The parasitic protozoa Toxoplasma gondii cause toxoplasmic encephalitis which is the most common opportunistic infection of the CNS in AIDS patients. Pyrimethamine plus sulfadiazine is currently the only acceptable therapy. However, it is limited by host toxicity. The increasing high incidence of toxoplasmic encephalitis and the lack of satisfactory drugs necessitate the search for new drugs and targets for chemotherapy. Studies on the uptake and metabolism of purine antimetabolites offer an excellent opportunity for this quest. Purines are required for the highly active nucleic acids synthesis essential for these rapidly replicating parasites. Since, T. gondii lack de novo purine biosynthesis and rely on their host for the salvage of purines, interference with the uptake and/or salvage of purines by purine antimetabolites should inhibit parasite replication. Our long range objective is to identify specific inhibitors of purine uptake and metabolism in T. gondii as potential chemotherapeutic agents.
Specific Aims are (1) Test purine analogues as substrates or inhibitors of adenosine kinase and purine phosphoribosyltransferase in cell free extracts, in order to formulate structure-activity relationships and identify compounds that selectively inhibit the T. gondii enzymes. (2) Evaluate the uptake and metabolic fate of promising purine analogues and their effects on nucleotide metabolism in infected cells and free parasites in vitro. (3) Determine the effect of nucleoside transport inhibitors on the uptake and metabolism of purine analogues in infected cells and free parasites in vitro. (4) Study the nature of nucleoside transport in infected cells and free parasites to determine if infection alters the sensitivity to and/or the number of binding sites of nucleoside transport inhibitors in infected cells; if there exist such binding sites on the parasite; and search for selective inhibitors of parasite purine transport. (5) Evaluate inhibitors as potential antitoxoplasmosis agents and possible protection against host-toxicity by nucleoside transport inhibitors in cell culture and in mice. The RH strain of T. gondii is maintained in mice. Free parasites and infected human foreskin fibroblasts will be used for in vitro investigations. HPLC will be used to analyze the metabolic effects. The in vivo pharmacological studies will be carried out in mice.
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