The CC chemokine receptor-1 (CCR1) is a prime therapeutic target for treating autoimmune diseases. Through high capacity screening followed by chemical optimization, we identified a novel non-peptide CCR1 antagonist, R-N-[5-chloro-2-[2-[4-[(4-fluorophenyl)methyl]-2-methyl-1-piperazinyl]-2-oxoethoxy]phenyl]urea hydrochloric acid salt (BX 471). Competition binding studies revealed that BX 471 was able to displace the CCR1 ligands macrophage inflammatory protein-1␣ (MIP-1␣), RANTES, and monocyte chemotactic protein-3 (MCP-3) with high affinity (K i ranged from 1 nM to 5.5 nM). BX 471 was a potent functional antagonist based on its ability to inhibit a number of CCR1-mediated effects including Ca 2؉ mobilization, increase in extracellular acidification rate, CD11b expression, and leukocyte migration. BX 471 demonstrated a greater than 10,000-fold selectivity for CCR1 compared with 28 G-protein-coupled receptors. Pharmacokinetic studies demonstrated that BX 471 was orally active with a bioavailability of 60% in dogs. Furthermore, BX 471 effectively reduces disease in a rat experimental allergic encephalomyelitis model of multiple sclerosis. This study is the first to demonstrate that a non-peptide chemokine receptor antagonist is efficacious in an animal model of an autoimmune disease. In summary, we have identified a potent, selective, and orally available CCR1 antagonist that may be useful in the treatment of chronic inflammatory diseases.It is clear that the inappropriate interaction of immune cells, such as T lymphocytes and monocytes, can lead to extensive inflammation and tissue destruction, which is a hallmark of several autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. Immune cells are sent on their destructive journey by chemoattractant molecules known as chemokines, which interact with and signal through specific cell surface chemokine receptors. Chemokine receptors belong to the GPCR 1 superfamily and have been viewed as attractive therapeutic targets by the pharmaceutical industry mainly because of their central role in regulating leukocyte trafficking. The premise that drugs that can inhibit the directed migration and activation of immune cells could be useful therapeutically has prompted the search for specific and highly potent chemokine receptor antagonists.Autoimmune diseases like multiple sclerosis and rheumatoid arthritis are characterized by interactions between invading T lymphocytes and tissue macrophages that result in extensive inflammation, tissue damage, and chronic disease pathologies. Numerous studies have demonstrated CCR1 expression in these cell types, and a variety of evidence provides strong in vivo concept validation for a role of this receptor in animal models of these diseases. For example, Karpus et al. (1, 2) were able to show in a mouse EAE model of multiple sclerosis that antibodies to MIP-1␣ prevented the development of both initial and relapsing paralytic disease as well as infiltration of mononuclear cells into the central nervous system. Treatment wit...

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...

Atherosclerosis develops and progresses spontaneously in apolipoprotein E-knockout (apoE-KO) mice. A direct consequence of atherosclerosis is an increase in vascular stiffness. Pulse wave velocity (PWV) has been used to assess the stiffness of large vessels and was found to be increased in patients with atherosclerosis. In the present study, aortic stiffness was assessed by PWV in 4- and 13-mo-old apoE-KO mice and age-matched controls (C57BL/6J). In 13-mo-old apoE-KO mice with extensive atherosclerotic lesions in the aorta (61 ± 4%), PWV increased significantly (3.8 ± 0.2 m/s) compared with controls (2.9 ± 0.2 m/s). Endothelial nitric oxide (EDNO)-mediated vasorelaxation in response to ACh was markedly diminished in the aortic rings isolated from 13-mo-old apoE-KO mice compared with age-matched controls. In contrast, in 4-mo-old apoE-KO mice with only moderate atherosclerotic lesions in the aorta (23 ± 5%), there were no significant changes in PWV and EDNO-mediated relaxation compared with controls. Blood pressure was not different among the four groups of mice. There were no significant differences in endothelium-independent vascular responses to sodium nitroprusside among different groups investigated. Histological evaluation revealed focal fragmentation of the elastic laminae in the aortic walls of 13-mo-old apoE-KO mice. These results demonstrate for the first time that aortic stiffness determined by PWV increases in 13-mo-old apoE-KO mice. Endothelial dysfunction and elastic destruction in vascular wall caused by atherosclerosis may have contributed.

This study investigated the role of endogenous nitric oxide (NO) in the progression of atherosclerosis in apolipoprotein E-deficient [apoE-knockout (KO)] mice. Mice were treated with N(omega)-nitro-L-arginine methyl ester (L-NAME) an inhibitor of nitric oxide synthase (NOS) or with the NOS substrate L-arginine for 8 wk. L-NAME treatment resulted in a significant inhibition of NO-mediated vascular responses and a significant increase in the atherosclerotic plaque/surface area in the aorta of apoE-KO mice. L-arginine treatment had no influence on endothelial function and did not alter lesion size. Mean arterial blood pressure and serum lipid levels were not altered by the treatments. At the beginning of the study impairment in endothelial function was only apparent in the case of N(G)-nitro-L-arginine-induced, NO-mediated contraction, whereas ACh-induced, NO-mediated relaxation was not different between age-matched apoE-KO and C57Bl/6J mice. After the 8-wk treatment with the NOS inhibitor, both NO-mediated responses were significantly inhibited. The acceleration in lesion size concomitant to the severely impaired NO-mediated responses indicates that lack of endogenous NO is an important progression factor of atherosclerosis in the apoE-KO mouse.

Lipoxin A(4) (LXA(4)) is a structurally and functionally distinct natural product called an eicosanoid, which displays immunomodulatory and anti-inflammatory activity but is rapidly metabolized to inactive catabolites in vivo. A previously described analogue of LXA(4), methyl (5R,6R,7E,9E,11Z,13E,15S)-16-(4-fluorophenoxy)-5,6,15-trihydroxy-7,9,11,13-hexadecatetraenoate (2, ATLa), was shown to have a poor pharmacokinetic profile after both oral and intravenous administration, as well as sensitivity to acid and light. The chemical stability of the corresponding E,E,E-trien-11-yne analogue, 3, was improved over 2 without loss of efficacy in the mouse air pouch model of inflammation. Careful analysis of the plasma samples from the pharmacokinetic assays for both 2 and 3 identified a previously undetected metabolite, which is consistent with metabolism by beta-oxidation. The formation of the oxidative metabolites was eliminated with the corresponding 3-oxatetraene, 4, and the 3-oxatrien-11-yne, 5, analogues of 2. Evaluation of 3-oxa analogues 4 and 5 in calcium ionophore-induced acute skin inflammation model demonstrated similar topical potency and efficacy compared to 2. The 3-oxatrien-11-yne analogue, 5, is equipotent to 2 in an animal model of inflammation but has enhanced metabolic and chemical stability and a greatly improved pharmacokinetic profile.

Increased nitric oxide (NO) production by inducible NO synthase (NOS2), an obligate homodimer, is implicated in the cardiovascular sequelae of sepsis. We tested the ability of a highly selective NOS2 dimerization inhibitor (BBS-2) to prevent endotoxin-induced systemic hypotension, myocardial dysfunction, and impaired hypoxic pulmonary vasoconstriction (HPV) in mice. Mice were challenged with Escherichia coli endotoxin before treatment with BBS-2 or vehicle. Systemic blood pressure was measured before and 4 and 7 h after endotoxin challenge, and echocardiographic parameters of myocardial function were measured before and 7 h after endotoxin challenge. The pulmonary vasoconstrictor response to left mainstem bronchus occlusion, which is a measure of HPV, was studied 22 h after endotoxin challenge. BBS-2 treatment alone did not alter baseline hemodynamics. BBS-2 treatment blocked NOS2 dimerization and completely inhibited the endotoxin-induced increase of plasma nitrate and nitrite levels. Treatment with BBS-2 after endotoxin administration prevented systemic hypotension and attenuated myocardial dysfunction. BBS-2 also prevented endotoxin-induced impairment of HPV. In contrast, treatment with NG-nitro-l-arginine methyl ester, which is an inhibitor of all three NOS isoforms, prevented the systemic hypotension but further aggravated the myocardial dysfunction associated with endotoxin challenge. Treatment with BBS-2 prevented endotoxin from causing key features of cardiovascular dysfunction in endotoxemic mice. Selective inhibition of NOS2 dimerization with BBS-2, while sparing the activities of other NOS isoforms, may prove to be a useful treatment strategy in sepsis.

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Activated protein C (APC) is a plasma serine protease with antithrombotic and cytoprotective functions. Based on the hypothesis that specific inhibition of APC's anticoagulant but not its cytoprotective activity can be beneficial for hemophilia therapy, 2 types of inhibitory monoclonal antibodies (mAbs) are tested: A type I active-site binding mAb and a type II mAb binding to an exosite on APC (required for anticoagulant activity) as shown by X-ray crystallography. Both mAbs increase thrombin generation and promote plasma clotting. Type I blocks all APC activities, whereas type II preserves APC's cytoprotective function. In normal monkeys, type I causes many adverse effects including animal death. In contrast, type II is well-tolerated in normal monkeys and shows both acute and prophylactic dose-dependent efficacy in hemophilic monkeys. Our data show that the type II mAb can specifically inhibit APC's anticoagulant function without compromising its cytoprotective function and offers superior therapeutic opportunities for hemophilia.