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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.

SUBMITTER: McMillan K 

PROVIDER: S-EPMC26464 | biostudies-literature | 2000 Feb

REPOSITORIES: biostudies-literature

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Allosteric inhibitors of inducible nitric oxide synthase dimerization discovered via combinatorial chemistry.

McMillan K K   Adler M M   Auld D S DS   Baldwin J J JJ   Blasko E E   Browne L J LJ   Chelsky D D   Davey D D   Dolle R E RE   Eagen K A KA   Erickson S S   Feldman R I RI   Glaser C B CB   Mallari C C   Morrissey M M MM   Ohlmeyer M H MH   Pan G G   Parkinson J F JF   Phillips G B GB   Polokoff M A MA   Sigal N H NH   Vergona R R   Whitlow M M   Young T A TA   Devlin J J JJ  

Proceedings of the National Academy of Sciences of the United States of America 20000201 4


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 mon  ...[more]

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