Abstract

The main objective of this proposal is to exploit the molecular knowledge of the Saccharomyces cerevisiae cell wall for high throughput screening of antimicrobial agents. To this end, a consortium of 10 laboratories (one company, one SME and 8 public research institutes) will convert the molecular data on essential gene targets involved in cell wall cross-linking, remodelling and chitin pathways into assays amenable for drug-discovery programmes and to apply genomics, proteomics and bio-informatics, to identify new targets through the characterisation of the cell wall compensatory mechanism which is induced when the cell wall is weakened by drug treatment, stress, or mutations. The deliverables will be the production of number of assays for high throughput screenings, and the goal is to find novel antifungal molecules acting on the designed targets.

Objectives

The scientific objectives of this proposal are:

• To characterise the cross-linking and remodelling pathways which are the basis for the modular structure of the cell wall in pathogen and non-pathogen fungi, using the yeast S. cerevisiae as the model system.
• To identify by an integrated approach involving genomics, proteomics and bio-informatics, the determinants of the cell wall compensatory mechanism which is induced when the cell wall is weakened by various treatments.
• The technological objective is to provide assays from selected cell wall targets for high throughput screening.

The socio-economic impact of the project is to speed up the search of novel effective antifungal drugs. These objectives will be achieved by a strong commitment between molecular and cellular identification of cell wall targets and their biochemical characterisation to design assays allowing development of drug-discovery programmes.

Description of the work

The objectives will be reached by an integrated programme which will be divided into four workpackages.

Workpackage I will deal with the molecular and biochemical characterisation of cell wall cross-linking and remodelling pathways, identifying the different transglycosidases, beta-1,3 and 1,6 glucantransferase and chitinolytic/transglycosidic enzymes which catalyse critical steps of linkage between the 3 essential macromolecules which compose the cell wall. This characterisation will be followed by the design of assays for high throughput screening.

Workpackage II will deal with a detailed molecular and biochemical characterisation of the complex chitin synthesis pathway. This work will result in the setting-up of a novel chitin synthase assay which is highly effective for high throughput screening since it will take into account all newly regulatory proteins implicated in control of chitin synthesis.

Workpackage III will be devoted to the identification of new potential cell wall targets through a global genomic and proteomic analysis of the cell wall compensatory mechanism which is activated when the cell wall is weakened by drugs treatment, stress or mutations.

Workpackage IV will deal with the administrative, financial and report-writing aspects. All interesting, non-confidential data arising during the work will be communicated through a WEB site.

To raise to this challenge, eight leading academic research groups in the fields of the biochemistry and molecular biology of the fungal cell wall, genomics, proteomics, and bio-informatics will pool their expertise and skills, to increase our knowledge on the molecular and biochemical mechanisms involved in cell wall assembly, and to provide industry with reliable assays on well-characterised cell targets for high throughput screening of antimicrobial agents. The discovery of an antifungal sample from at least two of the selected targets is expected at the end of the project.

Deliverables

• Provide specific assays of well-dedicated cell wall enzymes for high throughput screening (HTS).
• Evaluate enzymes of the cross-linking and remodelling pathways as novel putative targets, and identify new targets from genomic and proteomic analysis (Month 36).
• Identify novel antifungal activities.
• Provide progress reports every 6 months

Contacts

Coordinator

• Institut National des Sciences Appliquées Department Genie Biochimique et Alimentaire / FRANCE

Participant

• Jean-Luc ZUNDEL / Aventis Crop Science SA Centre de Recherche de la Dargoire / FRANCE
• Cesar RONCERO / Departamento de Microbiologia Eidificio Departamental H-219 / SPAIN
• Joerg HOHEISEL / Deutsches Krebsforschungscentrum Functional Genome Analysis / GERMANY
• Francisco del REY / Universidad de Salamanca Dept de Microbiologica y Genetica / SPAIN
• Laura POPOLO / Universita Degli Studi di Milano Dipt Fisiologia e Biochemica generali / ITALY
• Graham GOODAY / University of Aberdeen Deparment of Molecular and Cell Biol / UNITED KINGDOM
• Universität Regensburg Lehstuhl für Zell Biologie und Planzenphysiologie / GERMANY
• Lucia CARRANO / Vicuron Pharmaceuticals Microbial Technologies / ITALY