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 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. 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 reductase domain that contains binding sites for NADPH, FAD, FMN, and calmodulin, and an N-terminal oxygenase domain that contains binding sites for heme, tetrahydrobiopterin (H 4 B), and L-arginine (3-7). As in cytochrome P-450, the NOS heme iron coordinates to the protein through a cysteine thiolate (6 -8), binds O 2 as a sixth ligand (9), and participates directly in catalysis (9 -13). The heme ligands CO, Ϫ CN, imidazole, N-phenylimidazoles, and other imidazole-containing compounds all inhibit NO synthesis (10, 14 -17).The NOS isoforms are only active as homodimers (18 -20). For iNOS (5) and nNOS (21-23), only the oxygenase domains of two monomers interact to form the dimer (24). Dimerization of iNOS is required for fully coupled enzyme activity because the flow of electrons during catalysis occurs in trans from the reductase domain of one monomer to the oxygenase domain of the other monomer (25). Dimerization of NOS monomers is initiated by heme insertion, which results in rapid conformational changes (18,19,26). The heme-containing iNOS monomer is an intermediate in dimerization and, in the presence of H 4 B and L-arginine, forms a stable active dimer (18,24,(27)(28)(29).Highly potent and selective pyrimidineimidazole-based iNOS dimerization inhibitors were disc...

Monomeric inducible nitric oxide synthase localizes to peroxisomes in hepatocytes

“…6D). Similar findings were observed when complete monomerization of iNOS was achieved by using a mechanistic inhibitor that suppresses dimerization (data not shown) (17). Thus iNOS monomerization does not force a greater distribution of the enzyme to peroxisomes.…”

Regulation of inducible nitric oxide synthase by rapid cellular turnover and cotranslational down-regulation by dimerization inhibitors

“…23), acts by binding to the iNOS monomer. Crystallographic studies showed that BBS-1 coordinates directly to the heme at the sixth axial position and disrupts helices 7a and 8 in the active site (23,24). Thus, in occupying the iNOS active site, BBS-1 appears to allosterically prevent appropriate protein-protein interactions between monomers and prevents dimer formation.…”

“…BBS-1 binding to iNOS monomer is previously assumed to lead to the formation of a ''dead-end'' inhibitor-monomer complex (23,24). Therefore, it was logical to speculate that the inhibitor-monomer complex may be rapidly degraded compared with the active dimeric iNOS.…”

“…Thus, in occupying the iNOS active site, BBS-1 appears to allosterically prevent appropriate protein-protein interactions between monomers and prevents dimer formation. The inhibitor does not, however, bind to and has no effect on the catalytic activity of the already formed iNOS dimer, because it cannot gain access to the heme site in the dimer (23,24).…”

“…These compounds have been shown to inhibit iNOS dimerization by binding to its heme ligand. However, they have low affinity to NOS and lack specificity for the iNOS isoform (22)(23)(24). Recently, screening of an encoded combinatorial chemical library, designed based on a structurally related series of compounds of a pyrimidineimidazole core, resulted in the discovery of a class of allosteric high-affinity potent and selective iNOS inhibitors.…”