Abstract
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.
Highlights
The antifungal imidazoles miconazole, ketoconazole, and clotrimazole inhibit citrulline formation by nitricoxide synthase
Interaction at site 1 reduces the responsiveness of the enzyme to activation by calmodulin, whereas interaction at site 2 reduces the maximal velocity of citrulline formation
Miconazole, clotrimazole,and ketoconazole are antimycotic agents that inhibitergosterol biosynthesis in yeast by inhibiting14a-demethylase, amicrosomal cytochromeP-450-dependent enzyme system. l4a-Methyl sterols that accumulate intracellularly are incorporated into thleipid bilayer, disrupting its structure and cellular growth (1).Imidazole antimycotic agentshave been shown to inhibiat variety of cytochrome P-450-dependent enzyme activities including steroid aromatase(2) and ratliver microsomal cytochrome P 450 isoforms (3).The imidazole antimycotic agents,do not inhibit cytochrome-P-450 reductaseactivity
Summary
Materials-Miconazole, clotrimazole, PIPES, affinity-purified bovine intestinal mucosal alkaline phosphatase, dimethyl sulfoxide, guanosine 3’,5’-cyclicphosphate, and p-nitrophenyl phosphate were obtained from Sigma. CaM-dependent cyclic nucleotide phosphodiesterase was prepared from bovine brain by the procedure of Brostrom and Wolff (9). CaM-dependent phosphatase (calcineurin) was prepared from bovine brain by the procedure of Wolff and Sved (10). Assay of Citrulline Formation by Nitric-oxide Synthase-Standard incubations were prepared and conducted, and citrulline formation was assessed as described previously (8). Standard incubations were constructed in a reaction volume of 150 p1 containing 20 mM HEPES, pH 7.5, 5 mM MgC12, 30 p~ cGMP, 200 p~ Ca2+,2 p M CaM, 2 ng of bovine intestinal mucosal alkaline phosphatase,and 100,000cpm [3H]cGMP. The hydrolysis of pnitrophenyl phosphatewas measured in standard incubations of 1ml in disposable plastic cuvettes containing 50 mM HEPES, pH 7.5, 100 mM KC1, 20 mM p-nitrophenyl phosphate, and 1 mM MnC12with or without CaM. Methanol (5%)was found not to affect the myelin basic protein dephosphorylation rate
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