Purine Pathways and Inhibitor Design in Plasmodium

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DESCRIPTION (provided by the applicant): Plasmodium falciparum has been recognized as a purine auxotroph for more than three decades. Despite this difference from its human host, our understanding of its purine salvage pathways and the design of anti-metabolites have proven inadequate for new antimalarial agents. The purine salvage pathways will be targeted in an integrated Interactive Research Project Grant (IRPG) between the laboratories of C. Grubmeyer (phosphoribosyltransferases and inhibitor design), K. Kim (genetics and expression) and V.L. Schramm (metabolism and inhibitor design). The overall goals of the IRPG are to: 1) quantitate the pathways of purine salvage in Plasmodium falciparum, 2) characterize genetic knock-outs to define the pathway response to ablation of specific steps in major salvage pathways, 3) establish the transition states of the critical enzymatic steps, 4) design and synthesize uniquely powerful transition state inhibitors against the targets, 5) test the inhibitors in P. falciparum cultured in human erythrocytes, and 6) compare the salvage pathways in cultures with specific inhibiors to gene knock-outs at the same sites. The results will provide definitive knowledge of the purine pathways of P. falciparum, genetic tests of essential steps and powerful transition state inhibitors against vulnerable enzymes of the pathway. Purine salvage pathways will be quantitated in cultured P. falciparum by following 3H and 14C-labeled precursors into nucleic acids. High-sensitivity accelerator mass spectrometry (>106 more sensitive than counting) will be used to establish purine salvage flux without perturbing purine metabolite concentrations. Purine salvage flux will be compared in gene-disruptions of purine nucleoside phosphorylase (PNP), HGXPRT, APRT, and methylthioadenosine phosphorylase (MTAP). The hypothesis is that PNP and HGXPRT form the dominant purine salvage pathway. Ablation of this pathway may induce a secondary pathway of adenosine salvage. Transition state inhibitors will be designed, synthesized and tested for PNP and MTAP in this lab, and for APRT and HGPRT (Grubmeyer lab). The effects of transition state inhibitors will be tested on the growth of P. falciparum in erythrocytes with and without purine supplements to by-pass the metabolic block. The efficiency of pathway blocks will be evaluated by comparing metabolite incorporation patterns in genetic knockouts to those with transition state inhibitors. Validation of essential steps in purine salvage pathways will direct this and future programs in inhibitor design. Inducing inhibitor blocks at essential steps of purine salvage may provide an effective strategy for killing the malarial parasite.