EUROPEAN DRUG INITIATIVE ON CHANNELS AND TRANSPORTERS

Final Report Summary - EDICT (EUROPEAN DRUG INITIATIVE ON CHANNELS AND TRANSPORTERS)

Executive Summary:
Summary
The European Drug Initiative on Channels and Transporters, EDICT, allies for the first time, partners with world-class expertise in both the structural and functional characterisation of membrane channels and transporters. State-of-the-art facilities and personnel for X-ray Crystallography, Electron Microscopy and Nuclear Magnetic Resonance and the latest throughput technology, provides infrastructure support for scientists characterising channel and transport functions in man and pathogenic microorganisms. Our bioinformatics experts specialising in the molecular modelling of membrane proteins are working with our chemists and industrial partners on specific protein targets chosen for their potential to improve the health of European citizens, increase the competitiveness of European health-related businesses and address global health issues. EDICT will increase knowledge of biological processes and mechanisms involved in normal health and in specific disease situations, and transpose this knowledge into clinical applications. By combining computational and experimental analyses, existing detailed molecular models of channel and transporter proteins, and, importantly, novel structures derived by our partners, are being analysed to identify the critical regions constituting drug targets. These basic discoveries are being translated via in silico and experimental strategies with our chemists and industrial partners into the design of novel drugs that modify activities of the membrane proteins for the benefit of patients. The range of human proteins covered includes aquaporins, potassium channels, anion and cation transporters, neurotransmitter transporters, cation-transporting ATPases and mitochondrial transporters. Structures of bacterial homologues to the human proteins are exploited to inform the studies of their human counterparts.

Partners Involved
University of Leeds, UK; Université de Lausanne, Switzerland; Göteborg University, Sweden; Aston University, UK; Universität Basel, Switzerland; Max-Planck Institute, Germany; FMP Berlin, Germany; Frankfurt am Main, ermany; Universität Zu?rich, Switzerland; ETH Zu?rich, Switzerland; Heinrich-Heine Universität Du?sseldorf, Germany; University of Groningen, Netherlands; Commissariat à l'Energie Atomique, France; Medical Research Council, UK; University of Aarhus, Denmark; Imperial College London, UK; AstraZeneca, Sweden; University of Oxford, UK; Stockholm University, Sweden; Moscow State University, Russia; Hebrew University of Jerusalem, Israel; University of Milan , Italy; Institute for Research in Biomedicine Barcelona, Spain; Helsingin Yliopisto, Finland; Karolinska Institute, Sweden; University of Copenhagen, Demark; Xention, UK; University of Freiburg, Germany

Project Website: http://www.edict-project.eu

Project Context and Objectives:
Period 1
• Target selection has been accomplished, not just for the ABC and mitochondrial transporters due in Y1, but additionally for ion channels and ion-linked secondary active transport systems, metabolite/transporter families, aquaporins and ATPases/PPases as described in Annexe I.
• A membrane protein structure database has been created (TCOMP) as required in Y1 for the Scientific Objective of ‘Advancement of in silico analyses for modelling and structure prediction’, which has commenced as described in Annexe I.
• The annotation system for membrane proteins has been implemented in the web site.
• The following facilities have been established as required in Y1 for the Scientific Objective of ‘Application of leading technologies in structural biology’:
o automated crystallisation methods for membrane transport proteins, ion channels and their complexes;
o rational crystal optimal strategies for the crystallisation of transport proteins, ion channels and their complexes;
o novel crystallisation methods for membrane proteins;
o high through put crystallisation and screening methods;
• The first high expression of a functionally active eukaryotic channel and a transporter have been achieved in Y1 as required for the Scientific Objective ‘Expression of target proteins and of a continuous supply for analyses’.
• Subcloning of all the target cDNAs into various expression vectors has started .
• Crystals of the human Na+,K+-ATPase and fungal H+-ATPases are reproducibly obtained, so the Scientific Objective ‘Integrated structural and functional analyses to validate potential drug targets’ has been started early for this group of proteins.
• Subcloning of all the target cDNAs into various expression vectors has commenced as the first step in for the Scientific Objective ‘Expression of target proteins and of a continuous supply for analyses’.
• Overexpression and purification of target proteins is already achieved in Y1 for some of the targets as described in the database.
• Initial crystals of transport proteins, ion channels and their complexes is already achieved in Y1 for some of the targets as described in the database.
• Docking models of P-ATPases has been started in Y1.
• Project website and mailing lists heve been created as required in Y1.
• Recruitment of EDICT researchers has been accomplished as required in Y1.

Period 2
• All the functionalities of molecular modelling for the Scientific Objective of ‘Advancement of in silico analyses for modelling and structure prediction’ as described in Annexe I, have been accomplished and implemented in the web site.
• Subcloning of all the target cDNAs described in Annexe I into various expression vectors is completed.
• Functionally active eukaryotic channels and transporters are routinely expressed at levels for both functional and structural analyses required for the Scientific Objective ‘Expression of target proteins and of a continuous supply for analyses’.
• The following facilities are in continuing development and use as required for the Scientific Objective of ‘Application of leading technologies in structural biology’ as illustrated in a number of EDICT publications:
o automated crystallisation methods for membrane transport proteins, ion channels and their complexes;
o rational crystal optimal strategies for the crystallisation of transport proteins, ion channels and their complexes;
o novel crystallisation methods for membrane proteins;
o high through put crystallisation and screening methods;
o application of microfocus x-ray beamlines;
o electron crystallography;
o particle imaging and analysis by electron microscopy;
o high field solution state NMR;
o solid state NMR;
o cell-free expression of membrane proteins.
• Novel structures of both channel and transporter proteins are achieved (and published) so the Scientific Objective of ‘Integrated structural and functional analyses to validate potential drug targets’ has been implemented and continues with more targets.
• From these successful structural studies progress has resulted towards the Scientific Objective of the ‘Identification of protein domains for binding of drugs’.
• Diffracting crystals of a variety of additional target channel and transport proteins are now reproducibly obtained in several laboratories, towards expansion of the Scientific Objective of ‘Integrated structural and functional analyses to validate potential drug targets’
• A very substantial number of virtual ‘hits’ for a range of both channel and transporter targets of established structures have been generated. Sub-sets of these hits have been assayed and effective compounds identified towards the Scientific Objective of ‘Virtual design and experimental tests of pharmaceutical candidates to generate hits.’

Period 3
• All the functionalities of molecular modelling for the Scientific Objective of ‘Advancement of in silico analyses for modelling and structure prediction’ as described in Annexe I and accomplished previously have been routinely exploited by other Partners.
• Functionally active eukaryotic channels and transporters have continued to be routinely expressed at levels for both functional and structural analyses required for the Scientific Objective ‘Expression of target proteins and of a continuous supply for analyses’.
• The following facilities have been exploited in structural studies of over 50 target proteins as required for the Scientific Objective of ‘Application of leading technologies in structural biology’ as illustrated in a number of EDICT publications:
o automated crystallisation methods for membrane transport proteins, ion channels and their complexes;
o rational crystal optimal strategies for the crystallisation of transport proteins, ion channels and their complexes;
o novel crystallisation methods for membrane proteins;
o high through put crystallisation and screening methods;
o application of microfocus x-ray beamlines;
o electron crystallography;
o particle imaging and analysis by electron microscopy;
o high field solution state NMR;
o solid state NMR;
o cell-free expression of membrane proteins.
• More novel structures of both channel and transporter proteins have been achieved and published during the period using X-ray crsytallography, electron microscopy and nuclear magnetic resonance. Hence the Scientific Objective of ‘Integrated structural and functional analyses to validate potential drug targets’ has been implemented for over twenty proteins already and will continue with more targets.
• From these successful structural studies more progress has resulted towards the Scientific Objective of the ‘Identification of protein domains for binding of drugs’, resulting in at least twelve successes so far.
• Diffracting crystals of a variety of additional target channel and transport proteins are continuing to be obtained in several laboratories, towards future expansion of the Scientific Objective of ‘Integrated structural and functional analyses to validate potential drug targets’
• A very substantial number of virtual ‘hits’ for a range of both channel and transporter targets of established structures have been generated. Sub-sets of these hits have been assayed and at least twelve effective compounds identified towards the Scientific Objective of ‘Virtual design and experimental tests of pharmaceutical candidates to generate hits.’
• For three of the targets, publication of the nature of ‘hit’ compounds has already taken place or is in process.
• For five clinical targets, hit/lead compounds developed in EDICT are being pursued by translational development and/or spin-off companies.
• A final General Assembly (GA) was held in Portugal (Tavira), fulfilling a total of five GAs, the others being in Spain (Madrid), Tallin (Estonia), Eden Roc (Spain) and Dusseldorf (Germany).

Project Results:
THE SCIENTIFIC AND TECHNICAL OBJECTIVES AND THEIR OUTCOMES

Call text
HEALTH-2007-2.1.1-5: Structure-function and analysis of membrane transporters and channels for the identification of potential drug target sites
The project should focus on applying high throughput approaches for structure-function analysis of membrane transporters and channels. This large multidisciplinary and integrated effort should use different technology for structure determination (NMR, X-Ray crystallography and Electron microscopy) and should also combine computational and experimental analyses. This should help to identify potential new target sites for drugs to alleviate the burden of the diseases involving membrane-transporters and channels.

The overall aim of EDICT
Determination of the structure-function relationships of protein channels and transporters of clinical significance in biological membranes and initial drug candidate design and screening.

To achieve this aim there were six key scientific and technical objectives (SO):
SO1. Advancement of in silico analyses for modelling and structure prediction of membrane proteins.
SO2. Expression of target proteins and provision of a continuous supply for analyses.
SO3. Application of leading technologies in structural biology - X-ray crystallography (XRC), electron microscopy (EM) and nuclear magnetic resonance (NMR) - underpinned by high throughput screening (HTPS) to achieve full structural determination
SO4. Identification of protein domains for binding of drugs.
SO5. Integrated structural and functional analyses to validate potential drug targets.
SO6. Virtual design and experimental tests of pharmaceutical candidates to generate ‘hits’.

In order to achieve these objectives, a protocol of activities was established as illustrated in the PIRT chart contained in the supporting Annex. In brief, the operation was managed by the Coordinator and Scientific Steering Committee in a Work Package (WP12), which oversaw first the establishment of a web site for recording the target database, and then monitored and managed progress with Milestones and Deliverables throughout the project. After identification of the targets, proteins were modelled as far as possible with the then-available data, and selections were made of the proteins for functional and structural analyses. These proteins were then produced on a smaller scale for functional studies, particularly aimed at defining ligand specificity with a view to later design of drugs. In parallel, judiciously chosen proteins were produced on an appropriate scale for structure determination by X-ray crystallography (XRC), NMR, or electron microscopy (EM). Establshment of the 3d structure at resolutions below 3A was the aim, as the resulting knowledge of the structure-activity relationship (SAR) would inform the rational design of therapeutic candidates by the bioinformatacists and chemists associated with the project. Synthesis and/or purchase of these candidate compounds could then initiate iteration through the functional screens established in the parallel studies to identify ‘hits’, followed by structure determination of the protein-ligand complex and in silico ligand design to convert ‘hits’ to ‘leads’.

In order to exploit the phenomenal range of expertises covered by the Partners, the target proteins were grouped into six Work Packages WP 2-7, each containing experts in a particular evolutionary group of channels or transporters. In parallel, technical advances were implemented by WP1 and WP11 in protein modelling, ligand design and chemical syntheses, by WP8 in XRC, by WP9 in EM, and by WP10 in NMR. Throughout the project these technical advances were exploited horizontally by WP2-7 in elucidating the SAR of the target proteins.

The outcomes of EDICT are evidenced by the publications lists

The Work Packages
Each WP had a set of Objectives and Tasks to accomplish. In the course of the EDICT project virtually all of these were fulfilled and these outcomes are described in detail in the three Periodic Reports and summarised in the descriptions of the Deliverables, which are not yet in the public domain. Accordingly, here we summarise these Objectives and Tasks and follow this with a summary Table of the target structures achieved by the different WPs and the outcomes published so far in over 140 papers. In brief, WP2-7 have already determined structures of over twenty membrane proteins, with additional structures of conformational intermediates and protein-ligand complexes. This has resulted in, so far, eight hit/lead compounds developed in collaborations with WP1 and WP11, three of which are being published and five of which are being taken forward in drug-design programmes; the IPR involved for these is, of course, protected.

Work Package 1 - Database, annotations and structural modelling
Work Package leader: Stockholm University (Elofsson, von Heijne, Lindahl)
Other Work Package partners: All others
Objectives
• Develop the TCOMP database for classification and prioritisation of potential targets
Provide the interface for expert annotations of targets and proteins
• Continually update classification of ion-channels and transport genomes
• Provide accurate automatic 3D modelling of ion-channels and transport proteins
• Develop prediction of re-entrant regions in transporters/ channels
• Provide family wide computational cavity calculations
Tasks
1. Development of a structure-based classification of membrane channel and transporter proteins database, including ligand binding and drug susceptibilities.
2. Interface for annotation of potential target proteins.
3. Detection of new potential targets by searching for ion channels and transporters.
4. Identification and classification of re-entrant loops in channels and transporters by hidden Markov models.
5. Creation of accurate homology models of channels and transporters for potential use in ligand and drug design.
6. Computational generation of a cavity databases.

Work Package 2 - Ion channels and secondary active ion transporters
Work Package leader: AstraZeneca (Snijder)
Other Work Package partners: University of Zurich (Dutzler), Medical Research Council (Tate), Oxford University (Ashcroft, Sansom), Moscow University (Sokolova), Hebrew University Jerusalem (Kanner), University of Milan (Moroni), Copenhagen University (Gether), Xention (Madge)
Objectives
• Overexpress and purify eukaryotic members of different ion channel families [ClC chloride channels and transporters, voltage-gated cation channels, ATP-sensitive K+ channels and inward rectifier K+ (Kir) channels].
• Overexpress and purify prokaryotic and viral homologues of ion channels (KirBac channels, K+- channels) and secondary active ion transporters [cation chloride cotransporters (CCC), SLC26 transporters].
• Crystallize and determine the structures of prokaryotic and eukaryotic ion channels and secondary ion transporters, and of their regulatory domains.
• Characterize the function and pharmacology of ion channels and secondary ion transporters electrophysiology, transport assays and yeast complementation.
• Predict the interaction with small molecule effectors in silico.
• Screen for small molecule effectors and characterize their interactions on a structural and functional level.
Tasks
1. Overexpression and purification of eukaryotic ion channels and transporters
2. Overexpression and purification of prokaryotic and viral homologues of ion channels and transporters
3. Functional characterisation
4. Crystallization and structure determination by X-ray crystallography and structural investigations by electron microscopy and NMR
5. Computational analysis

Work Package 3 – Human metabolite/ion transporters and their homologues
Work Package leader: University of Leeds (Baldwin)
Other Work Package partners: University of Leeds (Henderson), University of Lausanne (Thorens), Max-Planck Institute (Michel, Kühlbrandt), Imperial College London (Iwata, Byrne, Cameron), Hebrew University Jerusalem (Padan), IRB (Palacin), Karolinska Institute (Nordlund), Freiburg University (Hunte)
Objectives
• To produce mg quantities of functional mammalian metabolite/ion transporters and their homologues.
• To implement conventional and high-throughput (HTP) transport and binding assays to facilitate structure-function analysis and screening of wild-type and mutant transporters.
• To utilize in vivo approaches including use of transgenic mice, to assess the physiological relevance of selected transporter families and their potential as drug targets.
• To determine the structures of at least 5 of these proteins at a resolution of at least 3.5 Å
• To provide structural information for in silico screening of ligand libraries to identify lead candidates for development of novel drugs.
• To identify and validate putative ligand/drug binding sites by mutagenesis, in vitro assays and in vivo approaches.
• To screen selected ligands in vitro and in vivo.
Tasks
1. Overproduction and purification of human metabolite/ion transporters and their homologues
2. Structure-Function Analysis and Assays
3. 3-D crystallography of prokaryote and eukaryote transporter
4. Identification and validation of permeant/drug binding sites

Work Package 4 – Structural, functional and computational analyses of human aquaporins
Work Package leader: Aston University (Bill)
Other Work Package partners: Goteborg University (Neutze), Basel University (Engel), Max-Planck Institute (de Groot),
Objectives
• Overproduce hAQP1, hAQP2, hAPP3, hAQP4, hAQP5, hAQP6, hAQP7, hAQP8 and hAQP9
• Crystallise and solve the X-ray or electron diffraction structures of at least four of these aquaporins to high resolution (> 3 Å resolution).
• Exploit high-resolution structures of human aquaporins to screen in-silico millions of virtual compounds for binding and inhibition and to optimise selected hits together with WP11.
• Screen top-ranking compounds in available in vivo aquaporin screening assays.
• Develop novel high-throughput assays for testing inhibition in additional human aquaporins inhibition.
• Co-crystallise and recover high resolution structures of human aquaporins in complex with validated inhibitors.
• Iteratively repeat the in-silico screening, assay validation, and co-crystallisation so as to rationally optimise candidate drugs.
Tasks
1. Protein overproduction and purification.
2. Development of reliable high-throughput assays for screening the inhibition of human aquaporins.
3. Crystallisation of human aquaporins for structural determination.
4. Modelling human aquaporins as drug target.
5. In-silico inhibitor design using structure-based computational methods.
6. In vivo validation of predicted inhibitors in cell-based assays.

Work Package 5 – Structural and functional analysis of ABC transporters
Work Package leader: Goethe University Frankfurt (Tampé)
Other Work Package partners: Basel University (Engel), ETH Zurich (Locher), Heinrich-Heine University Dusseldorf (Schmitt), Groningen University (Poolman), Stockholm University (Elofsson, von Heijne, Lindahl)
Objectives
• High throughput cloning, over-expression and purification of clinically relevant, human ABC transporters and of their prokaryotic homologues.
• Determine the functional properties of well-expressing transporters in terms of the specificity, the binding, and the translocation of substrates as well as ATP binding and hydrolysis.
• Identify interaction partners and modulation cellular networks
• Structure determination (2D and 3D crystallography) of ABC transporters.
• Mechanistic understanding of the substrate transport cycle of ABC transporters.
• Homology modelling of human ABC transporters using available structures of prokaryotic homologues and mapping drug-binding sites onto structures of ABC transporters.
Tasks
1. Protein overproduction and purification.
2. Functional characterization and assays.
3. Structure determination.
4. Map potential drug/inhibitor-binding sites of clinically relevant ABCs.

Work Package 6 – Structural, functional and computational analyses of P-type, F-type and PP-ATPases
Work Package leader: University of Helsinki (Goldman)
Other Work Package partners: Max-Planck Institute (de Groot), MRC (Walker), Aarhus University (Nissen), Stockholm University (Elofsson, von Heijne, Lindahl)
Objectives
• Overproduce human Na+,K+-ATPases (?1-?3 isoforms), fungal P-type H+-ATPases, trypanosomal H+-PPases and archaebacterial H+-PPases, bacterial (including E. coli) F-ATPases. Mitochondrial ATPases will be isolated from natural sources of mitochondria using a generic affinity chromatography method.
• Crystallise and determine the X-ray diffraction crystal structures of at least two of these targets in complex with identification of putative inhibitor binding sites typically by including ligands of medical interest such as inhibitors and modulators.
• Exploit atomic structures of human ATPase transporters, fungal H+-ATPases and trypanosomal H+-PPases to screen in-silico millions of virtual compounds for binding, and to optimise selected hits/leads.
• Co-crystallise and recover structures of bovine F-ATPase as a model for human ATPase. Same for bacterial F-ATPase in complex with ligands, similar for fungal P-type H+-ATPase.
• Iteratively repeat the in-silico screening, target validation, and co crystallisation so as to rationally optimise a candidate lead compound.
Tasks
1. Overproduction and purification of ATPase/PPase transport proteins and their homologues
2. Structure-Function analysis and assays
3. Determination of structures of the ion-linked ATP/PPase transport proteins by X-ray crystallography
4. Map potential drug/inhibitor-binding sites

Work Package 7 – Structural, functional and computational analyses of mitochondrial transport proteins
Work Package leader: Medical Research Council (Kunji)
Other Work Package partners: CEA (Pebay-Peyroula), Medical Research Council (Walker)
Objectives
• Develop procedures for the overproduction of mitochondrial transport proteins to high-levels.
• Crystallise and solve structures of at least two structures of mitochondrial carriers or intermediates by X-ray or electron crystallography.
• Exploit high-resolution structures of mitochondrial carriers to screen in-silico millions of virtual compounds for binding and inhibition and to optimise selected hits.
• Develop high-throughput assays for testing inhibition or activation of mitochondrial carriers, including the uncoupling protein.
• Co-crystallise and recover high resolution structures of mitochondrial carriers in complex with validated inhibitors and regulators.
• Iteratively repeat the in-silico screening, assay validation, and co-crystallisation so as to rationally optimise candidate drugs.
Tasks
1. Protein production
2. Assays
3. Determination of structures of the mitochondrial transport proteins by Xray crystallography and electron microscopy.
4. Map potential drug/inhibitor-binding sites.

Work Package 8 – X-ray crystallography methods for structure analysis of membrane channels and transporters
Work Package leader: Freiburg University (Hunte)
Other Work Package partners: Goteborg University (Neutze), Max-Planck Institute (Michel, Kühlbrandt), Zurich University (Dutzler), ETH Zurich (Locher), CEA (Pebay-Peyroula), Medical Research Council (Kunji, Tate), Aarhus University (Nissen), Imperial College London (Iwata, Bryne, Cameron), University of Milan (Moroni), Helsinki University (Goldman)
Objectives
• To establish high-throughput 3D crystallization (preliminary and optimization screening) of transport proteins, ion channels and their complexes for X-ray crystallography
• To establish high-quality X-ray data collection strategies for transport proteins, ion channels and their complexes.
• To obtain diffraction quality crystals of transport proteins, ion channels and their complexes for structural analysis.
• To determine the structures of transport proteins, ion channels and their complexes.
Tasks
1. Crystallisation of membrane transport proteins, ion channels and their complexes.
2. Crystal optimization for ion channels, membrane transport proteins, and their complexes.
3. X-ray data collection from crystals of membrane transport proteins, ion channels and their complexes.
4. Structure and mechanisms of membrane transport proteins, ion channels and their complexes.

Work Package 9 – Electron microscopy methods for structure analysis of membrane channels and transporters
Work Package leader: Max-Planck Institute (Kühlbrandt)
Other Work Package partners: Basel University (Engel), Medical Research Council (Kunji, Tate), Moscow (Sokolova)
Objectives
• High-throughput 2D crystallisation of channels and membrane transport proteins for electron crystallography
• Structure determination by electron crystallography
• Analysis of different functionally relevant states
• Analysis of structures of large membrane protein complexes by cryo-EM and single-particle image processing
• Development of software for crystallographic image processing
• Evaluation of electrostatic phase plate and electron detectors for high-resolution cryo-EM of membrane proteins
Tasks
1. 2D crystallisation of membrane proteins
2. Membrane protein structure and mechanisms by electron crystallography
3. Cryo-EM and image processing of membrane protein complexes
4. Instrumentation and software development

Work Package 10 – NMR technologies in the structural and functional analysis of membrane channels and transporters
Work Package leader: Goethe University Frankfurt (Dötsch, Bernhard)
Other Work Package partners: University of Leeds (Henderson), University of Berlin (Oschkinat)
Objectives
• Development of liquid state and solid-state NMR techniques for the structural calculation of larger ?-helical polytopic transporters.
• Cell-free expression for bacterial and eukaryotic transporters and porins.
• Structure determination of transporters and porins in detergent solution and natural lipid environment.
• Structure NMR-based ligand binding studies of transporters.
• Thermal melt, NMR and X-ray screening of fragment libraries
• Functional characterization of the ABC-transporters ArtMP in natural lipid environment by solid-state NMR
Tasks
1. Development of liquid state and solid-state NMR techniques for the investigation of transporters and channels.
2. Screening of membrane protein targets for structure determination by high-throughput sample preparation
3. Structure determination of transporters
4. Functional characterization of transporters using NMR
5. Low affinity ligand screening methods

Work Package 11 – Design of lead candidates for therapeutic ligands
Work Package leader: Max-Planck Institute (de Groot)
Other Work Package partners: University of Leeds (Fishwick & Johnson), Stockholm University (Elofsson, von Heijne, Lindahl), Xention(Madge)
Objectives
• High-throughput virtual screening assay for efficient hit identification for membrane proteins
• Full flexible molecular dynamics based docking toolbox for hit optimisation
• Inhibitor design targeted at the specific membrane proteins listed in the task list below.
Tasks
1. vHTS and fragment-based molecular design
2. Affinity predictions
3. Hit refinement
4. Efficient and accurate free energy simulation methods
5. Specific targets

Potential Impact:
THE IMPACT OF EDICT

The specific call for this project did not list expected impacts. However, the preamble for the overall section ‘2.1 Integrating biological data and processes: Large scale data gathering, systems biology’ had a number of expected impacts and the section below demonstrates how EDICT met the relevant expected impacts.

Expected impact: ‘Support European research excellence in fields related to genomics and proteomics by increasing understanding of key biological processes’

EDICT contribution to impact
The field of membrane protein research is still in its infancy on a global scale – the number of structures known is very low, particularly for those proteins linked to serious disorders. EDICT offers Europe the opportunity to accelerate significantly knowledge of protein structure and function on a scale unparalled in Europe. Not only would the structures themselves provide a significant boost to the understanding of biological processes but the functional data generated would provide a resource, all combined within the significant TCOMP database, not yet seen in European membrane protein research.

For an EC investment of just under €12m EDICT has already published 144 scientific and technical papers ‘related to genomics and proteomics’ that ‘increase understanding of key biological processes’ with at least 14 more in press. At least 29 structures of membrane proteins have already been completed by XRC, EM and NMR and there will be more. A key example of the integration of EDICT partners is the paper in Nature Biotechnology [Bill, R.M. Henderson, P.J. Iwata, S., Kunji, E.R. Michel, H., Neutze, R., Newstead, S., Poolman, B., Tate, C.G. and Vogel, H. (2011) Overcoming barriers to membrane protein structure determination. Nat Biotechnol 29 (4): 335-340] between seven different partners and cited already 26 times.

Steps required to bring about impact
These impacts were felt through publications as soon as EDICT commenced– it brought together such a significant number of leading membrane protein researchers in Europe, across all the major protein families, exploiting technologies that guaranteed results in terms of structure and function studies that became available very early in the project in 2008. One example is the paper in Science [Weyand, S. and Shimamura, T. and Yajima, S. and Suzuki, S. and Mirza, O. and Krusong, K. and Carpenter, E.P. and Rutherford, N.G. and Hadden, J.M. and O'Reilly, J. and Ma, P. and Saidijam, M. and Patching, S.G. and Hope, R.J. and Norbertczak, H.T. and Roach, P.C. and Iwata, S. and Henderson, P.J. and Cameron, A.D. (2008) Structure and molecular mechanism of a nucleobase-cation-symport-1 family transporter. Science 322 (5902): 709-713] between two partners and since cited over 100 times
This allowed researchers in connected clinical and academic fields to advance their understanding of the disease process during the lifetime of EDICT.

Expected impact: ’Provide the foundation for more extensive functional studies’

EDICT contribution to expected impact
There was no doubt that EDICT would make major contributions to understanding functions of membrane proteins in man as well as understanding structure. An integral part of establishing protein structure by expression in a variety of host cells was the confirmation of function. Functional studies must be undertaken before a structure can be used for drug design and to establish understanding of a protein’s role in a disease process. Extensive work on function was a central feature of EDICT as evidenced in the topics of the publications (see list) and their impacts measured by citations from sources outside EDICT.

Steps required to bring about impact
The steps required to bring out this impact were undertaken within the project, particularly through publications and presentations at conferences. These data from EDICT Partners but published and presented outside the consortium boosted significantly knowledge of function and structure within the lifetime of the project again evidenced by the numbers of citations in publications from outside EDICT. Over and above this EDICT papers of high impact received wider publicity through articles in the general scientific press including interviews of EDICT scientists, and even on a BBC radio programme ‘Material World’.

Expected impact ‘Aid design of drugs and treatments’ and ‘Detailed analysis of membrane-transporters structure/function will allow identification of critical regions…that constitute important drug targets and serve as template for structure-based drug design’

EDICT contribution to expected impact
As can be seen from the work package reports, drug design was undertaken within EDICT for some proteins from the first six months, fulfilling the expected impact immediately. The EDICT project laid the foundation for more extensive drug design through its capabilities in bioinformatics, medicinal chemistry, and structural biology as additional binding site structures became known, which occurred throughout the rest of the project. These included targets in humans, parasites and pathogenic microorgamisms.
For some of these targets, e.g. the aquaporin of the malarial parasite, the knowledge gained is to be published. For the majority involving three SME’s and MRCT, the IPR is currently protected.

Steps required to bring about impact
Initial drug design will be undertaken within the project itself as already stated. Steps required to rapidly expand drug design as the project draws to a close were presented in section B3.2 of the Annexe, but can be summarised here as: 1) identification and allocation of knowledge ownership within the consortium; 2) agreement of exploitation by all partners; and 3) identification of all exploitation routes for knowledge outside the consortium. This third step will be the point at which partners will open all exploitable routes not being pursued by themselves to external parties, primarily companies.

Expected impact ‘Each project should have a well-developed bioinformatics part’

EDICT contribution to expected impact
The database, TCOMP, generated in WP1 formed the central work tool for the consortium as partners were able to enter structural data as they were generated and partners involved in modelling were able to predict structures and function not possible before. These data then fed back into the structure determination process to allow more rapid and accurate structure determination, especially of bound ligands.The data were of course, the foundation of initial drug design within the project.

Requirement for a European approach
As was obvious from the list of partners and description of work, no one area of expertise exists in any one country. A project of this scale could only be carried out on a European level and it was fortunate that Europe had such an excellent foundation within membrane protein research, including the Nobel Prize winners John Walker and Hartmut Michel, who were leading members of this consortium.

EDICT contribution to expected impact
It was also gratifying to include over 60 young researchers with a substantial majority of women, in their fields from across Europe. These researchers operated on an international level during the EDICT project and their training cemented membrane protein research as a truly European rather than national expertise.

Plan for the use and dissemination of foreground resulting from the project
With a project of this significance, it was essential that dissemination was high profile, regular and captured all existing initiatives worldwide. Dissemination mechanisms included:

Project website
This formed a central tool for the project and consortium, not least because it hosted the database constructed in WP1 of the project. The database were fully interactive for consortium members and the website provided the interface for members to enter and obtain structure function data. Aside from a working tool for consortium members, it now provides a permanent data source for the project.

The project website included:
• Database (password protected)
• Clear project summary
• Member list
• Publications

Presentations at meetings
This is a well-established mechanism for dissemination. It was one of the primary mechanisms for dissemination to the global scientific community. Even before the launch of EDICT, each of the principal investigators was invited to present data at between 5 and 50 meetings a year around the world (even higher for researchers such as John Walker and Hartmut Michel, the Nobel Prize holders) and with the significant number of researchers in the project, dissemination potential is enormous.

Peer reviewed publications
All non-confidential data were published in peer reviewed journals. The consortium maintained an impressive track record by publishing in journals such as Nature, Science, EMBO Journal and Proceedings of the National Acadeny of Sciences USA – the most significant publications within this field. The following is the Foreword drafted by the members of the Advisory Board of EDICT to precede contributions to be published in a Special Volume of Molecular Membrane Biology devoted to reviewed articles from EDICT partners.

“The European Drug Initiative for Channels and Transporters - the EDICT Project
Genome sequencing exercises revealed that about 30% of all cellular proteins are membrane located or associated. The study of the structures and functions of these membrane proteins has proven to be extremely difficult as indicated by the fact that the unique structures of more than 20,000 soluble proteins have been solved compared with fewer than 400 of membrane transport and channel proteins. These membrane proteins perform crucial functions for every biological cell, ranging from signal transduction, generation of metabolic energy, transport of ions and essential solutes to the inside, excretion of waste products to the outside, and communication with the external milieu. The activities of a significant number are implicated in disease, and, being readily accessible to the outside of a cell, they are ideal targets for compounds that can modify their activities. In view of the rapidly increasing resistance of micro-organisms against antibiotics, and of cancer cells against chemotherapy, the development of new drugs is urgently needed since such diseases affect hundreds of millions of human beings. In order to be able to design these drugs, understanding the molecular structure and the mechanism of action of relevant target membrane proteins is of immense value. That has proven to be a very difficult requirement. General protocols to obtain sufficiently large quantities of solubilised stable membrane proteins that crystallize and diffract to a high resolution are not available. Each protein requires its own protocol, which explains the slow progress in understanding the structure and function of these proteins.
The Seventh Framework Programme of the European Community supported with 12 million Euro the European Drug Initiative on Channels and Transporters (EDICT) project for a period of four years. The aim of EDICT was the determination of the structure-function relationships of clinically significant membrane protein channels and transporters with special emphasis on initial drug design and screening. This project brought together prominent European research groups working on membrane proteins: structural biologists, molecular geneticists, physiologists, pharmacologists, bioinformatics specialists and industrial partners. No fewer than 25 universities and research institutes in 10 European countries plus Israel and the Russian States plus two industrial partners, comprising a total of 40 research groups, participated in EDICT. The coordination of the project was carried out excellently by Prof. Peter Henderson of the University of Leeds, UK.
EDICT started in 2008 and will formally end in July 2012. Owing to the intense interactions between the partners, including five General Assemblies in Germany, Spain, Estonia and Portugal and many smaller meetings and staff exchanges, the results obtained in EDICT are impressive. Advanced tools have been developed and applied for over-expression, solubilisation, reconstitution in artificial membranes, functional analysis, crystallization, structural analysis and ligand design, and these tools have been exchanged broadly between the partners and published. More than 20 high resolution structures of 16 membrane proteins have already been determined while the analyses of several more membrane proteins are at an advanced stage in the pipeline. Potential modulators or inhibitors of function have been tested for most of these proteins with several successful and promising hits already under further development.
The current issue of Molecular Membrane Biology devoted to EDICT clearly demonstrates that collaborative research between scientists with different disciplines and experiences is required to tackle effectively extremely difficult problems such as membrane proteins. The high-quality articles in this special issue, edited by EDICT partners Bernadette Byrne and Roslyn Bill, illustrate that scientific support from the European Community can substantially and successfully contribute to the advancement of European Health.
EDICT Scientific Advisory Board
Wim G. J. Hol, University of Washington, Seattle, WA, USA
Wil N. Konings, University of Groningen, Groningen, The Netherlands.”

Researcher Exchange Scheme
During the EDICT 12 project researchers successfully applied to the Researcher Exchange Scheme. Most made single visits and one researcher, Kamil Gotfryd, used his allocation to make a series of visits to Poul Nissen’s laboratory in Aarhus. The supporting reports from each of the researchers about their visits are recorded in the EDICT Final Project Report.

Spin-off companies
Two spin-off companies were created as a result of EDICT. The third company listed below was not a direct result of the project but the work performed in EDICT contributed to the improvement of the methodology of the patented product.
Partner 20, Stockholm University. Professors Arne Elofsson and Erik Lindhahl created a company called BinaryBio AB after obtaining results within the EDICT project. The company specialises in bioinformatics, molecular dynamics and high performing computing. BinaryBio currently has two full time employees and are in the process of preparing an FP7-Eurostar application with the EDICT SMe partner, Xention. http://www.binarybio.com/
Partner 16, Aarhus University. Professor Poul Nissen co-founded the company in 2009. Pcovery is a leader in structure based drug development against membrane protein drug targets with a broad portfolio of innovative technologies, drug targets and chemical products. The company currently employs six people.
Partner 14, MRC. Dr Edmund Kunji, has a start-up company called Cytoprom, which arose from a patent generated several years ago for Europe. The world-wide patent application process is still ongoing. The patents are for a bubble excluder to aid the monitoring of aerated cultures in fermentation. Although not directly a result of the EDICT project, the fermentations done for the EDICT project, were used to improve the methodology. The company has three employees.

List of Websites:
The EDICT website address is http://www.edict-project.eu

Coordinator Contact Details
Scientific Coordinator, Professor P J F Henderson. Project Manager, Mrs Helen Inman
p.j.f.henderson@leeds.ac.uk h.inman@leeds.ac.uk
University of Leeds
6.108 Astbury Building
Leeds, LS2 9JT
UK

Final Report Summary - EDICT (EUROPEAN DRUG INITIATIVE ON CHANNELS AND TRANSPORTERS)

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