The parasitic protozoa of the genus Trypanosoma are the causative agents of African sleeping sickness and of Chagas' disease in South America. These parasites cause significant mortality and drug therapy to combat these diseases is inadequate. Polyamines are essential growth factors, and inhibition of polyamine biosynthesis provides a mechanism to inhibit cell growth. The first committed step in the biosynthesis of polyamines is catalyzed by ornithine decarboxylase (ODC). This enzyme is one of the few validated targets for the treatment of parasitic diseases. A number of enzymes in the pathway are essential for cell growth, and inhibitors of several polyamine biosynthetic enzymes have anti-trypanosomal activity. In addition the parasites synthesize a unique cofactor that is a conjugate of spermidine and glutathione, termed trypanothione that functions to maintain the reduced thiol pool in the cell. In this proposal our planned studies focus on the three enzymes in the pathway (ODC, S-adenosylmethionine decarboxylase and gamma-glutamylcysteine synthetase) that catalyze the first committed steps in the biosynthesis of the polyamines and trypanothione. In the first two aims we will explore the structural basis for inhibition of these enzymes. These studies will lay the foundation for future inhibitor design.
In Aim one we plan to explore the role of active site interactions in the energetics of ligand binding and catalysis, using site-directed mutagenesis and X-ray crystallography.
In Aim two mechanisms of allosteric inhibition will be studied to explore the potential to develop novel mechanisms of inhibition that target regions outside of the active site. While ODC is a fully validated target, other enzymes in the pathway are not. The nature of the rate-determining steps in polyamine and trypanothione biosynthesis in T. brucei has not been fully elucidated. Understanding the metabolic flux through the pathway will provide insight into which additional enzymes are best targeted for drug design. Polyamine metabolism is highly regulated in most cells, yet no evidence for regulation has been described in trypanosomatids. If the parasite alters polyamine levels in response to pathway inhibitors this regulation may impact on the effectiveness of drugs targeted at these enzymes.
In Aim 3 we propose to study the effects of both genetic and metabolic perturbations in the pathway on the levels of key enzymes and metabolites.
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