The parasitic protozoa, Trypanosoma brucei and Trypanosoma cruzi, are the cause of major morbidity and mortality in Africa and South America, yet the available anti-trypanosomal drugs have limited effectiveness. A successful method for the development of new drugs to combat infectious diseases has been to take a structure-based approach to designing new inhibitors of protein function, followed by generation of these leads into clinically useful drugs. However, application of this method requires a detailed knowledge of the target proteins structure and function. We have identified three drug targets, ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (SAMDC) and gamma-glutamylcysteine synthetase (gammaGCS), which are essential for the biosynthesis of the polyamines, putrescine, spermidine and trypanothione. Data supporting all three enzymes as valid drug targets for the treatment of these parasites has been reported. Alpha- difluoromethylornithine (DFMO), an inhibitor of ODC is used clinically for the treatment of African sleeping sickness, while inhibitors of both gammaGCS and SAMDC cure T. brucei infections in mice. While this data is conclusive for ODC, only limited studies have been carried our for gammaGCS. Our goals are the detailed mechanistic and structural characterization of these enzymes. We recently solved the X-ray structure of T. brucei ODC and plan to continue our analysis by solving the structure of wild-type ODC bound to inhibitors. The structural basis for dimer formation, substrate binding and catalysis will be studied by site-directed mutagenesis and biophysical analysis of the mutant enzymes. Structure-based computational methods will be used to develop design strategies for novel ODC inhibitors. Studies on T. brucei and T. cruzi SAMDC will focus on kinetic analysis and initiation of X-ray structure determination. In addition, the regulatory mechanisms which control SAMDC expression in T. brucei will be characterized. To validate gammaGCS as a drug target, a knockout mutant of gammaGCS will be generated in T. brucei. The knockout strain will allow us to determine if the enzyme is required for cell viability and to study the regulatory mechanisms which control the levels of polyamines and trypanothione in T. brucei cells. Additional studies on gammaGCS will include kinetic analysis of the T. cruzi and T. brucei enzymes, including testing of a series of inhibitors. Studies to search for a possible regulatory subunit of the parasite enzyme are also planned. The results of these studies will be applied to the design of species selective inhibitors of these enzymes, while also contributing to our fundamental understanding of how structure dictates protein function.
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