Drug therapy studies imply that Pneumocystis carinii and Toxoplasma gondii possess the enzymes necessary for de novo folate synthesis. To verify this, incorporation of [3H]paraaminobenzoic acid [( 3H]PABA) into reduced folates by P. carinii and T. gondii was investigated. Both organisms synthesized tritiated reduced folates. In P. carinii, 10-formyltetrahydrofolate and tetrahydrofolate, and in T. gondii, 5-formyltetrahydrofolate were the major synthesized folates. P. carinii remained metabolically active in vitro for only a few days. Because current systems for screening antipneumocystis agents are cumbersome, the utility of this assay system for screening therapeutic agents was investigated. Sulfonamides and pentamidine efficiently inhibited de novo folate synthesis in P. carinii. Inhibitors of dihydrofolate reductase such as trimethoprim and trimetrexate were poor inhibitors for P. carinii but efficient inhibitors for T. gondii. This study demonstrates the first unambiguous evidence of metabolic activity in P. carinii, and provides a potential assay for efficiently screening antipneumocystis drugs in vitro.

Toxoplasma gondii is a common protozoan disease that often causes life-threatening disease, particularly among patients with the acquired immunodeficiency syndrome. This study demonstrates that the dihydropteroate synthase in T. gondii is kinetically distinct from the enzyme characterized from other sources and can be highly purified with a high yield using sequential dye-affinity chromatography. Conditions have been identified that allow for stabilization of the purified enzyme, and its physical characteristics have been elucidated. The molecular weight of the native protein was 125,000 and the protein appeared to contain both dihydropteroate synthase and 6-hydroxymethyl-dihydropterin pyrophosphokinase activities. The sulfonamide class of compounds vary in inhibitory potency by more than three orders of magnitude. Sulfathiazole, sulfamethoxazole, and sulfamethazine, with 50% inhibitory concentrations (IC50's) of 1.7, 2.7, and 5.7 MM, respectively, represent the most potent of this class of inhibitors. Several sulfone analogues, including dapsone, were identified as highly potent inhibitors with ICN's < 1 uM. The results of these cell-free experiments were corroborated by investigating the metabolic inhibition produced by the various inhibitors in intact organisms. The qualitative and quantitative relations among the inhibitors were preserved in both the cell-free and intact cell assay systems. These studies suggest that the sulfones may be important therapeutic agents for the treatment of toxoplasmosis. (J. Clin. Invest. 1990. 85:371-379.) antimetabolites.enzymology-protozoan * sulfonamides

No abstract

No abstract