The ultimate goal of this research is to identify biochemical differences between mammalian host and parasite that can be exploited for the development of new and better chemotherapeutic compounds against diseases caused by Trypanosoma cruzi (Chagas' disease), Leishmania spp. (cutaneous, mucocutaneous and visceral Leishmaniasis) and T.rhodesiense or T.gambiense (human African trypanosomiasis). The general approach relies on the identification of novel and unique pathways, essential for the survival of the parasite, but not the human host. By characterizing such biochemical pathways and, in particular, gaining an understanding at the molecular level of the chemical mechanisms of key enzymatic reactions, it is hoped to provide a rational basis for the design of new inhibitors. The present application is a continuation of a previous grant, which led to the discovery of the novel dithiol spermidine-peptide, trypanothione (bis(glutathionyl)-spermidine). Three unique and key target enzymes have been identified so far: trypanothione reductase, trypanothione synthetase and trypanothione peroxidase. During the next grant period, it is intended to focus on T.cruzi (using C.fascicufata as a model, where appropriate) in order to achieve the following specific aims: 1. To purify and characterize trypanothione synthetase from C.fasciculata, the enzyme required for biosynthesis of trypyanothione and glutathionylspermidine. To design and test analogs of glutathione, spermidine and glutathionylspermidine as inhibitors. 2. To clone and sequence the genes encoding enzymes related to trypanothione metabolism, particularly with a view to expressing and producing T.cruzi trypanothione reductase and trypanothione synthetase in large amounts for drug development and crystallographic studies. 3. To further elucidate the roles of glutathione, glutathionylspermidine and trypanothione in the metabolism of these parasites, with particular emphasis given to: a) how glutathionylspermidine modulates intracellular levels of free spermidine. b) purification and characterization of trypanothione-dependent peroxidase activity. c) a comprehensive search for other glutathione- or trypanothione-dependent enzymes.
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