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. Our proposed research builds upon preliminary results from our laboratories, as well as from other investigators, that demonstrate a parasite cysteine protease (cruzain) is essential for parasite replication and transformation between stages of the T. cruzi life cycle. We will take a multidisciplinary approach to identification of inhibitors of this protease as new leads for chemotherapy and probes to study the biologic and molecular function of the enzyme. We expect our proposed work will not only be relevant to the study and treatment of Chagas' disease, but will be a model strategy by which to study and target a number of other protozoan and helminth parasites that express structurally and biochemically similar enzymes. Two complementary approaches will be taken. In the first, 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 in a murine model of infection 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 by our synthetic chemistry group (Roush). A second approach will be to identify non- peptide inhibitors by computational screens of chemical databases (Cohen). This approach has been possible by our successful crystallization of the target protease and collection of a crystallographic data set (McGrath and Fletterick). 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 catalysis and structure- function relationships of this important family of enzymes (Craik, Fletterick and McGrath). 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 (Craik, Cohen and McGrath). In parallel, localization of the enzyme at the light and ultrastructural level in different stages of the parasite will be analyzed for clues to its function (Engel and McKerrow). A gene transfection strategy will be developed to study the roles of specific domains of the molecule in protease processing, catalysis and intracellular trafficking (Craik, McKerrow and Engel). 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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