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A proteomics and structural genomics approach to therapeutic targeting of glycosaminoglycan-binding proteins

   
Project

QLK3-2001-01976

Cell factory area

3.1.3

EU Contribution

2 027 836 Euro

Duration

36 months

Type

Research project

Starting date

01-10-2001

Keywords
angiogenesis GAG ligand library
glycosaminoglycan (GAG)
proteomics
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ABSTRACT
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Glycosaminoglycans (GAGs) are polysaccharides which are involved in many (patho)physiological processes such as wound healing, cell differentiation, inflammation, cancer, and angiogenesis. By concentrating bioinformatical, biochemical, synthetic chemistry and structural biology efforts on establishing two comprehensive databases, one containing all proteins which bind to GAG ligands, the other containing all the ligands themselves, maximum information concerning the sequence and structural relationships among these molecules will be obtained.

Based on a classification of the GAG-binding proteins, the 3-D structures of selected protein/GAG ligand complexes will be experimentally determined and the remaining complex structures will be predicted. The structural relationships obtained will aid the design and synthesis of GAG analogues and thus the development of pharmaceutical lead compounds directed against selected GAG-binding protein targets involved in GAG-mediated diseases.

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OBJECTIVES
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Since GAGs exert their function via specific binding to proteins, the major objective of this project is to understand the structure-function relationships of GAG-binding proteins with respect to their specific GAG ligands. For this purpose, a proteomic approach towards protein sequence and structure relationships and a glycomic approach towards GAG sequence and structure interrelations will be taken. Both of these approaches are intended to yield information that will enable pharmaceutical inhibitor design of GAG-mediated diseases.

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DESCRIPTION OF THE WORK
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In the initial phase of the project we will focus on the development of a GAG-binding protein database and a GAG ligand library. Members of the protein database will be identified by numerical sequence comparison and fold recognition. In addition, we will experimentally search for further GAG-binding proteins by affinity chromatography. A GAG ligand library will be obtained by three experimental approaches - desulphation and enzymatic resulphation of size-defined heparin oligosaccharides, chemical synthesis, and affinity chromatography - and will be extended by numerical prediction methods. The members of the databases will be classified according to their structural relationships and the structures of a few members of each GAG-binding protein class in complex with their specific GAG ligands will be determined by X-ray crystallography. The remaining proteins will be subjected to numerical structure prediction. In addition, biophysical characterisation of the protein/ligand interactions will be undertaken in order to obtain dissociation constants as well as on- and off-rates for the interactions. A prerequisite for performing structural and biophysical experiments is the immediate access to sufficient amounts of pure proteins. Therefore, subcloning into suitable protein expression systems and protein production and purification will be an essential part of the project. After one year of collecting and generating information concerning GAG-binding proteins and their ligands, targets will be selected for inhibitor development. This selection will depend on the research and development strategies of our industrial partners but also on the amount of structural information available for the protein targets and their ligands. The number of structural homologues in the databases represents valuable information for inhibitor design in our pharmacoproteomic approach.

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DELIVERABLES
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Three milestones will be delivered in the course of the project:

  • A comprehensive database containing as many GAG-binding proteins as possible (presented after the first year and to be extended throughout the project).
  • A comprehensive GAG ligand library (delivered at the end of year 2 since less information is available to date about specific GAG ligands).
  • Synthesis of lead compounds representing GAG analogues to be further developed for the treatment of GAG-mediated diseases. In addition, reports describing the major achievements will be delivered on top of the yearly reports.

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CONSORTIUM
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COORDINATOR
  Andreas J. Kungl
Institut für Pharmazeutische Chemie
Universität Graz
Universitätsplatz 1
8010 Graz, Austria
Tel: +43 3163805373 and +43 6642402753
Fax: +43 316382541
andreas.kungl@kfunigraz.ac.at

PARTNERS
  John T. Gallagher
Department of Medical Oncology
Paterson Institute for Cancer Research
Wilmslow Road
M20 4BX Manchester, United Kingdom
Tel: +44 1614463201
Fax: +44 1614463269
JGallagher@picr.man.ac.uk

Maurice Petitou
CV/T
Sanofi-Synthélabo
195, Route d'Espagne
31036 Toulouse Cedex, France
Tel: +33 561162390
Maurice.Petitou@sanofi-synthelabo.com

Robert Huber
Max-Planck-Institut für Biochemie
Am Klopferspitz 18 a
82152 Planegg-Martinsried, Germany
Tel: +49 8985782677/8
Fax: +49 8985783516
huber@biochem.mpg.de

Antal Rot
Novartis Forschungsinstitut
Brunnerstraße 59
1235 Wien, Austria
Tel: +43 186634234
antal.rot@pharma.novartis.com
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