The folate pathway is a validated drug target in the malaria parasite. In Plasmodium falciparum the parasite is dependant on endogenous folate synthesis for survival.Inhibition of this pathway by antifolates has been one of the cornerstones for the control of Malaria. Bifunctional Dihydropteroate synthase - Dihydro-hydroxymethylpterin pyrophosphokinase (DHPS-PPPK(HPPK)) is one of the key enzymes in the folate pathway of P. falciparum and the DHPS activity is responsible for converting dihydropteridine pyrophosphate to dihydropteroate. Antifolate drugs such as sulfadoxine and dapsone, which targets this enzyme, are failing due to resistance caused by mutations in DHPS. The wild type enzyme and all the known mutations are being modelled using homology-based techniques. Homology modeling was chosen since Plasmodium proteins are very difficult to crystallize due to A+T rich areas and areas of low complexity. The structure of Staphylococcus aureus DHPS was used as tem-plate for comparative modelling. The predicted active site compared favourably with those of the template and Mycobacterium tuberculosis. The in silico models were subsequently screened against a library of ligands to identify potential new inhibitors of the mutant and the wild type enzymes.
Two residues surrounding the active site will be mutated to verify the proposed model and to test the effect of these mutations. The exible loop covering the active site is being studied using molecular dynamics techniques. Recombinant DHPS-PPPK is being expressed using the pTrc vector in E. coli BL21 cells, followed by various column purifications. The activity may be measured by a radioactive assay using labelled p-aminobenzoic acid. Mutagenesis and screening of putative inhibitors are currently underway on recombinantly expressed DHPS-PPPK.