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 microgram/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.
Martinez
A
A
Critical Care Medicine Department, National Institutes of Health, Bethesda, Maryland 20892.
Kovacs
J A
JA
D000666:Amphotericin B / Q000494:pharmacology; D000890:Anti-Infective Agents / Q000494:pharmacology*; D000935:Antifungal Agents / Q000494:pharmacology; D053626:Atovaquone; D002463:Cell Membrane Permeability / Q000187:drug effects; D005656:Fungal Proteins / Q000096:biosynthesis / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000378:metabolism; D008715:Methionine / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D009285:Naphthoquinones / Q000494:pharmacology; D010419:Pentamidine / Q000494:pharmacology; D011010:Pneumocystis / Q000187:drug effects* / Q000378:metabolism; D011233:Precipitin Tests; D013462:Sulfur Radioisotopes; D015662:Trimethoprim, Sulfamethoxazole Drug Combination / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D000890:Anti-Infective Agents; D000935:Antifungal Agents; D005656:Fungal Proteins; D008562:Membrane Glycoproteins; D009285:Naphthoquinones; D013462:Sulfur Radioisotopes; D014409:Tumor Necrosis Factor-alpha; D010419:Pentamidine; D000666:Amphotericin B; D015662:Trimethoprim, Sulfamethoxazole Drug Combination; D008715:Methionine; D053626:Atovaquone