Pneumocystis carinii dihydrofolate reductase used to screen potential antipneumocystis drugs

M. C. Broughton, Sherry Queener

Research output: Contribution to journalArticle

101 Citations (Scopus)

Abstract

Pneumocystis carinii was obtained in high yield from the lungs of immunosuppressed rats by rupturing mammalian host cells, washing away the soluble mammalian dihydrofolate reductase, and harvesting intact organisms in association with the mammalian plasma membranes. P. carinii dihydrofolate reductase, measured in the 100,000 x g supernatant from sonicated organisms, was obtained in yields ranging up to 62 IU per rat. The enzyme prepared in the presence of protease inhibitors was stable when frozen in liquid nitrogen. P. carinii dihydrofolate reductase differed from the mammalian enzyme in that the former was slightly inhibited by 150 mM KCl, whereas the latter was stimulated over twofold by 150 mM KCl. The standard assay for P. carinii dihydrofolate reductase contained 0.12 mM NADPH and 92 μM dihydrofolic acid. Under these conditions, the 50% inhibitory concentrations of the known inhibitors trimethoprim, trimetrexate, and pyrimethamine were 12 μM, 42 nM, and 3.8 μM, respectively. These standard compounds were also tested against dihydrofolate reductase from rat liver to allow an assessment of the selectivity of the drugs. Although it was the least potent, trimethoprim was the most selective. Pyrimethamine was more potent but was nonselective. Trimetrexate was extremely potent but was selective for mammalian dihydrofolate reductase. A series of experimental compounds was obtained from the National Cancer Institute and other sources through the Developmental Therapeutics Branch of the Division of AIDS at the National Institute of Allergy and Infectious Diseases. Among the first 87 compounds tested, 11 had 50% inhibitory concentrations below that of trimetrexate and 3 were more selective than trimethoprim. The most promising compounds in this original group were chemically related to methotrexate.

Original languageEnglish
Pages (from-to)1348-1355
Number of pages8
JournalAntimicrobial Agents and Chemotherapy
Volume35
Issue number7
StatePublished - 1991

Fingerprint

Pneumocystis carinii
Tetrahydrofolate Dehydrogenase
Trimetrexate
Trimethoprim
Pharmaceutical Preparations
Pyrimethamine
Inhibitory Concentration 50
National Institute of Allergy and Infectious Diseases (U.S.)
National Cancer Institute (U.S.)
Enzymes
Protease Inhibitors
NADP
Methotrexate
Acquired Immunodeficiency Syndrome
Nitrogen
Cell Membrane
Lung
Acids
Liver

ASJC Scopus subject areas

  • Pharmacology (medical)

Cite this

Pneumocystis carinii dihydrofolate reductase used to screen potential antipneumocystis drugs. / Broughton, M. C.; Queener, Sherry.

In: Antimicrobial Agents and Chemotherapy, Vol. 35, No. 7, 1991, p. 1348-1355.

Research output: Contribution to journalArticle

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