title: Allosteric inhibitors of inducible nitric oxide synthase dimerization discovered via combinatorial chemistry.
abstract: Potent and selective inhibitors of inducible nitric oxide synthase (iNOS) (EC ) were identified in an encoded combinatorial chemical library that blocked human iNOS dimerization, and thereby NO production. In a cell-based iNOS assay (A-172 astrocytoma cells) the inhibitors had low-nanomolar IC(50) values and thus were >1,000-fold more potent than the substrate-based direct iNOS inhibitors 1400W and N-methyl-l-arginine. Biochemical studies confirmed that inhibitors caused accumulation of iNOS monomers in mouse macrophage RAW 264.7 cells. High affinity (K(d) approximately 3 nM) of inhibitors for isolated iNOS monomers was confirmed by using a radioligand binding assay. Inhibitors were >1,000-fold selective for iNOS versus endothelial NOS dimerization in a cell-based assay. The crystal structure of inhibitor bound to the monomeric iNOS oxygenase domain revealed inhibitor-heme coordination and substantial perturbation of the substrate binding site and the dimerization interface, indicating that this small molecule acts by allosterically disrupting protein-protein interactions at the dimer interface. These results provide a mechanism-based approach to highly selective iNOS inhibition. Inhibitors were active in vivo, with ED(50) values of <2 mg/kg in a rat model of endotoxin-induced systemic iNOS induction. Thus, this class of dimerization inhibitors has broad therapeutic potential in iNOS-mediated pathologies.
authors.affiliation: Pharmacopeia, Inc., Princeton, NJ 08512, USA.
authors.forename: K
authors.initials: K
authors.lastname: McMillan
authors.forename: M
authors.initials: M
authors.lastname: Adler
authors.forename: D S
authors.initials: DS
authors.lastname: Auld
authors.forename: J J
authors.initials: JJ
authors.lastname: Baldwin
authors.forename: E
authors.initials: E
authors.lastname: Blasko
authors.forename: L J
authors.initials: LJ
authors.lastname: Browne
authors.forename: D
authors.initials: D
authors.lastname: Chelsky
authors.forename: D
authors.initials: D
authors.lastname: Davey
authors.forename: R E
authors.initials: RE
authors.lastname: Dolle
authors.forename: K A
authors.initials: KA
authors.lastname: Eagen
authors.forename: S
authors.initials: S
authors.lastname: Erickson
authors.forename: R I
authors.initials: RI
authors.lastname: Feldman
authors.forename: C B
authors.initials: CB
authors.lastname: Glaser
authors.forename: C
authors.initials: C
authors.lastname: Mallari
authors.forename: M M
authors.initials: MM
authors.lastname: Morrissey
authors.forename: M H
authors.initials: MH
authors.lastname: Ohlmeyer
authors.forename: G
authors.initials: G
authors.lastname: Pan
authors.forename: J F
authors.initials: JF
authors.lastname: Parkinson
authors.forename: G B
authors.initials: GB
authors.lastname: Phillips
authors.forename: M A
authors.initials: MA
authors.lastname: Polokoff
authors.forename: N H
authors.initials: NH
authors.lastname: Sigal
authors.forename: R
authors.initials: R
authors.lastname: Vergona
authors.forename: M
authors.initials: M
authors.lastname: Whitlow
authors.forename: T A
authors.initials: TA
authors.lastname: Young
authors.forename: J J
authors.initials: JJ
authors.lastname: Devlin
mesh_terms: D000494:Allosteric Regulation; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D020650:Combinatorial Chemistry Techniques; D019281:Dimerization; D004791:Enzyme Inhibitors / Q000138:chemical synthesis* / Q000494:pharmacology; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D015394:Molecular Structure; D009569:Nitric Oxide / Q000097:blood; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors*; D052247:Nitric Oxide Synthase Type II; D011485:Protein Binding; D051381:Rats
chemical_list: D004791:Enzyme Inhibitors; D009569:Nitric Oxide; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; C496318:Nos2 protein, rat