The dual mode of inhibition of calmodulin-dependent nitric-oxide synthase by antifungal imidazole agents.
The antifungal imidazoles miconazole, ketoconazole, and clotrimazole inhibit citrulline formation by nitric-oxide synthase. These agents both increase the concentration of calmodulin required to activate the enzyme half-maximally and reduce the maximal velocity of citrulline formation. This inhibition was not reversed by increased concentrations of either the arginine substrate or (6R)-5,6,7,8-tetrahydro-L-biopterin. Miconazole, ketoconazole, and clotrimazole also inhibited the cytochrome-c reductase activity of nitric-oxide synthase competitively versus calmodulin concentration, with apparent Ki (IC50) values of 8, 20, and 0.8 microM, respectively. Miconazole, ketoconazole, and clotrimazole inhibited the activity of calmodulin-dependent cyclic nucleotide phosphodiesterase competitively versus calmodulin concentration, with apparent Ki values of 6, 18, and 25 microM, respectively. These observations are consistent with the proposal that the antifungal imidazoles inhibit citrulline formation by interaction with the nitric-oxide synthase at two sites. Interaction at site 1 reduces the responsiveness of the enzyme to activation by calmodulin, whereas interaction at site 2 (involving putative binding of the imidazole to the heme iron) reduces the maximal velocity of citrulline formation. The interactions of calmodulin antagonists at site 1 occur at substantially lower concentrations of drug than those at site 2 and are the principal determinant of enzyme inhibition.
Wolff
D J
DJ
Department of Pharmacology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway 08854.
Datto
G A
GA
Samatovicz
R A
RA
D000594:Amino Acid Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology*; D001708:Biopterins / Q000031:analogs & derivatives / Q000494:pharmacology; D001921:Brain / Q000201:enzymology*; D002147:Calmodulin / Q000494:pharmacology*; D002417:Cattle; D002956:Citrulline / Q000378:metabolism; D003022:Clotrimazole / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology*; D007654:Ketoconazole / Q000494:pharmacology; D007700:Kinetics; D008825:Miconazole / Q000494:pharmacology; D009245:NADH Dehydrogenase / Q000378:metabolism*; D019001:Nitric Oxide Synthase; D010911:Pituitary Neoplasms; D051381:Rats; D014407:Tumor Cells, Cultured
D000935:Antifungal Agents; D002147:Calmodulin; D007093:Imidazoles; D001708:Biopterins; D002956:Citrulline; D008825:Miconazole; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; D009245:NADH Dehydrogenase; C003402:sapropterin; D003022:Clotrimazole; D007654:Ketoconazole