The long-term objective of this proposal is to identify and study new chemotherapeutic targets and agents for the treatment of opportunistic infections caused by Toxoplasma gondii and Pneumocystis carinii. These parasites pose a major health risk for immunocompromised individuals, such as those suffering from acquired immunodeficiency syndrome (AIDS). Unfortunately, the effectiveness of the current therapies for these infections is limited, primarily due to toxicity associated with them. One potential target for chemotherapeutic agents in T. gondii and P. carinii is purine metabolism. When these parasites are dividing, they require purine nucleotides for the synthesis of nucleic acids. Thus, inhibition of purine nucleotide synthesis by purine analogs should result in inhibition of parasite replication. In order to identify potential targets for these compounds, however, the enzymatic pathways responsible for purine metabolism in T. gondii and P. carinii must be completely elucidated, and the enzymes involved in these pathways studied in detail. Therefore, the specific aims of this proposal are to: (1) elucidate the pathways involved in the salvage of purine deoxynucleosides by T. gondii by measuring the uptake and incorporation of radiolabelled purine deoxynucleosides into the nucleotide pools and nucleic acids of extracellular T. gondii and by identifying the enzymes involved in purine deoxynucleoside salvage in T. gondii; (2) identify purine analogs that are specific inhibitors or substrates of T. gondii purine phosphoribosyltransferase and adenosine kinase by formulating structure-activity relationships for the binding of purine analogs to T. gondii enzymes and comparing them to those for the corresponding mammalian (host) enzymes; (3) assess any promising purine analogs (i.e. those found to be specific inhibitors or substrates) as potential antitoxoplasmal agents by examining their inhibition of intracellular T. gondii replication in cell culture, uptake and incorporation by extracellular T. gondii, and inhibition of T. gondii nucleotide metabolism in vivo; and (4) elucidate the purine metabolic pathways in P. carinii by measuring the uptake and incorporation of radiolabelled purine precursors into the nucleotide pools and nucleic acids of P. carinii and by determining the metabolic fate of purine nucleobases and nucleosides (and purine analogs) incorporated by P. carinii.
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