The anti-inflammatory eicosanoid lipoxin A4 (LXA4), aspirin-triggered 15-epi-LXA4, and their stable analogs down-regulate IL-8 secretion and subsequent recruitment of neutrophils by intestinal epithelia. In an effort to elucidate the mechanism by which these lipid mediators modulate cellular proinflammatory programs, we surveyed global epithelial gene expression using cDNA microarrays. LXA4 analog alone did not significantly affect expression of any of the >7000 genes analyzed. However, LXA4 analog pretreatment attenuated induction of ∼50% of the 125 genes up-regulated in response to the gastroenteritis-causing pathogen Salmonella typhimurium. A major subset of genes whose induction was reduced by LXA4 analog pretreatment is regulated by NF-κB, suggesting that LXA4 analog was influencing the activity of this transcription factor. Nanomolar concentrations of LXA4 analog reduced NF-κB-mediated transcriptional activation in a LXA4 receptor-dependent manner and inhibited induced degradation of IκBα. LXA4 analog did not affect earlier stimulus-induced signaling events that lead to IκBα degradation, such as S. typhimurium-induced epithelial Ca2+ mobilization or TNF-α-induced phosphorylation of IκBα. To establish the in vivo relevance of these findings, we examined whether LXA4 analogs could affect intestinal inflammation in vivo using the mouse model of DSS-induced inflammatory colitis. Oral administration of LXA4 analog (15-epi-16-para-fluoro-phenoxy-LXA4, 10 μg/day) significantly reduced the weight loss, hematochezia, and mortality that characterize DSS colitis. Thus, LXA4 analog-mediated down-regulation of proinflammatory gene expression via inhibition of the NF-κB pathway can be therapeutic for diseases characterized by mucosal inflammation.

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-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 (Kd Ϸ 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 cellbased 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 mg͞kg in a rat model of endotoxininduced systemic iNOS induction. Thus, this class of dimerization inhibitors has broad therapeutic potential in iNOS-mediated pathologies.T he mammalian nitric ox ide synthase (NOS) (EC 1.14.13.39) enzyme family comprises three isoforms: inducible (iNOS), neuronal, and endothelial NOS. NOS isoforms are homodimers that catalyze NADPH-dependent oxidation of L-arginine to NO⅐ and citrulline (1-3). NOS monomers consist of an oxidoreductase domain and an oxygenase domain. The reductase domain is homologous to cytochrome P450 reductase and contains binding sites for NADPH, FAD, and FMN (4, 5). The oxygenase domain has binding sites for L-arginine, the heme prosthetic group, and tetrahydrobiopterin (H 4 B). Formation of stable NOS homodimers requires structural elements in the oxygenase domain and is an H 4 B-, substrate-, and heme-dependent process (6 -8). Dimerization of NOS is required for fully coupled enzyme activity because the f low of electrons during catalysis occurs in trans from the reductase domain of one monomer subunit to the oxygenase domain of the other monomer (9). The crystal structures of oxygenase domains of murine iNOS monomer (10), murine and human iNOS dimer (11-13), and human and bovine endothelial NOS dimer (13, 14) indicate a high degree of structural similarity within the critical catalytic center and dimer interface regions between NOS isoforms.NO⅐ plays a pivotal role in the physiology and pathophysiology of the central nervous, cardiovascular, and immune systems (15-17). The reactivity of NO⅐ toward molecular oxygen, thiols, transition metal centers, and other biological targets enables NO⅐ to function both as a rapidly reversible, specific, and local signal transduction molecule as well as a nonspecific mediator of tissue damage (1...

In this study we examined the role of inducible nitric oxide synthase (iNOS) in acute respiratory distress syndrome (ARDS) in sheep with severe combined burn and smoke inhalation injury. BBS-2, a potent and highly selective iNOS dimerization inhibitor, was used to exclude effects on the endothelial and neuronal NOS isoforms. Seven days after surgical recovery, sheep were given a burn (40% of total body surface, 3rd degree) and insufflated with cotton smoke (48 breaths, < 40 degrees C) under anesthesia. BBS-2 was provided by constant infusion at 100 microg/kg/hour, beginning 1 hour after injury. During 48 hours, control sheep developed multiple signs of ARDS. These included decreased pulmonary gas exchange, increased pulmonary edema, abnormal lung compliance, and extensive airway obstruction. These pathologies were associated with a large increase in tracheal blood flow and elevated plasma NO2-/NO3- (NOx) levels. These variables were all stable in sham animals. Treatment of injured sheep with BBS-2 attenuated the increases in tracheal blood flow and plasma NOx levels, and significantly attenuated all the pulmonary pathologies that were noted. The results provide definitive evidence that iNOS is a key mediator of pulmonary pathology in sheep with ARDS resulting from combined burn and smoke inhalation injury.

The crystal structures of the heme domain of human inducible nitric-oxide synthase (NOS-2) in zinc-free and -bound states have been solved. In the zinc-free structure, two symmetry-related cysteine residues form a disulfide bond. In the zinc-bound state, these same two cysteine residues form part of a zinc-tetrathiolate (ZnS 4 ) center indistinguishable from that observed in the endothelial isoform (NOS-3). As in NOS-3, ZnS 4 plays a key role in stabilizing intersubunit contacts and in maintaining the integrity of the cofactor (tetrahydrobiopterin) binding site of NOS-2. A comparison of NOS-2 and NOS-3 structures illustrates the conservation of quaternary structure, tertiary topology, and substrate and cofactor binding sites, in addition to providing insights on isoform-specific inhibitor design. The structural comparison also reveals that pterin binding does not preferentially stabilize the dimer interface of NOS-2 over NOS-3. Nitric-oxide synthases (NOS)1 are a family of enzymes that produce nitric oxide (NO) and L-citrulline from L-arginine in the presence of NADPH and O 2 (1). NOS maintains a bidomain structure (2) with the catalytic center residing in the heme domain utilizing electrons derived from the cytochrome P450 reductase-like biflavin domain. The heme domain also contains the binding site for enzyme cofactor, tetrahydrobiopterin (H 4 B).The NOS family currently consists of three mammalian isoforms (3, 4). The endothelial (eNOS or NOS-3) and neuronal (nNOS or NOS-1) isoforms are constitutive and are activated by Ca 2ϩ -dependent calmodulin binding. The inducible isoform (iNOS or NOS-2) entertains a tightly bound calmodulin subunit and is regulated at the level of transcription. Because overproduction of NO by NOS-1 and NOS-2 leads to pathological conditions in stroke (5) and shock (6), respectively, isoformspecific inhibition of NOS has wide therapeutic potential. As a result, there is considerable interest within the pharmaceutical community to develop isoform-specific NOS inhibitors (7). Crystal structures of the catalytic heme domain for the various NOS isoforms are essential if rational drug design is to be applied to NO up/down-regulation.Crane et al. (8) solved the crystal structure of the murine NOS-2 heme domain, which established the overall fold of NOS as well as the location and structure of the pterin and L-Arg binding sites. The structure of the NOS-3 heme domain also has been solved (9), which revealed a novel zinc tetrathiolate (ZnS 4 ) at the bottom of the dimer interface. In the structure of the heme domain of murine NOS-2 (8), a disulfide was identified in the zinc binding site, and no zinc was seen in the structure. To understand the structural consequences of the metal center in other NOS isoforms, we have solved the crystal structure of human NOS-2 in both zinc-free and -bound forms. The availability of a zinc-bound NOS-2 affords direct comparison with NOS-3 and provides a molecular basis for the development of isoform-specific inhibitors. MATERIALS AND METHODS Expression and Pur...

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...