Alternative forms of therapy are needed to treat acute toxoplasma gondii encephalitis and to prevent relapses after resolution of the event. Protein synthesis inhibitors of the macrolide and lincosamide class have activity against acute T. gondii infections in murine models and early clinical data suggests that these drugs may be efficacious. The compounds show limited and variable activity against T. gondii in vitro, however, and the mechanism of action remains undefined. Efficacy of protein synthesis inhibitors against T. gondii requires that the compounds reach the ribosomal target site within the intracellular parasite, bind to the parasite ribosome, and inhibit protein synthesis. No data is available on these events with T. gondii, precluding a systematic approach to drug development. This proposal outlines rational strategy for the selection, testing and design of protein synthesis inhibitors with activity against Toxoplasma gondii, both for the actively replicating tachyzoite stage present in acute infections and for the more dormant bradyzoite stage responsible for relapse. Drugs suggest to have in vivo activity will be assessed for uptake into extracellular and intracellular parasites, for inhibition of protein synthesis in T. gondii in vitro and for interaction with T. gondii ribosomes. Development of and in vitro method for production of cysts is necessary to carry out these experiments with bradyzoites, and forms an important part of the proposal. Initial experiments will be done with currently available protein synthesis inhibitors, concentrating on selected agents in the macrolide and lincosamide class. Subsequently, new compounds will be selected and/or designed, in conjunction with the Macrolide Discovery Core, based on structure-activity relationships defined in the initial experiments. The second major aim is the identification and molecular characterization of selected new therapeutic targets in T. gondii. This parasite must salvage purine from the host cell. Tachyzoite and later bradyzoite components involved in purine nucleoside transport will be characterized as possible targets for therapeutic intervention. Characterization of bradyzoite-derived cDNA libraries and bradyzoite-specific clones will allow the molecular characterization and analysis of these and other new pathways for parasiticidal drug development. Four projects and three core components are included. These component parts are extensively interwoven with one another to rapidly and efficiently accomplish the scientific goals set forth above.
