Mechanism-Based Drug Selection and Design: Nucleotide SA

This R01 application was submitted in response to PA AI-98-100, "National Cooperative Drug Discovery Groups-Opportunistic Infections" (NCDDG-OI) by a group of investigators from the Oregon Health Sciences University, the University of Pennsylvania, and the Yale University School of Medicine. It represents the continuation of a long-term formal collaboration among the investigators of these three institutions, supported since 1991 with a grant under the auspices of the National Cooperative Drug Discovery Group (NCDDG). The major scientific objective of the proposed studies is to integrate genetic, biochemical, and structural studies on key transporters and enzymes of the pyrimidine and purine salvage pathways in Toxoplasma gondii and related apicomplexan parasites, including Cryptosporidium, Sarcocystis, and Plasmodium. The long-term goal will be focused on developing better and more efficacious antiparasitic drugs for these parasites - particularly opportunistic pathogens associated with AIDS. Interference with pyrimidine synthesis has traditionally provided the most effective tool for management of clinical toxoplasmosis. However, despite the fact that (i) all of these pathogens are purine auxotrophs, (ii) the existing precedent of subversive purines as effective treatment for other parasitic diseases, and (iii) that the availability of antiparasitic lead compounds that target either purine or pyrimidine salvage pathways, the latter two pathways have not been extensively explored as targets for chemotherapeutic treatment of either T. gondii or other apicomplexans. Reagents previously developed through this research collaboration include: (1) a genetic map of nucleotide salvage pathways in Toxoplasma; (2) molecular clones encoding T. gondii UPRT, HGXPRT, AK, NTPase, and the parasite's major adenosine transporter; (3) milligram quantities of each of the above soluble enzymes purified to homogeneity, and heterologous systems for transporter expression; (4) high-resolution crystal structures for UPRT, HGXPRT, and AK; and (5) transgenic parasites harboring mutations in (or altered expression of) each of the above genes. These reagents are expected to permit structure-based discovery of new drug classes that target proteins necessary for parasite survival. Currently available molecular, biochemical, and cellular reagents and data that have become available through these studies include: (i) Molecular clones. Full length genomic and cDNA sequences for T. gondii uracil phosphoribosyl transferase (UPRT), nucleoside triphosphate hydrolyze (NTPase), hypoxanthine-guanine-xanthine phosphoribosyl transferase (HGXPRT), adenosine kinase (AK), and the parasite's major adenosine transporter (AT). (ii) Recombinant proteins. E. coli that overexpresses T. gondii UPRT, HGXPRT, AK, or NTPase and effectively provides unlimited quantities of these proteins purified to homogeneity. Functional expression of recombinant AT in a Xenopus oocyte assay system. (iii) Crystal structures. High-resolution three-dimensional crystal structures for the UPRT, HGXPRT, and AK proteins determined by x-ray crystallography. (iv) Transgenic parasites. UPRT-, HGXPRT-, and AK-knockout transgenics created by homologous gene replacement in otherwise syngeneic wild type parasites. AT and xanthine transporter (XT) transgenics isolated by imertional mutagenesis. NTPase-deficient transgenics in which the endogenous enzyme is down-regulated by antisense expression. Specific aims for the proposed studies include: (1) performing a detailed biochemical and structural characterization of T. gondii UPRT and AK enzymes. The resolution of the T. gondii UPRT and AK enzymes will be extended and enzyme-substrate and enzyme-product structures determined in order to provide a full understanding of their catalytic mechanisms, and to facilitate the discovery of novel inhibitors through computational methods (see Specific Aim 2). High-resolution crystal structures of a series of site-directed mutant UPRT and AK proteins will also be carried out to assess the roles of key residues in substrate specificity and catalysis. Mutant enzymes will be purified from E. coli for kinetic appraisal, and crystal structures will be determined by molecular modification or molecular replacement to ascertain structural changes. The phenotypic consequences of mutations of interest will be tested in intact parasites by replacement of either the wild type UPRT or AK allele with an appropriate targeting construct; (2) developing screens for identifying and evaluating novel classes of potential antiparasitic drugs that target either UPRT or AK. Small-molecule structural databases will be screened computationally using the crystallographically determined high-resolution apo- and substrate- and product-bound UPRT and AK structures to identify novel compounds that may interact with the active sites of either enzyme. Compounds that are computationally predicted to target the active site of the T. gondii UPRT or AK enzymes will be evaluated as potential lead compounds against the purified UPRT or AK enzymes; E. coli expressing T. gondii UPRT or AK cDNAs; and wild type, UPRT- or AK-T. gondii parasites in culture. The crystal structures will be solved for UPRT or AK co-crystallized with promising lead compounds; (3) functionally characterize, localize, and genetically dissect the T. gondii adenosine transporter (AT). AT ligand specificity and kinetic parameters will be determined in detail by functional expression of the AT cDNA in Xenopus laevis oocytes and/or nucleoside transport (NT)-deficient Leishmania donovani. The applicants also plan to use electrophysiologic approaches in the Xenopus expression system to ascertain whether AT is a proton- or Na(+)-coupled symporter that actively concentrates adenosine. Antibodies against AT will be used to determine the subcellular location of AT by immunofluorescence and immunoelectron microscopy. Finally, forward genetic approaches will be implemented to initiate a structure-function analysis of key amino acids of AT that participate in ligand recognition or that govern substrate specificity; (4) isolation of XT cDNA and characterizing the properties of T. gondii XT. The XT gene will be isolated from insertional mutants by marker rescue and used to obtain full-length cDNA clones. Functional properties of XT will be evaluated after heterologous expression of XT cDNA in Xenopus oocytes, and the transporter will be immunolocalized after generating monospecific antibodies; and (5) to crystallize and solve the x-ray structure of the T. gondii NTPase and determine how enzymatic activity is regulated. The NTPase cDNA has been overexpressed in E. coli providing ample and replenishable quantities of monomeric recombinant protein for initial crystallization trials. In parallel, the production, purification, and crystallization of enzymatically active oligomeric NTPase will be pursued. Ultimately, these crystallization experiments will lead to the x-ray structure determination of the NTPase by multiple isomorphous replacement. Concurrent experiments will determine how NTPase functions in AK HGXPRT-, and AT parasites, identify the protein(s) which regulate NTPase enzymatic activity, and evaluate the impact of abrogating NTPase expression on parasite viability.