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...

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...

Studies have been carried out to investigate aspects of the structure of thrombomodulin, an endothelial cell glycoprotein that binds thrombin and accelerates both the thrombin-dependent activation of protein C and the inhibition of antithrombin III. We have determined the shape of Solulin TM , a soluble recombinant form of human thrombomodulin missing the transmembrane and cytoplasmic domains, by electron microscopy of preparations rotary-shadowed with tungsten. Solulin appears to be an elongated molecule about 20 nm long that has a large nodule at one end and a smaller nodule near the other end from which extends a thin strand. About half of the molecules form bipolar dimers apparently via interactions between these thin strands. Electron microscopy of complexes formed between Solulin and human ␣-thrombin revealed that a single thrombin molecule appears to bind to the smaller nodule of Solulin, suggesting that this region contains the epidermal growth factor-like domains 5 and 6. Epidermal growth factor-like domains 1-4 comprise the connector between the small and large nodule, which is the lectin-like domain; the thin strand at the other end of the molecule is the carbohydrate-rich region. With chondroitin sulfatecontaining soluble thrombomodulin produced from either human melanoma cells Bowes or Chinese hamster ovary cells, a higher percentage of molecules bound thrombin and, in some cases, two thrombin molecules were attached to one soluble thrombomodulin in approximately the same region. These structural studies provide insight into the structure of thrombomodulin and its interactions with thrombin as well as aspects of the mechanisms of its actions.Human thrombomodulin is a single chain glycoprotein made up of 557 amino acids, as well as N-and O-linked carbohydrate, that acts as a co-factor in the activation of protein C by thrombin and a modulator for the specificity of thrombin (1-6). From analysis of the amino acid sequence, thrombomodulin is made up of five regions, a lectin-like domain, a group of six epidermal growth factor-like modules, a serine-, threonine-, and O-glycosylation-rich region, a transmembrane sequence, and a cytoplasmic domain. There is, however, little information on the spatial arrangement of these domains.In its actions as an anticoagulant, thrombomodulin has several functions: the binding of thrombin, resulting in the modulation of its substrate specificity; a co-factor in the activation of protein C; and a co-factor in the inhibition of thrombin by antithrombin III (1, 2, 4 -6). The binding of thrombomodulin to thrombin effectively destroys the specificity of this enzyme for catalyzing the conversion of fibrinogen to fibrin, the activation of factors V, VIII, and XIII, and the activation of platelets. At the same time, in the presence of calcium ions, thrombomodulin acts as a co-factor for the activation of protein C, which, together with protein

Platelet factor 4 (PF4) is an abundant platelet ␣-granule heparin-binding protein. We have previously shown that PF4 accelerates up to 25-fold the proteolytic conversion of protein C to activated protein C by the thrombin⅐thrombomodulin complex by increasing its affinity for protein C 30-fold. This stimulatory effect requires presence of the ␥-carboxyglutamic acid (Gla) domain in protein C and is enhanced by the presence of a chondroitin sulfate glycosaminoglycan (GAG) domain on thrombomodulin. We hypothesized that cationic PF4 binds to both protein C and thrombomodulin through these anionic domains. Qualitative SDS-polyacrylamide gel electrophoresis analysis of avidin extracts of solutions containing biotinylated PF4 and candidate ligands shows that PF4 binds to GAG؉ but not GAG؊ forms of thrombomodulin and native but not Gla-domainless protein C. Quantitative analysis using the surface plasmon resonance-based BIAcore TM biosensor system confirms the extremely high affinity of PF4 for heparin (K D ‫؍‬ 4 nM) and shows that PF4 binds to GAG؉ thrombomodulin with a K D of 31 nM and to protein C with a K D of 0.37 M. In contrast, PF4 had no measurable interaction with GAG؊ thrombomodulin or Gla-domainless protein C. Western blot analysis of normal human plasma extracted with biotinylated PF4 demonstrates PF4 binding to protein C in a physiologic context. Thus, PF4 binds with relative specificity and high affinity to the GAG؊ domain of thrombomodulin and the Gla domain of protein C. These interactions may enhance the affinity of the thrombin⅐thrombomodulin complex for protein C and thereby promote the generation of activated protein C.

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