The Inhibition of the Constitutive and Inducible Nitric-Oxide Synthase Isoforms by Indazole Agents

Abstract Citrulline formation by Ca 2+ -calmodulin (CaM)-dependent nitric oxide synthase from bovine brain is inhibited reversibly by indazole, 5-nitro-, 6-nitro-, and 7-nitroindazole with IC 50 values of 2.3 mM, 1.15 mM, 40 μM, and 2.5 μM, respectively. Inhibition of citrulline formation by 7-nitroindazole exhibited a K i value of 0.16 μM and was competitive versus both arginine substrate and (6 R )-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 IC 50 value of 0.6 μM. Citrulline formation by the interferon-γ/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 IC 50 values of 470, 240, 56, and 20 μM, respectively. Inhibition of citrulline formation by 7-nitroindazole exhibited a K i value of 1.6 μM and was noncompetitive versus arginine substrate but competitive versus (6 R )-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 IC 50 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.