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Volume 2
     

Research, selection and mechanism of action of potential therapeutic agents against Flaviviridae (Hepatitis C virus, Dengue virus, West Nile virus) (Flavitherapeutics)

   
Project

QLK3-2001-00506

Cell factory area

3.1.2

EU Contribution

1,902,308 Euro

Duration

36 months

Type

RS

Starting date

01 January 2002

Keywords
anti-viral chemotherapy
bone marrow
mitochondrial DNA
nucleoside analogues
ribonucleosides
ABSTRACT

Our objective is to provide new nucleoside analogues, modified on their base or sugar moieties, interfering specifically with the replication of Flaviviridae viruses.

These molecules will then be suitable for advanced preclinical and clinical development. Five leading European groups will join their efforts with Novirio sarl, a bio-pharmaceutical company. We intend to investigate the activity of newly synthesised lead compounds that inhibit the replication of various Flaviviridae viruses. Evaluation (using virology, enzymology and toxicopharmacology assays and 3D modelling) will provide data for structure activity relationship analysis (SAR) and will be used to direct further chemical orientation. The outcome is the development of the best new antiviral agents based on rational design with low toxicity and potent antiviral activity agents against this viral family as a whole.

OBJECTIVES

Significant recent advances in the ability to replicate and study in vitro and in vivo members of the Flaviviridae family, eg. hepatitis C (HCV), dengue (DENV) and West Nile (WNV) viruses, now bring the goal of developing successful anti-viral therapy for these pathogens within the grasp of biomedical research. Our objective is to provide a series of new nucleoside analogues, which interfere specifically with the replication of Flaviviridae family members, focusing on HCV, DENV and WNV. Among these molecules one or more lead compounds will be selected for advanced preclinical and clinical development.

It is a purpose of this consortium to:

  1. enable rational discovery process of potent anti-viral agents.
  2. provide structure-function analysis on selected leads.
  3. provide proof-of-principle assays for selected drug candidates.
  4. enable preclinical development, manufacturing and formulation of antiviral therapeutics.
DESCRIPTION OF THE WORK

The expertise of each laboratory will be strategically integrated to provide a cohesive rational approach in the areas of organic chemistry, molecular virology, enzymology and toxico-pharmacology. To achieve the objective, the structure of the work plan is organised in work-packages as follows:

WP 1 design and synthesis of novel sugar-modified ribonucleosides, and their mono and triphosphorylated derivatives.

WP 2 design and synthesis of novel base-modified ribonucleosides, and their mono and triphosphorylated derivatives.

WP 3 screening of nucleoside analogues for anti-viral activity against representative members of Flaviviridae.

WP 4 use of Flaviviridae RNA-dependent replicase as a target for new anti-viral chemotherapy. We will attempt to characterise the inhibition of replicase (polymerase and helicase) complex.

WP 5 cellular metabolism of novel nucleoside analogues and cytotoxicity studies. We plan to clone and express human dCK and UK and determine their effect on cellular DNA, RNA and protein syntheses.

WP 6 crystal structure determination, structure-function, and modelling studies of viral targets for anti-Flaviviridae therapeutics.

WP 7 determination of in vitro cellular toxico-pharmacology of anti-Flaviviridae agents, the myelosuppressive effects on human bone marrow clonogenic cells and mitochondrial DNA content will be tested.

WP 8 we plan to compare and complement the data obtained in all experimental models by regular phone conferences every two to four months and meetings every eight months.

DELIVERABLES
  • novel sugar-modified ribonucleosides synthesis and structure activity relationship (SAR) review.
  • DENV capping enzyme; activity, binding and inhibition assay and production of recombinant NS3 and NS5 and NS5B polymerase (Flaviviridae).
  • RNA polymerase NS5B/NS5 incorporation/inhibition assays.
  • cloning and expression of human dCK and UK.
  • interaction with cellular kinases.
  • quantitative analysis of enzyme substrate and enzyme/inhibitor interaction.
  • mechanism of interaction with cellular and/or viral enzymes.
  • structural studies on NS3 (DENV, WNV) NTPase/ helicase.
  • in vitro selection of YFV/BVDV resistant mutants.
  • crystal growth of DENV and WNV NS5 and X-ray analysis of full length NS5 (DENV, WNV).
  • in vitro mitochondrial toxicity and bone marrow toxicity using in vitro clonogenic assays.
CONSORTIUM
COORDINATOR
 
Dr Gilles Gosselin
CNRS UMR 5625
CC008, Université de Montpellier II
Place Eugène Bataillon
34095 Montpellier Cedex 5, France
Tel: +33-4-6714 3855
Fax: +33-4-6754 9610
gosselin@univ-montp2.fr
 
PARTNERS
 
Dr Frank Seela
Laboratorium für Organische und Bioorganische Chemie
Institut für Chemie
Universität Osnabrück
Barbarastrasse 7
49069 Osnabrück, Germany
Tel: +49-541-969 2791
Fax: +49-541-969 2370
Frank.Seela@uni-osnabrueck.de

Prof. Paolo La Colla
Dipartimiento di Biologio Sperimentale
Università di Cagliari
Cittadella Universitaria
SS 554- KM4.5
09133 Monserrato, Cagliari, Italy
Tel: +39-070-675 4147
Fax: +39-070-675 4210
placolla@unica.it

Dr Bruno Canard
CNRS UMR 6098
Laboratoire d'architecture et fonctions des macromolécules biologiques
ESIL-CNRS-AFMB
Case 925, 163 Avenue de Luminy
13288 Marseille Cedex 09, France
Tel: +33-4-9182 8644
Fax: +33-4-9182 8646
bruno@esil.univ-mrs.fr

Dr Silvio Spadari
Istitudo di Genetica Biochimica ed Evoluzionistica
Via Abbiategrasso 207
27100 Pavia, Italy
Tel: +39-0382-546 356

Fax: +39-0382-422 286
spadari@igbe.pv.cnr.it

Dr Martin Bryant
Novirio pharmaceuticals Ltd
125 Cambridge Park Drive
MA 02140 Cambridge, USA
Tel: +1-617-250 3100
Fax: +1-617-250 3101
bryant.martin@novirio.com

Dr Jean-Marc Allaire
Novirio Sarl
Immeuble "La Vigie"
170 rue Léon Blum
34 000 Montpellier, France
Tel: +33-4-9952 2252
Fax: +33-4-9952 2250