) discussedArena’s general approach to drug discovery and illustrated this with referenceto the development of an agonist with potential efficacy in Type II diabetes.

Keywords: dimerisation, G-protein-coupled receptors (GPCRs), high-throughput screens

Expert Opin. Ther. Targets (2004) 8(2):165-170

1. Introduction

G-protein-coupled receptors (GPCRs) influence every major biological function inall eukaryotic systems, including man. Approximately 60% of current drugs targetGPCRs and global sales are estimated to be ∼ US$85 billion. Identifying morepotent drugs with greater specificity is a major goal for pharmaceutical companies,and most major companies have extensive drug development programmes aimed atGPCRs. Furthermore, the absolute number of GPCRs that are targets for currentmedicines represents only a small subset of the total number of receptors encoded bythe human genome. Recent analysis associated with the human genome sequencingproject has predicted a total of 367 non-sensory GPCRs in humans. The endog-enous ligands for a large number of these GPCRs remain unknown and deorphani-sation projects to pair receptors with ligands are a major focus of many companies.IBC’s meeting on GPCRs covered both known and orphan receptors, includingtalks on the search for new drugs for existing targets and attempts to validate orphanreceptors as new targets.

2. G-protein-coupled receptors and their interacting proteins

The meeting opened with a Keynote Address from J Bockaert (Laboratory of Func-tional Genomics, CNRS Montpellier, France) describing his work on proteins thatinteract with GPCRs [1].

There are several well-characterised proteins, such as the receptor kinases andarrestins, that regulate receptor activity. But there are also a large number of

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166 Expert Opin. Ther. Targets (2004) 8(2)

cytosolic adaptor and scaffolding proteins, all of which affectthe GPCR response by helping to recruit different signaltransduction complexes (signal transductosomes). These, intheory, could provide different molecular pharmacologicalphenotypes, which, although not currently included in phar-maceutical drug design, could have enormous potential asdrug targets. The first proteins described by Bockaert were thePDZ-containing family, which interact with the extremeC-terminal tail of various receptors (specifically anExxSxV sequence motif ). Several such proteins were identi-fied using a proteomic approach in which an immobilisedC-terminal tail derived from the 5-hydroxytryptamine(5-HT)2C receptor was used to ‘pull-down’ interacting pro-teins from brain extract. The pulled-down proteins, whichwere identified by two-dimensional gel electrophoresis, tryp-tic fragmentation and mass spectrometry, included Veli3,CASK, Mint1, Munc18, Dynamin, PSD95, nNOS andMUPP1. The experiment was repeated with the 5-HT2A and5-HT2B receptors and several splice variants of the5-HT4 receptor. Each receptor pulled down slightly differentcomplexes of proteins; some proteins were pulled down by allreceptors but some only interacted with certain receptors.Using different tissue extracts (brain, lung and heart) also pro-duced different complexes. It is not yet known whether or notthe various PDZ-containing proteins affect the activity of thereceptors. In other examples described by Bockaert, interact-ing proteins had measurable effects on the activity and/or spe-cificity of their associated GPCRs. The Homer 3 protein, forexample, appears to maintain the metabotropic glutamatereceptor (mGluR) in an inactive conformation, since ablationof Homer 3 through RNAi antisense technology made thereceptor constitutively active. In contrast, ablation of PICK1,a protein that interacts with the C-terminal tail of themGlu7a receptor, abolished the ability of the receptor toinhibit the P/Q Ca2+ channel. The challenge to the pharma-ceutical industry will be to discover whether or not there isany therapeutic benefit in interfering with the interactionsbetween GPCRs and their associated proteins and to thendevelop strategies to achieve this.

3. An integrated approach to drug discovery

The second Keynote Address on day 1 was from W Thomsen(Director of GPCR Drug Screening at Arena Pharmaceuti-cals, USA). Thomsen discussed Arena’s integrated approach todrug discovery [2] and illustrated this with reference to theirdevelopment of an agonist with efficacy in Type II diabetes.

Many companies have used bioinformatic analysis of thehuman genome to identify most of the likelyGPCR sequences. Arena Pharmaceuticals has taken this min-ing exercise to a more detailed level with its Hidden MarkovMethods (HMMs). HMMs are probabilistic sequence repre-sentations built on known GPCR family sequence hallmarks(primarily the transmembrane domains of the receptors) thatcan then be used to screen the genome sequence to identify

potential GPCRs. Microarray (gene chip) analysis revealswhich targets represent expressed GPCRs. The Arenaapproach has identified 359 olfactory GPCRs, 258 non-sen-sory GPCRs with known ligands and 190 orphan GPCRs.Target validation of these receptors is a multistage processinvolving expression mapping (at a tissue and cellular level inboth normal and diseased states), signal transduction analysisusing wild-type and constitutively activated receptors andknockin and knockout mice.

Very detailed expression analysis was presented with eachGPCR being monitored in every tissue. One of the most sur-prising findings from the expression analysis data was thatmost human tissues express between 40 and 100 differentGPCRs. To investigate expression at the cellular level, individ-ual groups of cells were isolated from whole tissues by posi-tional ablation with laser microbeams (PALM). It isapparently possible to perform the gene chip analysis on RNAextracted from a very small group of cells.

Once a GPCR is identified as a valid target, Arena under-takes drug screening using its constitutively activated receptortechnology (CART). Mutations introduced into the GPCRmimic the conformational change induced by the agonist andprovide ligand-independent screens for both known andorphan receptors. Several different screening assays weredescribed in outline (cAMP, GTPγS, receptor upregulation)but Thomsen focused on Arena’s principle screening platform,the frog melanophores. Melanophores are the skin cells thatallow frogs and other animals to change their colour as part ofa defence mechanism. The colour is due to pigment-contain-ing organelles called melanosomes and the colour change isachieved by altering the distribution of the melanosomesthrough the cell; the cells appear light when the melanosomesaggregate but become dark when they are dispersed through-out the cell. The movement of the melanosomes is controlledby GPCRs that work through either Gs and Gq (dispersion) orGi (aggregation). Suitable for use in high-throughput formats,the melanophores can screen two GPCR targets againstArena’s full library (150,000 compounds) each week.

Thomsen illustrated Arena’s approach using the orphanGPCR 19AJ, which has been used to develop potent andselective agonists with efficacy in rodent models of Type IIdiabetes (non-insulin-dependent diabetes mellitus[NIDDM]). Expression of 19AJ is restricted to the pancre-atic β cells, co-localising with the expression of insulin. Itstimulated cAMP production and the consequent disper-sion of melanosomes was used as an assay in the melano-phore system. A number of potential agonists and inverseagonists were identified from the Arena chemical libraryand structure–activity studies led to the development ofCompound 7, a potent and selective 19AJ agonist withfavourable pharmacokinetic properties. In vivo testing inmice demonstrated that Compound 7 improved glucosetolerance and Thomsen concluded his presentation by con-firming that Compound 7 was currently undergoing in vivotoxicology tests.

Davey

Expert Opin. Ther. Targets (2004) 8(2) 167

4. G-protein-coupled receptor dimerisation

Day 2 began with a Keynote Address from G Milligan (Pro-fessor of Molecular Pharmacology and Biochemistry, Univer-sity of Glasgow, UK). It has become increasingly apparent inrecent years that GPCRs probably function as dimers [3,4].The first well-characterised dimers were the covalently associ-ated heterodimers of the Family C receptors. The functionalGABA Type b receptor, for example, is a heterodimer of twodistinct gene products in which trafficking to the plasmamembrane requires interaction between the partner polypep-tides. In other cases, dimerisation is non-covalent but is stillimportant for the normal functioning of the receptors. Het-erodimerisation, of course, would increase the potential forcrosstalk between different GPCRs and provide even greaterpharmacological diversity for this family than is already appre-ciated. It also raises the exciting possibility that heterodimerscould provide new drug targets. Demonstrating the existenceof such heterodimers formed the basis of Milligan’s presenta-tion. He described the co-immunoprecipitation of differen-tially epitope-tagged receptors and several resonance energytransfer approaches including FRET (fluorescence resonanceenergy transfer) BRET (bioluminescence resonance energytransfer) and BRET2. Several dimer pairs were described andthe use of time-resolved FRET and sucrose density centrifuga-tion to resolve intracellular compartments confirmed that thedimers are present throughout the cell.

Having established that dimers exist, Milligan turned hisattention to whether or not they are functional and describedan elegant series of experiments in which he has been able touse two inactive receptor monomers to reconstitute a func-tional dimer. Fusion proteins in which G protein α subunitsare linked in-frame to the C terminus of a GPCR have becomewidely used tools to study the details of information transferbetween these proteins. Since these can be considered asbifunctional proteins containing the sequence and function ofboth GPCR and G protein, they can be used to generate con-trasting pairs of non-functional mutants. A wild-type opioidreceptor was fused to an inactive Gi α subunit and expressed incells containing a non-functional opioid receptor fused to anactive Gi α subunit. A mutation in the Gi α subunit fused tothe non-functional opioid receptor rendered it insensitive topertussis toxin and allowed the endogenous Gi α subunits tobe inhibited by pertussis toxin. Neither receptor alone was ableto signal (even if they can form homodimers) but cells express-ing both receptors respond to opioid agonists, presumablythrough the formation of ‘heterodimers’ and the transactiva-tion of the functional Gi α subunit (attached to the non-func-tional receptor) by the functional opioid receptor (attached tothe non-functional Gi α). The approach was also applied to aheterodimer containing the α1b-adrenergic receptor and thehistamine H1 receptor.

One of the next challenges of understanding GPCR het-erodimerisation will be to investigate whether heterodimersdisplay distinct pharmacology and/or function compared to

the individual GPCRs expressed alone, which thus presuma-bly exist as homodimers. There have been some excitinginsights from the analysis of opioid receptors but much moredetailed analysis is required before the true potential of het-erodimers as new pharmaceutical targets will be revealed.

5. A selection of agonists and antagonists

Chemokine receptor antagonists offer potential benefits in thetreatment of a range of indications and many pharmaceuticalcompanies have compounds in various stages of development.R Horuk (Director of Immunology, Berlex Biosciences, USA)discussed his company’s attempts to develop antagonists forchemokine receptor (CCR)1. CCR1 is believed to play a rolein the pathophysiology of multiple sclerosis (MS), transplantrejection and renal fibrosis. BX-471 was identified as aCCR1-specific antagonist in 1996, shown to have efficacy in amouse MS model in 1997 and to be effective in transplanta-tion models in 1999. Human Phase I clinical trials between2000 and 2002 have been followed by Phase II trials in 2003with indications for MS and psoriasis.

Once an orphan GPCR has been paired with potential lig-ands, the challenge is to extract value from this information.Such functional validation was the theme of the presentationby M Fidock (Head of Molecular Pharmacology, Pfizer Glo-bal R&D, UK) who described both ‘hypothesis-driven’ stud-ies, in which knowledge about the endogenous ligand canprovide clues about the receptor’s function, and the ‘test andsee’ approach, in which the receptor and ligand are screenedthrough many assays. Much of the presentation focused onthe MRGX1 receptor, a member of the dorsal root receptor(DRR) family. This potential pain target is expressed in thedorsal root ganglia and can be activated by the Bam22 ligand.Although this ligand also activates opioid receptors, a trun-cated version (Bam8-22) is specific for MRGX1. Electrophys-iology experiments in rats found that Bam8-22 increased theresponse when neurons were stimulated by pinch, suggestingthat Bam8-22 could potentiate the pain response throughMRGX1. The mechanism for this action remains unclear.

6. Single nucleotide polymorphisms in G proteins as determinants of drug responsiveness

SNiP Biotech is a recently founded spin-off company fromthe University of Essen Medical School. Its research is basedon the principle that single nucleotide polymorphisms (SNPs)modulate the functions of genes involved in signalling. Suchgenetic variations in prime drug targets could have profoundeffects on pharmacodynamics and may explain why a propor-tion of the population often fails to respond to frequentlyused drugs. U Frey (Institute of Pharmacology, UniversityHospital Essen and SNiP Biotech, Inc., Essen, Germany)described his work on SNPs in two G protein subunits,GNB3 (the Gβ3 subunit) and GNAS (the Gs α subunit).

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168 Expert Opin. Ther. Targets (2004) 8(2)

A C/T polymorphism in exon 10 of the GNB3 gene leads toalternate splicing and production of a truncated protein thatcontains only six out of the seven blades of the Gβ propellerstructure. Assays showing that the truncated Gβ3 increasesthe sensitivity of transfected cells to stimulation by carbacholled to speculation that the shortened protein has increased sig-nalling capability. A number of in vivo studies were consistentwith this suggestion.

7. Molecular pathological screening

The use of molecular pathological screening in target identifi-cation, validation and prioritisation through molecular patho-logical screening was described by J Beesley (Vice President,European Business Development, for LifeSpan Biosciences,Seattle, USA). The rationale behind the approach is that theexpression profile of a GPCR may account for the pharmaco-logical specificity of the ligand. By documenting receptor lev-els and expression patterns in normal and diseased tissues itshould be possible to link receptors with cells relevant to thedisease process. Tissue localisation studies at LifeSpan include320 GPCRs in 30 normal and 20 diseased human tissues.

The screening process begins with the production of affin-ity-purified rabbit polyclonal antibodies raised against syn-thetic peptides. These are screened for activity on wax-embedded tissues. LifeSpan’s human tissue bank containsnearly two million specimens with defined pathological diag-nosis and covering all stages of all major diseases. Slides aretraditionally analysed by pathologists experienced in theappropriate disease process but this imposes significant costand time restrictions on the screening process. Beesley endedhis presentation by describing the newly developed Auto-mated LifeSpan Imaging and Analysis System (ALIAS), whichprovides a fully automated assessment of marker localisationand quantification in a high-throughput format.

8. Target validation through gene disruption

Genomic analysis of GPCRs has delivered a large number ofpotential drug targets but there is likely to be a high attritionrate if these enter the drug pipeline without biological valida-tion. S Aparicio (Chief Scientific Officer, Paradigm Thera-peutics Ltd, UK) presented the case for using knockout micestudies to generate data on in vivo mammalian function. Micehave a similar physiology to man and an orthologous (1-to-1matching) genome, whilst embryonic stem (ES) cell technol-ogy has provided reliable tools for the precise genetic manipu-lation required to generate the assay animals. The process atParadigm Therapeutics takes ∼ 35 weeks to generate ahomozygous mutant mouse and the company can producebetween 50 and 75 knockout animals per year. All animals arethen subjected to a staged phenotype analysis that investigatesanatomy, physiology, metabolism, immunology, behaviourand neurology. Some primary screening of animal behaviourhas been automated through the introduction of LABORAS

(Laboratory Animal Behaviour Observation Registration andAnalysis System).

Paradigm’s orphan GPCR targets are named after famousorphans and Aparicio briefly described ‘Kim’ (a target forhypertension) and ‘Bach’ (a target for pain). The final part ofthe talk focused on GPR54. Analysis of knockout mice indi-cated that GPR54 is required for puberty in both males andfemales. The group were also able to uncover a rare humancondition in which mutations in GPR54 cause autosomalrecessive idiopathic hypogonadotropic hypogonadism (IHH).Possible therapeutic applications of GPR54 include sex hor-mone-dependent cancers (prostate, breast, ovarian), preco-cious/delayed puberty and issues related to fertility/libido/contraception. The company has recently identified a poten-tial hit compound that is undergoing further analysis.

An alternative to the costly and time-consuming creation ofknockout mice is the use of RNAi technology to knock downexpression of GPCR targets in selected tissues, and M Hoff-mann (Senior Scientist, Galapagos Genomics BV, The Neth-erlands) described the ScienceSelect platform that Galapagosuses to identify and validate GPCR targets. Adenoviral vectorsare created that express a short hairpin section of siRNA,which leads to degradation of the target mRNA. One advan-tage of using adenoviral delivery is that it can be applied toboth cultured cells and whole animals. The approach wasillustrated by Galapagos’ osteoporosis programme to identifytargets that stimulate osteoblast differentiation. Screening of3325 transcripts identified 212 isolates that were positive inan osteoblast differentiation assay. Of the positives, 43 wereGPCRs and included a receptor previously implicated in oste-ogenesis. As part of their investigation into Alzheimer’s dis-ease, Galapagos have also screened for transcripts that affectβ-amyloid production. Of the 35 positive hits generated inthe screen, 5 were activators of β-amyloid production and 30were repressors and are the subject of further analysis.

9. Selection, validation and prosecution of inflammatory targets

Not all GPCRs are expressed equally in all tissues, and it isreasonable to expect that those receptors that are highlyexpressed in particular tissues are likely to play a key role inregulating the activity of that tissue. This was certainly whatM Hodge (Director of Immunology and Pharmacology, Mil-lennium Pharmaceuticals, USA) found with GPCR-2871,a receptor that is significantly upregulated on T cells. Fur-thermore, receptor expression is elevated in tissue from asth-matic lungs. A concerted drug discovery process was initiatedand led to the identification of a nucleotide-like molecule asthe probable ligand. Mouse model studies revealed that themRNA was increased during experimental allergic inflamma-tion and that knockout animals had reduced mononuclearcell counts in bronchial alveolar lavage following ovalbumin-induced allergic airway disease. In a second study, Hodgebriefly described Millennium’s CCR9 research programme.

Davey

Expert Opin. Ther. Targets (2004) 8(2) 169

10. Structural genomics on G-protein-coupled receptors

Despite much effort, bovine rhodopsin remains the onlyGPCR structure to be solved. It is therefore used as the modelfor all other GPCRs, regardless of whether or not it is appro-priate. K Lundstrom (Chief Scientific Officer, BioXtal,Lausanne, Switzerland) coordinates the MePNet project(Membrane Protein Network), the first global structuralgenomics initiative on membrane proteins. Starting in Sep-tember 2001 and with financial support of US$4 millionfrom approximately 30 pharmaceutical and biotech compa-nies, a group of European laboratories have tried to express,purify and crystallise 100 GPCR targets. Expression has beenobtained in bacteria, yeast and cultured cells but the refolding,solubilisation and crystallisation processes have proved moredifficult and no new structures have been obtained – yet.Expansion of the programme to MePNet2 has recentlyreceived additional industrial funding as well as €10 millionfrom the European Union.

11. The analysis of human G-protein-coupled receptors in fission yeast

Bioinformatic analysis suggests that each human cell expressesbetween 40 and 100 different GPCRs. Given the potentialoverlap between similar receptors, the ability of each receptor tointeract with multiple G proteins and the variety of effectorproteins within the cell, studying GPCR activity in such cellscan be complicated. Attempts to highlight a particular signal-ling pathway are only partly successful and an alternativeapproach may be to study each GPCR and its associated signal-ling machinery in isolation. This is the approach that wasdescribed by J Davey (Chief Executive Officer, Septegen Ltd,UK). Yeast cells contain only two GPCRs and these can be eas-ily removed to provide a clean background in which to studythe activity of an expressed human receptor. The yeast G α sub-unit can be modified to provide a series of chimeric G proteinsthat resemble the different mammalian G proteins. These chi-meric G proteins exhibit specific binding to their appropriatereceptors (for example, the Gs chimaera interacts with receptorsthat normally function through G, and the Gi chimaera inter-acts with receptors that normally function through Gi) butretain the ability to activate the yeast signalling machinery andlead to expression of a reporter gene to provide simple readoutssuch as colour (β-galactosidase) or growth (nutritional mark-ers). Each yeast strain contains only one GPCR and one G pro-tein and it is possible to investigate each interaction in isolation.This has allowed Septegen to uncover some surprising observa-tions, including examples of where a single receptor can interactwith different G proteins in response to different agonists. Theyeast can be used in a variety of assay formats to identify ago-nists, antagonists and inverse agonists and the robustness of thecells makes them ideal for use with crude tissue extracts as partof a deorphanisation programme.

12. Screening technologies and library design

The application of confocal fluorescence microscopy for high-throughput screening was described by R Heilker (Senior Scien-tist, Department of Integrated Lead Discovery,Boehringer Ingelheim Pharma KG, Biberach, Germany). If thefluorescence signal from a ligand does not depend upon bindingto its GPCR, then the slower diffusion of the vesicles and thehigh number of receptors on the vesicles can be used to distin-guish between free and bound ligand. Confocal microscopy candetect such fluorescence fluctuations in a volume of only 1 fl andcan provide single molecule sensitivity. The receptor is incorpo-rated into large lipid vesicles or VLiPs (virus-like particles) gener-ated from virus-infected cells. Ultra high-throughput screeningof GPCRs was described by B Kalthof (Director of Cell BasedAssay Systems, Molecular Screening Technology, Bayer AG, Ger-many). Using aequorin as an intracellular calcium indicator,ultra-high throughput (uHTS) assays are performed in the1536 well microplate format on a fully automated robotic systemwith a throughput of more than 200,000 compounds per day.

Although much of the meeting concentrated on the biologi-cal aspects of GPCRs there was some inclusion of medicinalchemistry, and three presentations discussed different aspects oflibrary design. P Dean (Chief Scientific Officer, De novo Phar-maceuticals Ltd, UK) described de novo drug design methodsthat have been developed almost entirely from a structure-baseddesign perspective. If available, coordinate data for the targetbinding site is used to create computer algorithms that drive themolecular assembly process. A variety of chemotypes can beproduced that fit the site. A comparison between de novo-gener-ated chemotypes with active compounds shows good corre-spondence. Dean extended the design to a case where detailedinformation about the binding site was not available. A GPCRaminergic library is currently being synthesised. C Barker (Prin-cipal Scientist, Pfizer Global R&D, Sandwich, UK) describedhow analysis of known active compounds can be used to directthe synthesis of GPCR-focused libraries. A Stevens (SectionHead, Computational Chemistry Group, BioFocus, UK) dis-cussed how genomics have impacted on library design, withparticular emphasis on the emergence of chemogenomicapproaches such as the knowledge-based ligand design strategyat Novartis, the physicogenomic approach at 7TM Pharma andthe thematic analysis at BioFocus.

13. Conclusion

GPCRs are valuable drug targets and the recent identification ofmany new orphan receptors will ensure that they remain a majorfocus for pharmaceutical companies. Recent discoveries related toreceptor dimerisation and the possible effects that interactionswith other proteins may have on receptor pharmacology furtherincrease the opportunities for trying to find compounds that con-trol signalling pathways. Fortunately, as discussed at this meeting,we now have more sophisticated and effective tools to help usproduce the next generation of GPCR drugs.

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2. HAKAK Y, SHRESTHA D, GOEGEL MC, BEHAN DP, CHALMERS DT: Global analysis of G protein-coupled receptor signaling in human tissues. FEBS Lett. (2003) 550:11-17.

3. CARRILLO JJ, PEDIANI J, MILLIGAN G: Dimers of Class A G protein-coupled receptors function via agonist-mediated trans-activation of associated G proteins. J. Biol. Chem. (2003) 278:42578-42587.

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AffiliationJohn Davey BSc PhDDepartment of Biological Sciences, University of Warwick, Coventry CV4 7AL, UKTel: +44 (0)2476 524204; Fax: +44 (0)2476 523701;E-mail: J.Davey@Warwick.ac.uk