Mechanistic Studies with Potent and Selective Inducible Nitric-oxide Synthase Dimerization Inhibitors

Eric Blasko, Charles B. Glaser, James J. Devlin, Wei Xia, Richard I. Feldman, Mark A. Polokoff, Gary B. Phillips, Marc Whitlow, Douglas S. Auld, Kirk McMillan, Sanjay Ghosh, Dennis J. Stuehr (+1 others)
2001 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.
more » ... 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-
doi:10.1074/jbc.m105691200 pmid:11689556 fatcat:ywwnw7j7gnh63cob2s7vzougsm