Molecular basis of ALK1-mediated signalling by BMP9/BMP10 and their prodomain-bound forms.

Activin receptor-like kinase 1 (ALK1)-mediated endothelial cell signalling in response to bone morphogenetic protein 9 (BMP9) and BMP10 is of significant importance in cardiovascular disease and cancer. However, detailed molecular mechanisms of ALK1-mediated signalling remain unclear. Here, we report crystal structures of the BMP10:ALK1 complex at 2.3 Å and the prodomain-bound BMP9:ALK1 complex at 3.3 Å. Structural analyses reveal a tripartite recognition mechanism that defines BMP9 and BMP10 specificity for ALK1, and predict that crossveinless 2 is not an inhibitor of BMP9, which is confirmed by experimental evidence. Introduction of BMP10-specific residues into BMP9 yields BMP10-like ligands with diminished signalling activity in C2C12 cells, validating the tripartite mechanism. The loss of osteogenic signalling in C2C12 does not translate into non-osteogenic activity in vivo and BMP10 also induces bone-formation. Collectively, these data provide insight into ALK1-mediated BMP9 and BMP10 signalling, facilitating therapeutic targeting of this important pathway.

Oral Anti-Tumour Necrosis Factor Domain Antibody V565 Provides High Intestinal Concentrations, and Reduces Markers of Inflammation in Ulcerative Colitis Patients.

V565 is an engineered TNFα-neutralising single domain antibody formulated into enteric coated mini-tablets to enable release in the intestine after oral administration as a possible oral treatment for inflammatory bowel disease (IBD). Following oral administration, ileal recovery of V565 was investigated in four patients with terminal ileostomy. Intestinal and systemic pharmacokinetics were measured in six patients with Crohn's disease and evidence of target engagement assessed in five patients with ulcerative colitis. Following oral administration, V565 was detected at micromolar concentrations in ileal fluid from the ileostomy patients and in stools of the Crohn's patients. In four of the five ulcerative colitis patients, biopsies taken after 7d dosing demonstrated V565 in the lamina propria with co-immunostaining on CD3+ T-lymphocytes and CD14+ macrophages. Phosphorylation of signalling proteins in biopsies taken after 7d oral dosing was decreased by approximately 50%. In conclusion, enteric coating of V565 mini-tablets provided protection in the stomach with gradual release in intestinal regions affected by IBD. Immunostaining revealed V565 tissue penetration and association with inflammatory cells, while decreased phosphoproteins after 7d oral dosing was consistent with V565-TNFα engagement and neutralising activity. Overall these results are encouraging for the clinical utility of V565 in the treatment of IBD.

Preclinical Development of a Novel, Orally-Administered Anti-Tumour Necrosis Factor Domain Antibody for the Treatment of Inflammatory Bowel Disease.

TNFα is an important cytokine in inflammatory bowel disease. V565 is a novel anti-TNFα domain antibody developed for oral administration in IBD patients, derived from a llama domain antibody and engineered to enhance intestinal protease resistance. V565 activity was evaluated in TNFα-TNFα receptor-binding ELISAs as well as TNFα responsive cellular assays and demonstrated neutralisation of both soluble and membrane TNFα with potencies similar to those of adalimumab. Although sensitive to pepsin, V565 retained activity after lengthy incubations with trypsin, chymotrypsin, and pancreatin, as well as mouse small intestinal and human ileal and faecal supernatants. In orally dosed naïve and DSS colitis mice, high V565 concentrations were observed in intestinal contents and faeces and immunostaining revealed V565 localisation in mouse colon tissue. V565 was detected by ELISA in post-dose serum of colitis mice, but not naïve mice, demonstrating penetration of disrupted epithelium. In an ex vivo human IBD tissue culture model, V565 inhibition of tissue phosphoprotein levels and production of inflammatory cytokine biomarkers was similar to infliximab, demonstrating efficacy when present at the disease site. Taken together, results of these studies provide confidence that oral V565 dosing will be therapeutic in IBD patients where the mucosal epithelial barrier is compromised.

Anaesthetic impairment of immune function is mediated via GABA(A) receptors.

BACKGROUND: GABA(A) receptors are members of the Cys-loop family of neurotransmitter receptors, proteins which are responsible for fast synaptic transmission, and are the site of action of wide range of drugs. Recent work has shown that Cys-loop receptors are present on immune cells, but their physiological roles and the effects of drugs that modify their function in the innate immune system are currently unclear. We are interested in how and why anaesthetics increase infections in intensive care patients; a serious problem as more than 50% of patients with severe sepsis will die. As many anaesthetics act via GABA(A) receptors, the aim of this study was to determine if these receptors are present on immune cells, and could play a role in immunocompromising patients. PRINCIPAL FINDINGS: We demonstrate, using RT-PCR, that monocytes express GABA(A) receptors constructed of α1, α4, ß2, γ1 and/or δ subunits. Whole cell patch clamp electrophysiological studies show that GABA can activate these receptors, resulting in the opening of a chloride-selective channel; activation is inhibited by the GABA(A) receptor antagonists bicuculline and picrotoxin, but not enhanced by the positive modulator diazepam. The anaesthetic drugs propofol and thiopental, which can act via GABA(A) receptors, impaired monocyte function in classic immunological chemotaxis and phagocytosis assays, an effect reversed by bicuculline and picrotoxin. SIGNIFICANCE: Our results show that functional GABA(A) receptors are present on monocytes with properties similar to CNS GABA(A) receptors. The functional data provide a possible explanation as to why chronic propofol and thiopental administration can increase the risk of infection in critically ill patients: their action on GABA(A) receptors inhibits normal monocyte behaviour. The data also suggest a potential solution: monocyte GABA(A) receptors are insensitive to diazepam, thus the use of benzodiazepines as an alternative anesthetising agent may be advantageous where infection is a life threatening problem.

Highly potent, orally available anti-inflammatory broad-spectrum chemokine inhibitors.

A series of 3-acylaminocaprolactams are inhibitors of chemokine-induced chemotaxis. Branching of the side chain alpha-carbon provides highly potent inhibitors of a range of CC and CXC chemokines. The most potent compound has an ED(50) of 40 pM. Selected compounds were tested in an in vivo inflammatory assay, and the best compound reduces TNF-alpha levels with an ED(50) of 0.1 microg/kg when administered by either subcutaneous injection or oral delivery.

The MHP36 line of murine neural stem cells expresses functional CXCR1 chemokine receptors that initiate chemotaxis in vitro.

Chemokines help to establish cerebral inflammation after ischemia, which comprises a major component of secondary brain injury. The CXCR4 chemokine receptor system induces neural stem cell migration, and hence has been implicated in brain repair. We show that CXCR1 and interleukin-8 also stimulate chemotaxis in murine neural stem cells from the MHP36 cell line. The presence of CXCR1 was confirmed by reverse transcriptase PCR and immunohistochemistry. Interleukin-8 evoked intracellular calcium currents, upregulated doublecortin (a protein expressed by migrating neuroblasts), and elicited positive chemotaxis in vitro. Therefore, effectors of the early innate immune response may also influence brain repair mechanisms.

Broad-spectrum chemokine inhibition reduces vascular macrophage accumulation and collagenolysis consistent with plaque stabilization in mice.

BACKGROUND: A major determinant of the risk of myocardial infarction is the stability of the atherosclerotic plaque. Macrophage-rich plaques are more vulnerable to rupture, since macrophages excrete an excess of matrix-degrading enzymes over their inhibitors, reducing collagen content and thinning the fibrous cap. Several genetic studies have shown that disruption of signalling by the chemokine monocyte chemoattractant protein 1 reduced the lipid lesion area and macrophage accumulation in the vessel wall. METHODS: We have tested whether a similar reduction in macrophage accumulation could be achieved pharmacologically by treating apolipoprotein-E-deficient mice with the chemokine inhibitor NR58-3.14.3. RESULTS: Mice treated for various periods of time (from several days to 6 months) with NR58-3.14.3 (approximately 30 mg/kg/day) consistently had 30-40% fewer macrophages in vascular lesions, compared with mice treated with the inactive control NR58-3.14.4 or PBS vehicle. Similarly, cleaved collagen staining was lower in mice treated for up to 7 days, although this effect was not maintained when treatment time was extended to 12 weeks. The vascular lipid lesion area was unaffected by treatment, but total collagen I staining and smooth muscle cell number were both increased, suggesting that a shift to a more stable plaque phenotype had been achieved. CONCLUSIONS: Strategies, such as chemokine inhibition, to attenuate macrophage accumulation may therefore be useful to promote stabilization of atherosclerotic plaques.

Broad spectrum chemokine inhibitors related to NR58-3.14.3.

The chemokine family consists of more than 50 structurally-related small proteins which signal through type 1 G-protein coupled receptors (GPCRs) to regulate a range of immune functions, with particular focus on regulating leukocyte trafficking. They have been implicated both in normal physiological leukocyte traffic, and in recruitment of leukocytes to sites of pathological inflammation. As a result, chemokine inhibitors may have useful anti-inflammatory therapeutic properties in vivo. Compounds with chemokine-inhibitory properties that have been described to date, fall into two broad categories: receptor-specific antagonists which block the action of one or a small number of related chemokines, and broad-spectrum chemokine inhibitors (BSCIs) which block leukocyte migration in response to many, if not all, chemokines simultaneously. Since many chemokines apparently show functional redundancy in vivo, the BSCI class are attractive candidates for development as anti-inflammatory therapies. Here, we review the development of BSCIs, with particular focus on the design and characterisation of non-peptide compounds. The key structural requirements for BSCI activity are discussed, together with their implications for the mechanism of BSCI action.

Identification of 3-(acylamino)azepan-2-ones as stable broad-spectrum chemokine inhibitors resistant to metabolism in vivo.

3-(acylamino)glutarimides, a class of broad spectrum chemokine inhibitors, are rapidly hydrolyzed in serum, despite being stable in aqueous solution. Synthesis and high-performance liquid chromatography analysis of the proposed N-acyl-glutamate and -glutamine metabolites establish the enzyme-catalyzed breakdown pathways. In vitro assays suggest that despite their short half-life in vivo, the parent acylamino-glutarimides, not the ring-opened hydrolysis products, are the source of the antiinflammatory activity. Identification of this metabolic pathway has led to the development of 3-(acylamino)azepan-2-ones that are also broad spectrum chemokine inhibitors and act as stable, orally available powerful antiinflammatory agents in vivo with doses of 1 mg/kg.

Apolipoprotein E modulates clearance of apoptotic bodies in vitro and in vivo, resulting in a systemic proinflammatory state in apolipoprotein E-deficient mice.

Apolipoprotein E (apoE) is a 34-kDa glycoprotein involved in lipoprotein transport through interaction with the low-density lipoprotein receptor and related receptors. Recently, it has become clear that apoE binding to its receptors plays a role both in development and in control of the immune system. In this study, we show that apoE modulates the rate of uptake of apoptotic cells by macrophages. In vitro, apoE-deficient macrophages ingest less apoptotic thymocytes (but not latex beads) than wild-type macrophages, and this defect can be corrected by addition of exogenous apoE protein. In vivo, the number of dying macrophages is increased in a range of tissues, including lung and brain. Possibly in response to the larger numbers of persistent apoptotic bodies, the number of live macrophages in these tissues are also increased compared with those of wild-type control mice. In addition to the significant changes in macrophage population dynamics we observed, levels of the proinflammatory cytokine TNF-alpha and the positive acute phase reactant fibrinogen are also elevated in the livers from apoE-deficient mice. In contrast, neither deletion of the gene encoding the LDL receptor nor cholesterol feeding of wild-type mice affected either the number of apoptotic bodies or the number of live macrophages. We conclude that apoE deficiency results in impaired clearance of apoptotic cell remnants and a functionally relevant systemic proinflammatory condition in mice, independent of its role in lipoprotein metabolism. Any similar reduction of apoE activity in humans may contribute to the pathogenesis of a wide range of chronic diseases including atherosclerosis, dementia, and osteoporosis.

Tools for anti-inflammatory drug design: in vitro models of leukocyte migration.

Inhibiting leukocyte recruitment is now a major focus in the design of novel anti-inflammatory drugs. Following the identification of lead compounds from conventional high-throughput screens using appropriate receptors or enzymes, it is important to validate the action of the compounds in a suitable in vitro model of leukocyte migration. Here, we review a range of different experimental approaches to modelling leukocyte migration, and identify the multi-well filter migration assay as the best compromise between the amount of resources required to screen multiple compounds and the amount of information gained about the effects of the compounds on cell movement behavior. However, there are pitfalls in the interpretation of data obtained using the multi-well filter migration assay, which arise from the imperfect correlation between the number of cells undergoing migration and the inhibitory activity of the test substances. We examine a number of such pitfalls and provide practical approaches to mitigate these problems as far as possible. We recommend a general strategy for screening inhibitors of cell migration using in vitro functional assays. While being more resource intensive than surrogate measures such as calcium flux, functional approaches nevertheless provide superior correlations with anti-inflammatory activity in vivo.

Broad-spectrum chemokine inhibitors (BSCIs) and their anti-inflammatory effects in vivo.

Inappropriate inflammation is a component of a wide range of human diseases, including autoimmune disease, atherosclerosis, osteoporosis and Alzheimer's disease. Chemokines play an important role in orchestrating leukocyte recruitment during inflammation, and therefore represent an important target for anti-inflammatory therapies. Unfortunately, the chemokine system is complex, with about 50 ligands and 20 receptors, often acting with redundancy, making selection of appropriate specific antagonists difficult. One approach to overcoming this difficulty may be the development of broad-spectrum chemokine inhibitors (BSCIs). Here we review the present state of knowledge on BSCIs, including their activity in vitro and their anti-inflammatory effects in vivo, and discuss the future development of BSCIs as anti-inflammatory therapies for use in the clinic.

Design, synthesis, and preliminary pharmacological evaluation of N-acyl-3-aminoglutarimides as broad-spectrum chemokine inhibitors in vitro and anti-inflammatory agents in vivo.

A series of N-substituted 3-aminoglutarimides have been synthesized and tested for inhibitory activity against a range of chemokines in vitro and for suppression of lipopolysaccharide-induced inflammation in vivo. The results show that they represent the first class of small molecules with broad-spectrum chemokine inhibitory effects. Among the compounds studied, 10 (NR58,4) was the most potent, being active at doses between 5 and 15 nM in vitro and at 0.3 mg kg(-1) in vivo.