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Volume 2
     

Deoxyuridine triphosphate nucleotidohydrolase as drug target for the control of protozoal and bacterial infections (dUTPase)

   
Project

QLK3-2001-00305

Cell factory area

3.1.3

EU Contribution

1,116,561 Euro

Duration

36 months

Type

RS

Starting date

01 January 2002

Keywords
anti-infective agents
crystallography
enzyme characterisation
pathogen inhibitors
ABSTRACT

This proposal concerns the evaluation of the enzyme deoxyuridine triphosphate nucleotidohydrolase as a drug target against bacterial, protozoan and mycobacterial infections. The diseases of particular interest are malaria, leishmaniasis, African trypanosomiasis (sleeping sickness), Chagas' disease, bacterial infections and mycobacterial infections such as tuberculosis. This enzyme may also have application as an anti-cancer target. The enzyme is essential for the viability of the organism and represents a novel drug target. The following is planned: cloning and overexpression of the enzyme from key pathogens; crystallisation and X-ray crystallography of the enzymes; drug design using molecular modelling; screening of a large database of compounds to provide novel leads; synthesis of potential inhibitors; screening of compounds against pathogen and human enzymes; screening of compounds against protozoa, bacteria and mycobacteria in vitro.

OBJECTIVES

The overall objective of the proposal is to develop potential anti-infective agents which act at the enzyme deoxyuridine triphosphate nucleotidohydrolase (dUTPase). The therapeutic targets will be bacterial, protozoan and mycobacterial diseases and cancer. Objectives are characterisation of the enzymes from pathogens by cloning, overexpression, purification, kinetic analysis and X-ray crystallography; developing lead compounds; designing and optimising inhibitors using computer-aided design; synthesising potential inhibitors; and evaluating inhibitors by enzyme assays, and assays against the protozoa and bacteria. The aim is to develop lead compounds which selectively act on the pathogen for the treatment of malaria, leishmaniaisis, African trypanosomiasis, Chagas disease, bacterial infections and mycobacterial infections such as tuberculosis.

DESCRIPTION OF THE WORK

There is an urgent need for the development of new drugs to treat infectious diseases. We propose to target the enzyme dUTPase in an approach to develop new agents for the treatment of bacterial infections, mycobacterial infections such as tuberculosis and diseases caused by parasitic protozoa such as malaria or leishmaniasis. This enzyme is a novel drug target, and little has been done to investigate it. The enzyme has already been cloned and over expressed from a number of organisms, including bacteria, Leishmania major (which causes leishmaniasis), Trypanosoma cruzi (which causes Chagas' disease), Plasmodium falciparum (which causes malaria) Mycobacterium tuberculosis, Campylobacter jejuni (a major cause of food-borne bacterial disease) and humans. The enzyme from Trypanosoma brucei (which causes African sleeping sickness) will also be cloned and over-expressed. Mutants will be prepared to improve understanding of the mechanism of action of the enzyme. Crystal structures have already been obtained for the T. cruzi, human and Escherichia coli enzymes. Crystallisation studies will be undertaken with new enzymes; L. major, P. falciparum, M. tuberculosis, C. jejuni, T. brucei. In addition attempts will be made to prepare crystalline complexes of the enzymes with substrates and inhibitors for structure determination. In particular the leishmanial, trypanosome and camplyobacter enzymes are very different from the others. Lead compounds have been discovered against the leishmanial and E. coli enzymes and work will be undertaken to improve their activity and selectivity by preparation of new analogues. In addition further novel compounds will be prepared for evaluation, using the crystal structures, modelling and structure activity data. Compounds will then be assayed against the enzyme and against pathogens in vitro and in vivo.

DELIVERABLES

The principal milestones and expected achievements are cloning, overexpression and purification of dUTPase from various pathogenic organisms, crystal structure data on the enzymes from a number of species (including structures with inhibitors complexed into the active site), compounds to be prepared that are selective inhibitors of the enzyme from the pathogens of interest, activity data of a large number of compounds against the pathogens and some preliminary data on toxicity.

CONSORTIUM
COORDINATOR
 
Ian Gilbert
Welsh School of Pharmacy
Cardiff University
Cardiff, CF10 3XF, United Kingdom
Tel: +44-29-2087 5800
Fax: +44-29-2087 4149
gilbertih@cf.ac.uk
 
PARTNERS
 
Dolores Gonzalez Pacanowska
Instituto de Parasitologia y Biomedicina
Consejo Superior de Investigaciones Cientificas
18001 Granada, Spain
Tel: +34-958-203 802
Fax: +34-958-203 802
dgonzalez@ipb.csic.es

Keith Wilson
Department of Chemistry
University of York
York, YO10 5DD, United Kingdom
Tel: +44-1904-432 575
Fax: +44-1904-410 519
keith@yorvic.york.ac.uk

Reto Brun
Medical Parasitology
Swiss Tropical Institute
4002 Basel, Switzerland
Tel: +41-61-284 8231
Fax: +41-61-271 8654
reto.brun@unibas.ch

Nils Gunnar Johansson
Medivir AB
14144 Hudinge, Sweden
Tel: +46-8-608 3100
Fax: +46-8-608 3199
nils.gunnar.johansson@medivir.se