Program Director/Principal Investigator (Last, First, Middle): Ullman, Buddy 2R01 AI041622-11A1 Amalgamating the tools and techniques of molecular biology, genetics, and pharmacology, this competing renewal application offers an experimental paradigm to validate key components of the polyamine pathway of Leishmania donovani and to implement a strategy directed toward drug discovery for the treatment of leishmaniasis. The proposal will focus primarily on the L. donovani ornithine decarboxylase (LdODC) enzyme but will also impact upon other components of the polyamine pathway as well, including arginase (LdARG), Sadenosylmethionine decarboxylase (LdADOMETDC), and spermidine synthase (LdSPDSYN). During the course of our investigations on polyamine metabolism in Leishmania we have: 1) isolated genes encoding all four proteins from either L. donovani or L. mexicana;2) created and characterized ?ldodc, ?ldadometdc, and ?ldspdsyn knockouts in L. donovani and a ?lmarg knockout in L. mexicana;and 3) generated monospecific antisera against LdODC, LdADOMETDC, and LdSPDSYN. The majority of our studies thus far have been performed with the insect vector form of the parasite, and this application will now extend our characterization of the polyamine pathway to the mammalian stage of the parasite, primarily in rodent models. This proposal follows up upon our pivotal finding that ?ldodc L. donovani are profoundly incapacitated in their ability to infect mice and initiates studies that will pharmacologically exploit the genetically validated LdODC enzyme. The proposal that is under consideration for support from the American Recovery and Reinvestment Act of 2009 has one Specific Aim I with four components: 1) we will determine whether putrescine, the product of LdODC, can reverse the compromised infectivity phenotype of ?ldodc parasites in mice;2) we will attempt to pharmacologically simulate the deleterious effects of a ?ldodc lesion on parasite infectivity in mice by administration of a-difluoromethylornithine (DFMO), an inhibitor of LdODC, in the drinking water;3) we will expand our infectivity studies on the ?ldodc knockout to Syrian golden hamsters, a rodent model of visceral leishmaniasis that more closely resembles the human disease;and 4) we will establish whether the virulence defect of the ?ldodc null mutant extends to LdARG, LdADOMETDC, and LdSPDSYN deficiencies by assessing parasite burdens after infection of mice with ?ldarg, ?ldadometdc and ?ldspdsyn gene deletion mutants. The experiments in this aim will confirm the validity of LdODC as a drug target for the treatment of visceral leishmaniasis and will extend our characterization of LdARG, LdADOMETDC, and LdSPDSYN as potential drug targets to the infectious form of the parasite.
The overall purpose of this proposal is to genetically and pharmacologically validate components of the polyamine biosynthetic pathway of Leishmania donovani, the causative agent of visceral leishmaniasis, a parasitic disease that is invariably fatal if untreated. There is currently no vaccine and no consistently effective chemotherapy for the disease, so there is an urgent need for new drugs and new drug targets. Because polyamine biosynthesis is essential for Leishmania donovani and inhibition of polyamine biosynthesis in humans does not appear to have serious consequences, our studies address the vital issues of validating potential novel drug targets and eventually discovering new and better drugs to treat this disease.
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