T. gondii is a major cause of neurologic disease in AIDS patients. Currently very few drug regimens have been shown to be effective in treating T. gondii infection, and thus alternative agents are needed. Potential therapeutic agents are screened in standard in vitro culture assays, as well as by an assay specific for folate metabolism in T. gondii that utilizes incorporation of tritiated para-aminobenzoic acid into reduce folates. By these techniques, we have screened agents that interfere with folate metabolism, including those that will inhibit specific enzymes in the folate metabolism pathways. In collaboration with investigators at UCSF, we have identified a DHFR inhibitor, epiroprim, with potent anti-toxoplasma activity, that is also selective for protozoan compared to mammalian DHFR. In animal studies, we have previously shown that this inhibitor is able to prolong survival of mice acutely infected with T. gondii, and in combination with sulfadiazine and dapsone will prolong survival of acutely infected animals for the entire six week observation period. We have recently shown that very low doses of sulfadiazine combined with epiroprim can also prolong survival. To facilitate our studies, we have also developed a recombinant cDNA library using T. gondii RNA, and have attempted to clone the dihydropteroate synthase gene of T. gondii using anti-epitope antibodies as well as DHPS clones from other organisms, although to date these attempts have been unsuccessful. The goal of such studies would be to identify the genes coding for target enzymes, and to produce such enzymes in large quantities so that specific chemotherapeutic agents can be assayed in vitro prior to proceeding to some of the more cumbersome techniques outlined above. The ultimate goal of these studies is to identify agents that would be alternatives to the currently available regimens for treating T. gondii infection in immunosuppressed patients, including patients with AIDS.
