Plasmodium falciparum purine salvage: From genetics to biochemistry
Madrid, Dennis Carlos
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The aim of this thesis is to further characterize and elucidate the role of purine salvage in Plasmodium falciparum. It was apparent from the analysis of the P. falciparum genome that purine salvage in the human host and the malaria parasite are different. (1) Since immucillins, transition state inhibitors of purine nucleoside phosphorylase (PNP), had been shown to kill P. falciparum in vitro, we asked the question, "Could a transition state inhibitor selective for the malaria purine salvage enzymes do the same?" Drug assays using various inhibitors of ADA and PNP were carried out, and the work presented here shows that MT-ImmH and MT-Coformycin, specific inhibitors of PfPNP and PfADA respectively, kill or alter the growth of P. falciparum in vitro. Unfortunately, in vivo testing of the PfADA inhibitors showed no delay to death. (2) In light of the additional specificity of PfADA and PfPNP for methylthio-purines, a byproduct of polyamine synthesis, we screened a library of polyamine analogs. The screen identified thirteen compounds with activity in vitro and three with activity in vivo. (3) Since many drugs have off-target interactions, we used single crossover homologous recombination to genetically disrupt pfpnp in P. falciparum. Immucillin drug sensitivity profiles of both wild type parasites and transgenic parasites lacking PfPNP (Deltapfpnp) were altered, confirming the importance of the inhibition of PfPNP for immucillin efficacy. Furthermore, we showed that the Deltapfpnp lines have a greater requirement for exogenous purines and are unable to thrive at physiological concentrations of hypoxanthine. These results illustrate the importance of purine salvage enzymes for malaria viability. (4) We wanted to determine if there are differences in purine metabolism within the Plasmodium species. ADA and PNP were cloned from various species of Plasmodium and substrate and inhibitor kinetics were determined. The goal of this work was to establish the purine salvage pathway, in particular PfADA and PfPNP, as targets for the development of specific chemotherapeutics for malaria chemotherapy. These data and the important role that Plasmodium purine salvage will have in the development of novel chemotherapeutics are discussed.