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

Potent and selective inhibitors of inducible nitric oxide synthase (iNOS) ( EC 1.14.13.39) 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-nanomola r IC50 values and thus were >1,000-fold more potent than the substrate-base d direct iNOS inhibitors 1400W and N-methyl-L-arginine. Biochemical studies confirmed that inhibitors caused accumulation of iNOS monomers in mouse ma crophage RAW 264.7 cells. High affinity (K-d approximate to 3 nM) of inhibi tors for isolated iNOS monomers was confirmed by using a radioligand bindin g 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 coordi nation and substantial perturbation of the substrate binding site and the d imerization interface, indicating that this small molecule acts by alloster ically disrupting protein-protein interactions at the dimer interface. Thes e results provide a mechanism-based approach to highly selective iNOS inhib ition. Inhibitors were active in vivo, with ED50 values of <2 mg/kg in a ra t model of endotoxin-induced systemic iNOs induction. Thus, this class of d imerization inhibitors has broad therapeutic potential in iNOS-mediated pat hologies.