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.
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.
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.
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.
Thailand National Science and Technology Development Agency
Biotec Centre, NSTDA
Tel: +66 2644 8002
Fax: +66 2844 8022
- The Chancellor, Masters and Scholars of the University of Oxford - Dyson
Perrins Laboratory Oxford University, UK-Oxford, United Kingdom
- Dipartimento di Science Farmaceutiche, Universita di Modena, IT-Modena,
- International Centre for Genetic Engineering and Biotechnology - Malaria
Research Group, New Delhi, India