Development and characterization of a rapid screening assay for identifying antipneumocystis agents

We developed a rapid assay for screening of compounds with potential antipneumocystis activity on the basis of incorporation of [³⁵S]methionine into proteins newly synthesized by Pneumocystis carinii. Unambiguous evidence that P. carinii synthesizes proteins in vitro was provided by immunoprecipitation studies demonstrating the incorporation of [³⁵S]methionine into the major surface glycoprotein. Treatment with two clinically active antipneumocystis agents, atovaquone (10^-4 M) or pentamidine (10^-4 M), prevented this incorporation. Total [³⁵S]methionine incorporation paralleled incorporation into the major surface glycoprotein, permitting rapid assessment of anti-P. carinii activity by scintillation counting. Treatment with pentamidine (1 x 10^-4 M), atovaquone, trimethoprim (1 x 10^-4 M)-sulfamethoxazole (7.9 x 10^-4 M), piritrexim (1 x 10^-7 M), RO11-8958 (1 x 10^-4 M), and amphotericin B (1 μg/ml) resulted in a greater than 67% inhibition (P < 0.05) of [³⁵S]methionine incorporation. No decrease in [³⁵S]methionine incorporation was seen with dapsone (10^-5 M), trimethoprim (10^-4 M), recombinant mouse tumor necrosis factor (500 ng/ml), or gamma interferon. This rapid in vitro assay should be a useful adjunct in the development of new antipneumocystis agents.