Important legal notice
Contact   |   Search on Europa   

Infectious Diseases Graphic element print icon Graphic element
  Homepage
Graphic element General information
  RTD Infectious Diseases Unit
  Developing countries
  EDCTP
   
Graphic element Projects
  About FP6 Funding
  FP6 projects
  About FP5 Funding
  FP5 projects
   
   
Graphic element Addressed Diseases
  HIV/AIDS
- News
  Malaria
  Tuberculosis
   
Graphic element Calls for proposals
  FP7 Calls
   
Graphic element Contact corner
  Unit
  Scientific officers
  Subscribe to our mailing list
   
 
PVPFDHFR
MALARIA
Framework programme: 5
Project number:
ICA4-CT-2001-10077
EC contribution: € 589 995
Duration: 36 months
Type: RS
Starting date: 1 November 2001
Graphic element Dihydrofolate Reductase-Thymidylate Synthase from Plasmodium Vivax and P. falciparum as Targets for Antimalarial Chemotherapy

Summary:

Both dihydrofolate reductase-thymidylate synthase (DHR-TS) and DHFR from P. vivax will be cloned and expressed from E. coli. The kinetic properties of the expressed enzyme and the profiles of inhibition by various antifolates will be studied in order to find out whether it is inherently resistant to inhibition. Mutants of P. vivax DHFR, both found in the field and artificially designed, as well as mutants of P. falciparum, will be made and studied. The results will be used to model the three-dimensional structure of the enzymes, especially around the active site. Both DHFR and DHFR-TS from the two sources will be prepared in sufficient amounts for crystallisation and structure determination by X-ray diffraction. Newly designed antifolates and those synthesised previously will be tested for their inhibitory activities. The mode of binding of such agents to the enzyme will be studied from both modelling and X-ray studies. Such agents will be important as leads to new antifolate drugs against both types of malaria. Agents with activity against both P. vivax and P. falciparum DHFR will be of special interest, in view of the common occurrence of mixed infections.

Objectives:

The objectives of this project are:

1) to clone, express and purify P. vivax DHFR and DHFR-TS and then mutants, both found in the field and artificially created, in sufficient amounts for characterisation and crystallisation purposes

2) to study the properties of the P. vivax enzyme, to find out whether it is prone to inhibition by antifolates and whether mutant DHFRs found in the field have even lower sensitivity to such inhibition

3) to model the structure of P. vivax DHFR through sequence alignment and homology modelling with other DHFRs whose three-dimensional structure is known from crystallography, and compare with the P. falciparum model

4) to obtain the structure of the enzymes from both sources by X-ray diffraction, and to delineate the model of binding of inhibitors to the enzymes from kinetic, modelling and X-ray studies

5) to test the inhibitory activities of newly designed and synthesised antifolates, with the aim of finding effective inhibitors, and to provide leads to new antifolate drugs against vivax malaria, preferably with cross activity against falciparum malaria, which will be useful in mixed infections commonly found.

Description:

This project aims to examine the possibility of developing antifolates as agents against vivax malaria, in conjunction with ongoing works on falciparum malaria. Although it has been generally presumed that Plasmodium vivax is inherently resistant against such common antifolate antimalarials as pyrimethamine and cycloguanil, this has not been rigorously proven, nor the nature of the presumed resistance understood. It is suspected that the presumed inherent resistance of P. vivax against antifolates, if true, may not be very high, since a number of mutant DHFRs have been found in the field. These mutant DHFRs have changes in amino acids equivalent to those that cause antifolate resistance in P. falciparum. It should therefore be possible to develop antifolates against P. vivax malaria. Preferably, such agents should also work against falciparum malaria to be able to deal with commonly occurring mixed infections. The gene of DHFR- TS will be obtained from genomic DNA prepared from vivax-infected blood samples. After PCR amplification, the genes for both DHFR and DHFR-TS will be cloned and expressed from E. coli. The kinetic properties or the expressed enzyme and the profiles of inhibition by antifolates will be studied and compared with P. falciparum DHFR, in order to find out whether it is inherently less susceptible to inhibition by these antifolates. Furthermore, a number of mutants of P. vivax DHFR previously reported from the field will be constructed, expressed, and their sensitivity to antifolate inhibition studied. Mutant enzymes will also be made artificially through site-specific mutagenesis of pvdhfr gene, in order to identify key residues in inhibitor binding. Hybrid enzymes with different parts derived from P. vivax and P. falciparum will be made and studied in order to find out key regions involved in inhibitor binding. The structure of P. vivax DHFR will be obtained through sequence alignments and homology modelling techniques, and the results will be compared with existing models for P. falciparum DHFR. DHFR and DHFR-TS from both sources will be prepared in sufficient amounts for crystallisation and structure determination by X-ray diffraction. A number of antifolates synthesised earlier for P. falciparum DHFR, and new antifolates designed from modelling of the P. vivax DHFR, will be tested for their inhibitory activities. The mode of binding of such agents to the enzyme will be studied from both modelling and X-ray studies. In the ongoing work, a number of new antifolates effective against drug-resistant P. falciparum have already been obtained. More compounds against P. vivax DHFR will be designed and synthesised, putting emphasis on families other than diaminopyrimidines and dihydrotrialines.

Milestones:

1. Cloning, expression and purification of P. vivax DHFR and DHFR-TS, as wild type, mutants and hybrid with P. falciparum counterpart. Expression plasmids and pure wild type and mutant enzymes (mg quantities for each) are expected.

2. Homology modelling of the structures of wild type and mutant P. vivax DHFRs. Docking of inhibitors into the binding site and new lead design should be achieved.

3. Modelling of the wild type and mutant P. vivax DHFRs. Models of the enzymes complexed with substrates and inhibitors are expected.

4. Crystallisation and structure determination of P. vivax and P. falciparum DHFR and DHFR-TS.

Coordinator:

Yongyuth Yuthavong
Thailand National Science and Technology Development Agency
Biotec Centre, NSTDA
Bangkok
Thailand
Tel: +66 2644 8002
Fax: +66 2844 8022
E-mail: yongyuth@nstda.or.th

Partners:

  1. The Chancellor, Masters and Scholars of the University of Oxford - Dyson Perrins Laboratory Oxford University, UK-Oxford, United Kingdom
  2. Dipartimento di Science Farmaceutiche, Universita di Modena, IT-Modena, Italy
  3. International Centre for Genetic Engineering and Biotechnology - Malaria Research Group, New Delhi, India

 
 
top
Graphic element