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Volume 2
     

Prevalidation of novel alternative pharma-/toxicological screening based on yeast expression technology (MULTIPLEX)

   
Project

QLK3-2001-00401

Cell factory area

3.1.4

EU Contribution

1,655,161 Euro

Duration

36 months

Type

RS

Starting date

01 January 2002

Keywords
animal experimentation reduction
K channel mutations
mutant isoforms
yeast strains
ABSTRACT

The project aims to the prevalidation of novel alternative pharma-/toxicological screening on potassium channels involved in human diseases based on yeast expression. Ion channels conduct the nerve impulses and mediate the effects of many signal molecules. Potassium (K) channels are by far the most diverse group of ion channels known and participate in a wide range of physiological impulses. In most cells they are responsible for maintaining the resting potential. In excitable tissue currents mediated by K channels they underly the repolarisation of the action potential. Severe diseases connected to K channel dysfunction are ventricular tachycardia, hypertension, deafness and metabolic disorders. The yeast based screening technology will provide a reliable system for pharma-/ toxicological analysis and a bioassay for rapid identification of chemical and pharmaceutical compounds and thus has the potential to reduce the need for animal experimentation.

OBJECTIVES

The overall objective is the prevalidation of novel alternative pharma/-toxicological screening based on the yeast based technology for mammalian K channels and their mutant isoforms. Application and prevalidation of this yeast based technology for the analysis of disease-causing mutant isoforms addresses several goals of the 5th FP.

The project will enable novel large-scale screens for modulators of human K channels; at the present time no yeast based HTS for K channels exist. This system increases the likelihood of discovery of novel therapeutic agents and thus has the potential of improving the life of patients suffering from a variety of diseases linked to defective.

K channel activity. Finally, the yeast based technology will provide bioassays for rapid screening of chemical and pharmaceutical compounds that have the capability to reduce animal experiments.

DESCRIPTION OF THE WORK

An extensive number of diseases causing mutations in human K channels have been identified. Thus the first and continuing WP will be the set-up of a comprehensive annotated database to assemble all available molecular and clinical information regarding K channel mutations and to further foster and integrate results from five other WPs. WP2 concerns the construction of a plasmid library necessary for yeast expression. Since many K channels are heteromultimers composed of channel forming and accessory subunits in WP3 the functional expression of such a complex ion-channel will determine the feasibility of the yeast based system. In WP4 a transgenic yeast strain library comprising wild type and mutant alleles will be constructed; growth phenotypes, inhibitor sensitivity, K transport activity, localisation and quantification of the heterologous proteins provide the basis for WP8. Electro-/pharmacological properties of K channels expressed in yeast are characterised in WP5. In WP6 the prevalidation of the generated yeast strains by standardised procedures and protocols is encountered. The technology transfer within WP7 will provide and optimise SOPs as evaluation criteria for the rational design of the yeast-based assays adapted to altered K channel function in WP8 with subsequent development of automated screening concepts in WP9. Project accompanying evaluation towards quality assurance by identification and implementation of respective indicators is the task of WP10.

DELIVERABLES
  1. public database with annotated information of K channel mutations.
  2. plasmid library of wild-type and mutant K channel cDNAs that enable expression in yeast.
  3. characterised library of transgenic yeast strains.
  4. standardised protocols for an easy-to-handle expression system for the K channel function-based assays.
  5. prevalidation of novel primary procedures for HTS approaches.
  6. evaluation criteria of the system within an industrial context proving phase.
CONSORTIUM
COORDINATOR
 
PD Dr Hella Lichtenberg
Botanisches Institut
Universität Bonn
Kirsehalle 1
53115 Bonn, Germany
Tel: +49-228-735 518
Fax: +49-228-735 504
h.lichtenber@uni-bonn.de
 
PARTNERS
 
Dr Jost Ludwig
Medizinisch-Naturwissenschaftliches Forschungszentrum
Physiologisches Institut II
Ob dem Himmelreich 7
72074 Tübingen, Germany
Tel: +49-7071-297 5286
Fax: +49-7071-878 15
jost.ludwig@uni-tuebingen.de

Dr Dale Sanders
The Plant Laboratory
Biology Department
University of York
PO Box 373
York Y01 5YW, United Kingdom
Tel: +44-1904-432 825
Fax: +44-1904-434 317
ds10@york.ac.uk

PD Dr med Thomas Hartung
Biochemische Pharmakologie
Universität Konstanz
78457 Konstanz, Germany
Tel: +49-7531-884 116/915382
Fax: +49-7531-884 117
Thomas.Hartung@uni-konstanz.de

Dr Jörgen Drejer
Director of Cell Biology
NeuroSearch A/S
93 Pederstrupvej
2750 Ballerup, Denmark
Tel: +45-44-608 000
Fax: +45-44-608 080
Direct. 45-44-608 236
jd@neurosearch.dk

Prof. Niels Tommerup, MD, PhD
Department of Medical Genetics, IMBG
The Panum Institute
University of Copenhagen
Blegdamsvej 3
2200 Copenhagen N, Denmark
Tel: +45-353 27 826
Fax: +45-353 27 845
tommerup@imbg.ku.dk