* To test the validity of the combinatorial synthesis approach to antiparasitic drug development, based on the inhibition of the parasite dihydrofolate reductase domain of the bifunctional enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS), and to develop new drug candidates especially against malaria from this approach.
* To understand the relation between structure and function of the parasitic enzymes, the basis for differential inhibition of the enzymes by candidate agents, and the basis for development of resistance.
* The validity of using combinatorial libraries for development of antifolate antimalarials will be tested.
* Combinatorial libraries based on 2,4-diaminopyrimidines and dihydrotriazines in solution or on solid supports will be made, as will milligram quantities of recombinant wild-type and mutant malarial dihydrofolate reductases ready for screening.
* Appropriate screening methodologies for combinatorial libraries against the malarial enzyme will be established.
* Better understanding of inhibitor binding to the enzyme will be achieved.
Selected libraries of 2,4-diaminopyrimidines and dihydrotriazines which include known inhibitors of malarial dihydrofolate reductase will be created and screened against recombinant parasite and host enzymes. Libraries will be made both in solution and on solid supports. The second type of libraries will also be encoded with appropriate molecular tags for easy identification of structures. In the first type of libraries, screening will be made against the enzyme, either in free form or linked to an appropriate solid support, while in the second type, screening of the resin-bound libraries will be made against the enzyme linked to an appropriate fluorescent molecule.
Enzyme-bound drug candidates can be separated from the other compounds by centrifugal dialysis after addition of the enzyme. Alternatively, resin-bound enzyme can be used to trap the drug candidates. Identification of bound inhibitors from the first type of libraries will be made after extraction into organic solvent by high-performance liquid chromatography coupled with mass spectrometry and other analytical methods. Identification of bound inhibitors from the second type of libraries will be made, using a confocal laser microscope with a micromanipulator and an adapted fluorescent activated cell sorter.
After identification of these compounds, they can be synthesized in solution in bulk for further studies. This is important in order to ensure that the binding observed in the screening was not influenced by linking to solid supports or synergistic or antagonistic interaction of the molecules in the mixture. The characteristics of enzyme inhibition will be studied in solution to ensure that tight-binding components are indeed good inhibitors of enzyme activity. Emphasis will be made on discovery of compounds which effectively inhibit mutant enzymes from drug-resistant parasites.
Testing of inhibition of P. falciparum in culture will also be done as well as testing of enzyme inhibition. After identification of promising candidate molecules, focused sublibraries will be made to cover various structural possibilities related to the identified molecules. In parallel, molecular modelling of the dihydrofolate reductase of the malarial and trypanosomal enzyme will be made, using information from known structures of the Leishmanial enzyme as well as bacterial and mammalian enzymes. Site-directed mutagenesis of the malarial enzyme will allow us to create a number of enzymes with mutations at sites that are suspected to be important for binding of substrates and inhibitors. This will allow us to establish a model of binding between the inhibitors and the enzyme, further allowing optimisation of the inhibitor structures to obtain better drug candidates for both the wild-type enzyme and the mutant enzymes arising from resistance.
Funding SchemeCSC - Cost-sharing contracts
OX1 3QY Oxford