This grant application pertains to a critical issue in the treatment and control of toxoplasmosis, the need for better chemotherapies. Amalgamating techniques of molecular biology, biochemistry, structural biology, and computational chemistry, this proposal offers a multidisciplinary dissection of the hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) enzyme from Toxoplasma gondii, an enzyme that renders an important nutritional function for the parasite and that catalyzes the phosphoribosylation of certain cytotoxic purine base analogs that are not substrates for the human HGPRT counterpart. The proposed investigations constitute a logical step in the implementation of a rational strategy of drug discovery, and ultimately drug design, for the treatment and prevention of toxoplasmosis. Reagents available for these studies include: i. two biochemically distinct T. gondii hgxprt cDNAs ii., E. coli that overproduce each of the T. gondii HGXPRT proteins; and iii effectively unlimited amounts of the two T. gondii HGXPRT proteins that appear homogeneous by SDS-PAGE and iv. hgxprt populations of T. gondii that were generated by insertional mutagenesis. In addition, an homology-based 3-D molecular model of the T. gondii HGXPRT has been computationally constructed and serves as a cornerstone for our structural studies. The first specific aim of this project will be to perform a thorough biochemical characterization of the T. gondii HGXPRT protein. This will involve kinetic, mechanistic, and physicochemical studies on the recombinant protein and the generation of antibodies to determine which of the HGXPRT isoforms is/are physiologically relevant.
Specific Aim II will be to evaluate the 3-D model of the HGXPRT protein by site-directed mutagenesis of key amino acid residues that are postulated to participate in catalytic activity or govern substrate specificity and biochemical characterization of the genetically altered proteins. The second aspect of Specific Aim II will be to introduce crystallographic methods to the structural studies for the ultimate purpose of determining the structure of the T. gondii HGXPRT protein itself. The third and final specific aim will involve computational screens of 3-D small molecule structural databases with our molecular models, and ultimately with resolved structures, to discover novel 'lead' compounds that target the active site pocket of the T. gondii HGXPRT protein. Computationally identified compounds from the database screens, as well as approximately 40 procured purine base analogs, will be evaluated as potential antitoxoplasmal compounds using a simple, yet multifaceted, screen comprising of purified recombinant HGXPRT enzymes, E. coli that overexpress hgxprt genes, and intact parasites.
