Trypanosoma cruzi, a protozoan parasite, is the causative agent of Chagas' disease (American trypanosomiasis), an infection of cardiac muscle cells and nerve ganglia that affects over 20 million persons in South and Central America, with over 90 million at risk. For practical purposes, chronic Chagas' disease is incurable, and drugs used for early infections can produce adverse side effects. Preliminary results from our laboratories, and other investigators, demonstrate a parasite cysteine protease (cruzain) is essential for parasite replication and transformation between stages of the T. cruzi life cycle. We are taking a multidisciplinary approach to identification of inhibitors of this protease. We will build upon preliminary studies, which have shown that fluoromethyl ketone-derivatized peptides and pseudopeptides can arrest early amastigote replication in host cells. Some of these analogues have already been tested and have been shown to reduce parasitemia at doses which are not toxic to the host. Analogues to enhance half-life, maximize binding to the target enzyme, and minimize toxicity will now be produced. We will also identify non-peptide inhibitors by computational screens of chemical databases. In addition to providing a three-dimensional structure upon which computational screens for inhibitor leads can be made, solving the structure of cruzain will provide new insights into the mechanism of structure-function relationships of this important family of enzymes. A multilevel analysis of the mechanism of action of the enzyme, and its biologic function, will be carried out utilizing crystallographic analysis, molecular modeling, and site-directed mutagenesis. Localization of the enzyme at the light and ultrastructural level in different stages of the parasite will be analyzed for clues to its function. A gene transfection strategy will be developed to study the roles of specific domains of the molecule in protease processing, catalysis and intracellular trafficking. Finally, characterization of the effects of inhibitors on the parasite at the light microscopic and ultrastructural level will provide another approach to analyzing enzyme function as well as aid in the design and modification of inhibitor leads.
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