Toxoplasma gondii RH was obtained in high yield from culture in RPMI medium on a line of Chinese hamster ovary cells lacking dihydrofolate reductase activity (ATCC 3952 dhfr-; American Type Culture Collection). Dihydrofolate reductase preparations from harvested organisms had specific activities of 22.9 +/- 2.1 nmol/min/mg. The 50% inhibitory concentrations against reference compounds were 0.014 microM for methotrexate, 0.24 microM for pyrimethamine, 2.7 microM for trimethoprim, and 0.010 microM for trimetrexate. The Km value for NADPH was 11 microM and followed Michaelis-Menten kinetics; the Km for dihydrofolate was ca. 11 microM, but substrate inhibition appeared to occur at high substrate concentrations. Dihydrofolate reductase from T. gondii was used to screen 130 compounds from the National Cancer Institute repository. Thirteen compounds were > 100-fold more potent than pyrimethamine toward T. gondii dihydrofolate reductase; six compounds with various potencies were 8 to 46 times as selective as pyrimethamine for the protozoal form of the enzyme over the mammalian form. Four trimetrexate analogs were more potent than trimetrexate, and two were significantly more selective. Representative compounds were also tested in a culture model of T. gondii employing uracil incorporation as an index of growth. One pyrimethamine analog was as effective as pyrimethamine in inhibiting T. gondii in culture (50% inhibitory concentration, 0.45 microM). Three other compounds were also effective at micromolar concentrations.
Sulfanilanilides with 3',5'-halogen substitutions had Ki values 6- to 57-fold lower than the Ki of sulfamethoxazole when tested against dihydropteroate synthase from Toxoplasma gondii. The compounds acted as competitive inhibitors. These compounds were also active against dihydropteroate synthase from Pneumocystis carinii, Mycobacterium avium, and Escherichia coli but were not significantly more active than sulfamethoxazole. The compounds were significantly more active in culture than were standard agents. Against T. gondii in culture, 50% inhibitory concentrations were 7- to 30-fold lower than that of sulfadiazine; against P. carinii in culture, a concentration of 100 microM caused 33 to 95% inhibition of growth, compared with 9% inhibition with 100 microM sulfamethoxazole.
