Amalgamating the tools and techniques of molecular biology, genetics, biochemistry, and pharmacology, this competing renewal application offers an experimental paradigm to further validate key components of the polyamine biosynthetic pathway of Leishmania donovani, the causative agent of visceral leishmaniasis. The application focuses on the role of polyamine acquisition from the host in the establishment of parasite infection] and implements a strategy directed toward drug discovery for the treatment of visceral and perhaps other forms of leishmaniasis. The polyamine biosynthetic pathway of Leishmania consists of four enzymes: arginase (LdARG), ornithine decarboxylase (LdODC), S-adenosylmethionine decarboxylase (LdADOMETDC), and spermidine synthase (LdSPDSYN). [The parasite can also salvage host polyamines and their precursors via uptake mechanisms, one of which is the POT1 putrescine-spermidine transporter. The application will address this intricate and largely uncharacterized relationship between the host and parasite polyamine pathways] and is a logical extension of the fundamental observations that ?ldarg, ?ldodc, ?ldadometdc, and ?ldspdsyn lesions, all of which confer polyamine axotrophy, impact the capacity of L. donovani to infect the mammalian host to dramatically different extents. Key reagents available for these investigations include: 1) genes encoding each of the polyamine enzymes and [the POT1 permease];2) ?ldarg, ?ldodc, ?ldadometdc, and ?ldspdsyn null mutants of L. donovani;and 3) Arg1 flox/flox;Tie2cre mice that lack ARG1 in macrophages and their Tie2cre Cre deleter controls. [Specific Aim I has three components: 1) to ascertain the role of polyamine or polyamine precursor salvage from the host by comparing parasite loads in livers and spleens of both control and Arg1 flox/flox;Tie2cre mice that have been inoculated with either wild type, ?ldarg, ?ldodc, or ?ldspdsyn L. donovani followed by appropriate nutritional supplementation protocols to test mechanism;2) to perform complementary infectivity studies with the wild type and mutant parasites in peritoneal macrophages derived from the Arg1flox/flox;Tie2cre and Tie2cre strains;3) to establish the role of POT1 in modulating parasite infection by testing whether POT1 overexpression boosts parasite burdens when wild type mice are inoculated with either ?ldodc or ?ldspdsyn L. donovani and by ascertaining whether LdPOT1 is the sole polyamine permeation mechanism in L. donovani via the creation and phenotypic assessment of a ?ldpot1 knockout.] Specific Aim 2 is a logical step in drug development and offers a blueprint to discover small molecule inhibitors of the genetically validated polyamine pathway of L. donovani. A simple, cost-effective, and inclusive reverse chemical genetic screen of 5,000 - 6,000 proven anti-leishmanial compounds that have emerged from two comprehensive high throughput forward chemical genetic screens of structurally and chemically diverse small molecule libraries will be performed in order to identify chemicals that target any o the four enzymes of the L. donovani polyamine pathway.
Visceral leishmaniasis is an all-too-common and potentially fatal parasitic disease for which better and more effective drug treatments are desperately needed. Over the last several years, my laboratory has discovered that the pathway for synthesizing polyamines in Leishmania donovani, a protozoan parasite that is the causative agent of visceral leishmaniasis, includes a number of enzyme components that appear to be excellent candidates for new drugs. The research described in this application will lead to a further characterization and validation of the polyamine pathway, will delineate the involvement of the host in parasite polyamine metabolism, and offers an explicit strategy to discover inhibitors of polyamine biosynthesis that is expected to provide a foundation for future drug development strategies against this devastating disease.
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