The long range goal of this project is to employ molecular biological, biochemical, microbiological, and biophysical means to discover an effective and non toxic agents for the treatment of malaria. The specific approach will involve the identification of lead compounds and the eventual design of a specific, potent inhibitor for the hypoxanthine phosphoribosyltransferase (hprt) of Plasmodium falciparum. This approach will require the identification of differences in the biochemical properties and three dimensional structures of the human and malarial hprt's. The main reasons for selecting the hprt as a target for anti malarial chemotherapy stems from the knowledge that malarial parasites lack de novo pathways for the synthesis of purine nucleotide and therefore must rely upon hprt for salvage of purine bases from their host to replenish guanine nucleotide. Thus, a specific potent inhibitor of this enzyme should be lethal for parasite. Full length cDNA, encoding the hprt of P. falciparum has been acquired, amplified, and sub cloned into a recombinant expression system specifically designed for the synthesis of high levels of soluble, enzymatically active hprt's. Earlier success in the expression, purification, crystallization, and generation of high resolution X-ray diffraction patterns form the hprt's of Schistosoma mansoni and Homo sapiens indicate that similar results are likely to be achieved for the malarial enzyme. However, to insure success in generating crystal forms that are suitable for 3-dimensional analysis, hprt encoding cDNA from species of Plasmodium responsible for rodent malarias also will be expressed at high levels in bacteria for subsequent purification and crystallization. Also, strains of Escherichia coli, that are dependent on the salvage of guanine or hypoxanthine for their growth on defined media, have been transformed either with expression plasmid encoding the hprt of H. sapiens or P. falciparum. These recombinant microbes are being used to screen for lead compounds, that specifically inhibit the malarial hprt. Lead compounds can be modeled into the active site of the malarial hprt to determine molecular contacts between enzyme and inhibitor. The information generated by this analysis can be used to direct modifications of the lead compounds to enhance the binding specificity for the malarial enzyme. Also, a benefit of working with malarial parasites from rodents is that we will have the ability to begin testing the effectiveness of lead compounds in an animal model of the disease. This project will generate new screening systems and data that will have a probability of leading to the discovery of new drugs for the treatment of malaria.
