THE INHIBITION OF THE CONSTITUTIVE AND INDUCIBLE NITRIC-OXIDE SYNTHASE ISOFORMS BY INDAZOLE AGENTS

Citrulline formation by Ca2+-calmodulin (CaM)-dependent nitric oxide synthase from bovine brain is inhibited reversibly by indazole, 5-nitro-, 6-nitro-, and 7-nitroindazole with IC,, values of 2.3 mM, 1.15 mM, 40 mu M, and 2.5 mu M, respectively. Inhibition of citrulline formation by 7-nitroindazole exhibited a K-i value of 0.16 mu M and was competitive versus both arginine substrate and (6R)-5,6,7,8-tetrahydrobiopterin cofactor. The NADPH oxidase activity of bovine brain CaM-dependent nitric oxide synthase was inhibited by 7-nitroindazole with an IC50 value of 0.6 mu M. Citrulline formation by the interferon-gamma/lipopolysaccharide-inducible nitric oxide synthase of murine macrophages (264.7 cell line) is inhibited reversibly by indazole, 5-nitro-, 6-nitro-, and 7-nitroindazole with IC50 values of 470, 240, 56, and 20 mu M, respectively. Inhibition of citrulline formation by 7-nitroindazole exhibited a K-i value of 1.6 mu M and was noncompetitive versus arginine substrate but competitive versus (6R)-5,6,7,8-tetrahydrobiopterin cofactor. None of the indazoles tested inhibited the cytochrome c reductase activity of either nitric oxide synthase isoform at concentrations up to 1000-fold higher than their IC50 values for inhibition of citrulline formation. These observations are consistent with the proposal that the indazoles exert their inhibitory actions by interaction with the heme-iron of nitric oxide synthase such that oxygen does not bind. (C) 1994 Academic Press,Inc.