A purine salvage enzyme, hypoxanthine guanine phosphoribosyltransferase (HGPRT), has been identified as a potential targets of drugs for the treatment of a number f diseases cause by parasites, including Chagas' disease. Described herein in a structure-based approach to the discovery of potent inhibitors of HGPRT from Trypanosoma cruzi. Most of the compounds currently being studied that target the HGPRT's of parasites act as """"""""subversive substrates"""""""" that are converted by the enzyme into modified nucleotides that block subsequent enzymes, rather than directly inhibiting the activity of the HGPRT itself. For this study, the focus will be on the structural analysis of the trypanosomal enzyme and interactions with inhibitors, rather than subversive substrates. The most potent types of inhibitors of enzymes often are mimics of the predicted transition state of the reaction. Using recombinant enzyme produced in bacteria, the 3-D structure of the trypanosomal HGPRT will be determined by X-ray crystallography. The initial structure will be in conformation with a product analog bound to the active site provide direct comparisons with the currently available structure of the human enzyme bound to product, GMP. Several approaches will subsequently be employed to provide information relevant to the transition state of the enzyme. Site-directed mutagenesis of the cloned gene will be used to create mutant enzymes that may be able to bind both substrates but may be incapable of catalysis. These mutants will be used to co-crystallize the enzyme with both natural substrates bound. A complimentary strategy will employ a nonsalvageable purine analog (in which the reactive nitrogen is replaced with a carbon atom) in co-crystallization experiments with the trypanosomal enzyme and the second substrate, phosphoribosylphyrophospate. Also cocrystallization experiments with the recombinant enzyme will be performed using currently available transition state analogs. In addition to the structural studies, the mechanism for the enzyme catalyzed reaction will be investigate using steady state kinetics. These studies will provide details with regard to relative reaction rates., Km 's, substrate binding order and apparent K i's for inhibitors. The structural and kinetic experiments proposed herein for the trypansosomal HGPRT will contribute valuable information for the design of potent inhibitors specifically targeted to this enzyme. In addition, the elucidation of structural changes during substrate bind and catalysis will assist inhibitor design efforts targeting the HGPRT's of other parasites and will provide new information to increase our general knowledge about enzyme catalysis and structure/function relationships.
