Toxoplasma gondii is the leading cause of focal central nervous system infections in patients with the acquired immunodeficiency syndrome (AIDS). The limited repertoire of therapeutic agents for T. gondii is further confounded by the toxicity and variable efficacy of these compounds. New treatment strategies must be developed based on the basic biology of Toxoplasma. Fulfilling the primary goals outlined for the initial funding cycle of this grant, we have used a combination of pharmacological screening, cell biological, and genetic techniques to characterize potential therapeutic targets and identify novel classes of parasiticidal drugs. Our research has coalesced around three major target areas for new drug development: (i) the mechanism of action and target(s) of protein synthesis inhibitors, (ii) nucleoside/nucleobase transport and salvage pathways, and (iii) novel strategies for intervention, of which the most promising are cyclosporin analogs and dinitroaniline herbicides. The major goal of this application for renewal is to extend the substantial progress we have made in these focus areas, facilitating the development of drugs effective against AIDS-toxoplasmosis. Three projects and three cores are proposed. (Roos) seeks to investigate the basis of parasiticidal activity for compounds whose targets or mechanism of action remains unknown. We will explore the recently identified plastid-like extrachromosomal genome of Toxoplasma (suspected to be a target for macrolide antibiotics and other inhibitors of protein synthesis),, and use genetic techniques to identify the targets of cyclosporin A, dinitroanilines, and anticoccidials. The biology of parasite cyclophilins will also be dissected. Having identified parasite-specific pathways in nucleoside metabolism and transport, (Joiner) seeks to validate the potential of these targets. In particular, we hope to exploit the parasite's nucleoside triphosphate hydrolase and adenosine transport pathways for parasiticidal drug design. The versatile genetic tools developed in the course of this research have proved effective in cloning genes encoding several key enzymes involved in nucleoside metabolism. (Ullman) therefore seeks to characterize a critical purine salvage enzyme, the hypoxanthineguanine-xanthine phosphoribosyltransferase, and investigate the potential of structure-based drug discovery and design. A molecular genetics core (Roos) will expand the existing arsenal of available tools for probing the molecular genetics of T. gondii, and apply these tools for the validation of drug targets and mechanisms of action. The animal core (Luft) will continue to provide experimental support and carry out animal testing (where not otherwise feasible through existing contracts). The administrative core (Joiner) manages the structure necessary for continuing the highly interactive research programs established in the first funding cycle.
