title: Mechanistic studies with potent and selective inducible nitric-oxide synthase dimerization inhibitors.
abstract: 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 3H-labeled inhibitor bound to full-length human iNOS monomer with apparent Kd approximately 1.8 nm and had a slow off rate, 1.2 x 10(-4) x s(-1). Inhibitors also bound with high affinity to both murine full-length and murine oxygenase domain iNOS monomers. Spectroscopy and competition binding with imidazole confirmed an inhibitor-heme interaction. Inhibitor affinity in the binding assay (apparent Kd values from 330 pm to 27 nm) correlated with potency in a cell-based iNOS assay (IC50 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-arginine-independent 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.
authors.affiliation: Cardiovascular Research, Berlex Biosciences, Richmond, California 94804-0099, USA.
authors.forename: Eric
authors.initials: E
authors.lastname: Blasko
authors.forename: Charles B
authors.initials: CB
authors.lastname: Glaser
authors.forename: James J
authors.initials: JJ
authors.lastname: Devlin
authors.forename: Wei
authors.initials: W
authors.lastname: Xia
authors.forename: Richard I
authors.initials: RI
authors.lastname: Feldman
authors.forename: Mark A
authors.initials: MA
authors.lastname: Polokoff
authors.forename: Gary B
authors.initials: GB
authors.lastname: Phillips
authors.forename: Marc
authors.initials: M
authors.lastname: Whitlow
authors.forename: Douglas S
authors.initials: DS
authors.lastname: Auld
authors.forename: Kirk
authors.initials: K
authors.lastname: McMillan
authors.forename: Sanjay
authors.initials: S
authors.lastname: Ghosh
authors.forename: Dennis J
authors.initials: DJ
authors.lastname: Stuehr
authors.forename: John F
authors.initials: JF
authors.lastname: Parkinson
mesh_terms: D019281:Dimerization; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007093:Imidazoles / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D052247:Nitric Oxide Synthase Type II; D011743:Pyrimidines / Q000494:pharmacology; D011869:Radioligand Assay
chemical_list: D004791:Enzyme Inhibitors; D007093:Imidazoles; D011743:Pyrimidines; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II