Mechanistic Studies with Potent and Selective Inducible Nitric-oxide Synthase Dimerization Inhibitors
Journal of Biological Chemistry
A series of potent and selective inducible nitric-oxide synthase (iNOS) inhibitors was shown to prevent iNOS dimerization in cells and inhibit iNOS in vivo. These inhibitors are now shown to block dimerization of purified human iNOS monomers. A 3 H-labeled inhibitor bound to full-length human iNOS monomer with apparent K d ϳ1.8 nM and had a slow off rate, 1.2 ؋ 10 ؊4 s ؊1 . Inhibitors also bound with high affinity to both murine full-length and murine oxygenase domain iNOS monomers.
... nomers. Spectroscopy and competition binding with imidazole confirmed an inhibitor-heme interaction. Inhibitor affinity in the binding assay (apparent K d values from 330 pM to 27 nM) correlated with potency in a cellbased iNOS assay (IC 50 values from 290 pM to 270 nM). Inhibitor potency in cells was not prevented by medium supplementation with L-arginine or sepiapterin, but inhibition decreased with time of addition after cytokine stimulation. The results are consistent with a mechanism whereby inhibitors bind to a heme-containing iNOS monomer species to form an inactive iNOS monomer-heme-inhibitor complex in a pterin-and L-arginineindependent manner. The selectivity for inhibiting dimerization of iNOS versus endothelial and neuronal NOS suggests that the energetics and kinetics of monomer-dimer equilibria are substantially different for the mammalian NOS isoforms. These inhibitors provide new research tools to explore these processes. The mammalian nitric-oxide synthase (NOS) 1 family consists of three isoforms as follows: cytokine-inducible (iNOS), neuronal (nNOS), and endothelial NOS (eNOS). NOS isoforms are homodimers that catalyze NADPH-dependent oxidation of L-arginine to nitric oxide (NO) and L-citrulline (1-3). Each monomer subunit of the dimer consists of a C-terminal reduc-