MICROCULTURE SCREENING ASSAY FOR PRIMARY INVITRO EVALUATION OF DRUGS AGAINST PNEUMOCYSTIS-CARINII

Pneumocystis carinii inoculated into 96-well filtration plate assemblies was shown to synthesize radiolabeled folates de novo from [para-H-3]aminobenzoic acid ([H-3]pABA). At the end of each incubation with [H-3]pABA, a vacuum manifold was used to remove the medium and wash P. carinii. The membrane at the base of each well was dried and punched out, and the level of H-3 retained was determined by direct scintillation counting. High-pressure liquid chromatography analysis of duplicate filters confirmed that direct counting of H-3 retained on membranes (after correction for unmetabolized [H-3]pABA) was an accurate reflection of total [H-3]pABA incorporation by P. carinii. Greater than 95% of the H-3 recovered was shown to be present as polyglutamated species. After digestion with rat plasma folic acid gamma-glutamyl hydrolase, para-aminobenzoylglutamate, N-10-formyltetrahydrofolate, and tetrahydrofolate were identified as the major H-3-labeled components. para-Aminobenzoylglutamate was presumed to have arisen from folylpolyglutamates synthesized by P. carinii and was therefore included in the calculation of total [H-3]pABA incorporation. P. carinii incorporation of [H-3]pABA under optimal conditions was used as a selective measure of in vitro viability against which the inhibitory effects of some antipneumocystis agents (pentamidine, sulfamethoxazole, 566C80, and piritrexim) were quantitated. The concentrations of pentamidine, sulfamethoxazole, 566C80, and piritrexim required for 50% inhibition in this assay were 7.3, 0.1, 1.4, and approximately 100-mu-M, respectively. The results suggest that this 96-well [H-3]pABA incorporation assay has considerable potential for objective in vitro drug screening against P. carinii.