Identification of highly potent and selective inhibitors of Toxoplasma gondii dihydrofolate reductase

L. C. Chio, S. F. Queener

Research output: Contribution to journalArticle

92 Scopus citations

Abstract

Toxoplasma gondii RH was obtained in high yield from culture in RPMI medium on a line of Chinese hamster ovary cells lacking dihydrofolate reductase activity (ATCC 3952 dhfr-; American Type Culture Collection). Dihydrofolate reductase preparations from harvested organisms had specific activities of 22.9 ± 2.1 nmol/min/mg. The 50% inhibitory concentrations against reference compounds were 0.014 μM for methotrexate, 0.24 μM for pyrimethamine, 2.7 μM for trimethoprim, and 0.010 μM for trimetrexate. The K(m) value for NADPH was 11 μM and followed Michaelis-Menten kinetics; the K(m) for dihydrofolate was ca. 11 μM, but substrate inhibition appeared to occur at high substrate concentrations. Dihydrofolate reductase from T. gondii was used to screen 130 compounds from the National Cancer Institute repository. Thirteen compounds were >100-fold more potent than pyrimethamine toward T. gondii dihydrofolate reductase; six compounds with various potencies were 8 to 46 times as selective as pyrimethamine for the protozoal form of the enzyme over the mammalian form. Four trimetrexate analogs were more potent than trimetrexate, and two were significantly more selective. Representative compounds were also tested in a culture model of T. gondii employing uracil incorporation as an index of growth. One pyrimethamine analog was as effective as pyrimethamine in inhibiting T. gondii in culture (50% inhibitory concentration, 0.45 μM). Three other compounds were also effective at micromolar concentrations.

Original languageEnglish (US)
Pages (from-to)1914-1923
Number of pages10
JournalAntimicrobial Agents and Chemotherapy
Volume37
Issue number9
DOIs
StatePublished - Jan 1 1993

ASJC Scopus subject areas

  • Pharmacology
  • Pharmacology (medical)
  • Infectious Diseases

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