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 are in the process of screening 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 one DHFR inhibitor with potent antitoxoplasma activity that is also selective for protozoan compared to mammalian DHFR. In animal studies, we have been able to show that this inhibitor is able to prolong survival of mice acutely infected with T. gondii, and in combination with sulfadiazine will prolong survival of acutely infected animals for the entire six week observation period. To facilitate our studies, we have also developed a recombinant DNA library using T. gondii RNA. We are in the process of screening this library with a degenerate oligonucleotide probe that is based on the dihydropteroate synthase (DHPS) of cryptococcus, pneumocystis and a variety of bacteria, to try to identify the gene coding for the DHPS of T. gondii. The goal of such studies would be to identify the genes coding of 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.
