Purine salvage as a chemotherapeutic target in malaria

DESCRIPTION (provided by the applicant): Plasmodium falciparum purine salvage will be investigated as a chemotherapeutic target in a new Interactive Research Program Grant (IRPG) program between the laboratories of Vern. L. Schramm (purine metabolism and inhibitor design), Charles Grubmeyer (enzymatic mechanisms), and Kami Kim (genetic analysis of purine salvage pathways). Together these three projects will 1) quantitate the purine salvage pathways of P. falciparum 2) characterize purine salvage in parasites with disruptions in selected purine salvage enzyme genes 3) establish transition states of critical enzymatic steps 4) design and synthesize transition state inhibitors for these enzymatic targets 5) test inhibitors for effects upon P. falciparum growth in culture and in the P. yoelii mouse malaria model 6) determine compensatory pathways for purine salvage in parasites with genetic or inhibitor-induced ablation of selected purine salvage enzyme activity. This proposal based in the Kim laboratory will focus upon determining the importance of purine salvage enzymes using a genetic approach. Inosine metabolism via purine nucleoside phosphorylase (PNP) and hypoxanthine guanine xanthine phosphorylase (HGXPRT) is thought to represent the primary pathway for purine salvage in malaria parasites. PNP and HGXPRT will be genetically disrupted in P. falciparum. Mechanisms of compensation for loss of PNP and HGXPRT will be examined using purine uptake studies and real time PCR analysis of purine salvage enzyme gene expression. Purine uptake and purine salvage enzyme gene expression will be compared in knockout parasites and parasites treated with specific purine salvage enzyme inhibitors. The significance of alternative purine salvage enzymes methylthioadenosine phosphorylase (MTAP) and adenine phosphoribosyltransferase (APRT) will be examined using genetic disruption and inhibitor ablation of activity. Inhibitors designed in the Schramm and Grubmeyer laboratories will be tested against P. falciparum cultured in human erythrocytes and in mice infected with P. yoelii, a lethal form of mouse malaria.