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Development and characterization of a rapid screening assay for identifying antipneumocystis agents
1Critical Care Medicine Department, National Institutes of Health, Bethesda, Maryland 20892.
Abstract:
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.
Insights
A new assay detects anti-Pneumocystis carinii drug activity by measuring protein synthesis. This rapid method effectively screened compounds, identifying effective agents like pentamidine and atovaquone.
Area of Science:
- Infectious Diseases
- Drug Discovery
- Biochemistry
Background:
- Pneumocystis carinii pneumonia (PCP) is a significant opportunistic infection.
- Developing new antipneumocystis agents requires effective screening methods.
Purpose of the Study:
- To develop and validate a rapid in vitro assay for screening antipneumocystis compounds.
- To assess the utility of [35S]methionine incorporation for measuring drug efficacy.
Main Methods:
- Developed an assay measuring [35S]methionine incorporation into proteins synthesized by Pneumocystis carinii.
- Used immunoprecipitation to confirm [35S]methionine incorporation into the major surface glycoprotein.
- Validated the assay by testing known antipneumocystis agents like atovaquone and pentamidine.
Main Results:
- The assay demonstrated that [35S]methionine is incorporated into newly synthesized P. carinii proteins, particularly the major surface glycoprotein.
- Clinically active agents (atovaquone, pentamidine) significantly inhibited [35S]methionine incorporation.
- Several agents, including pentamidine, atovaquone, trimethoprim-sulfamethoxazole, piritrexim, RO11-8958, and amphotericin B, showed >67% inhibition.
Conclusions:
- A rapid, reliable in vitro assay for screening antipneumocystis activity based on protein synthesis was established.
- This assay is a valuable tool for the discovery and development of novel antipneumocystis therapies.