Discovery of GW870086: a potent anti-inflammatory steroid with a unique pharmacological profile.

BACKGROUND AND PURPOSE: Glucocorticoids are highly effective therapies for a range of inflammatory diseases. Advances in the understanding of the diverse molecular mechanisms underpinning glucocorticoid action suggest that anti-inflammatory molecules with reduced side effect liabilities can be discovered. Here we set out to explore whether modification of the 17α position of the steroid nucleus could generate molecules with a unique pharmacological profile and to determine whether such molecules would retain anti-inflammatory activity. EXPERIMENTAL APPROACH: The pharmacological properties of GW870086 were compared with fluticasone propionate (FP) using a range of cellular and in vivo model systems, including extensive gene expression profiling. KEY RESULTS: GW870086 repressed inflammatory cytokine release from lung epithelial cells in a similar manner to FP but antagonized the effect of dexamethasone on MMTV-driven reporter gene transactivation. GW870086 had a strong effect on the expression of some glucocorticoid-regulated genes (such as PTGS2), while having minimal impact on the expression of other known target genes (such as SGK). GW870086 retained the ability to strengthen tight junctions in epithelial cell culture but, unlike FP, was unable to protect the culture from elastase-mediated damage. In murine models of irritant-induced contact dermatitis and ovalbumin-induced allergic inflammation, GW870086 showed comparable anti-inflammatory efficacy to FP. CONCLUSION AND IMPLICATIONS: GW870086 is a potent anti-inflammatory compound with a unique ability to regulate only a subset of those genes that are normally affected by classical glucocorticoids. It has the potential to become a new topical steroid with a different safety profile to existing therapies.

Mometasone furoate is a less specific glucocorticoid than fluticasone propionate.

Fluticasone propionate (FP) and mometasone furoate (MF) are potent synthetic corticosteroids that are widely used as anti-inflammatory agents to treat respiratory diseases. As part of the assessment of the potential for side-effects associated with their use, their activities, not only at the glucocorticoid receptor (GR) but also at the other members of the steroid nuclear receptor family, have been compared. Cell-based functional systems were established to measure different aspects of GR function, as well as the activity at all the other steroid nuclear receptors. The effects of MF and FP on the GR were potent and indistinguishable. Neither corticosteroid showed any activity at the oestrogen receptor, while both were weak antagonists of the androgen receptor. FP was a relatively weak agonist of the progesterone receptor but MF was a very potent agonist of the progesterone receptor, giving activity at similar concentrations to those that stimulate the GR (concentration generating 50% maximal effect (EC50)=50 pM). Moreover, while FP was a weak antagonist of the mineralocorticoid receptor (concentration generating 50% maximal inhibitory effect=80 nM), MF displayed potent partial agonist activity (EC50=3 nM, 30%). Mometasone furoate is considerably less specific for the glucocorticoid receptor than fluticasone propionate, showing significant activity at other nuclear steroid receptors.

Glucocorticoid-induced osteopenia in the mouse as assessed by histomorphometry, microcomputed tomography, and biochemical markers.

Glucocorticoids are potent anti-inflammatory molecules used in the treatment of asthma, rheumatoid arthritis, inflammatory bowel disease, and other inflammatory and dermatological diseases, as well as in posttransplantation immunotherapy. Although glucocorticoids have been prescribed for many years, their potential side effects, when administered orally, can prevent their long-term use. The most serious side effect observed in the clinic is glucocorticoid-induced osteoporosis (GIOP). To develop a small animal model to characterize glucocorticoid-induced bone loss, we carried out a series of experiments using BALB/c mice given daily intraperitoneal doses of the synthetic glucocorticoid, dexamethasone. Following dexamethasone treatment, the mice became osteopenic, with highly significant decreases in bone formation rate and mineral apposition rate, as assessed by standard histomorphometry. Moreover, 3 week treatment with dexamethasone resulted in a decrease in trabecular thickness and trabecular number with an increase in surface-to-volume ratio of trabeculae in the distal femur, as measured using microcomputed tomography (micro-CT). The serum bone formation marker, osteocalcin, was dose-dependently decreased in all mice treated with dexamethasone and showed a parallel extent of regulation to the bone formation rate changes. In addition, serum levels of leptin, recently identified as playing a role in the regulation of bone mass, increased following dexamethasone treatment. BALB/c mice therefore represent a useful model system in which the detrimental effects of glucocorticoids on bone can be studied.

Functional analysis of the interleukin-1-receptor-associated kinase (IRAK-1) in interleukin-1 beta-stimulated nuclear factor kappa B (NF-kappa B) pathway activation: IRAK-1 associates with the NF-kappa B essential modulator (NEMO) upon receptor stimulation.

The interleukin-1 (IL-1)-receptor-associated kinase (IRAK-1) is essential for IL-1-stimulated nuclear factor kappa B (NF-kappa B) activation. To study the role of IRAK-1 in IL-1 beta signalling, we have generated a set of IRAK-1 variants that express distinct domains of IRAK-1 either alone or in combination and have examined their effects on an NF-kappa B-responsive reporter in HeLa cells. Unlike full-length IRAK-1, the deletion mutants were unable to activate NF-kappa B in the absence of cytokine stimulation. However, an IRAK-1 variant lacking only the N-terminal domain retained the ability of the full-length protein to potentiate both IL-1 beta and tumour necrosis factor alpha (TNF alpha)-induced NF-kappa B activation. In contrast, expression of the N-terminus or the C-terminus of IRAK-1, or a fusion protein incorporating both domains, inhibited both IL-1 beta- and TNF alpha-induced effects. Expression of an IRAK-1 variant lacking only the C-terminal domain preferentially inhibited IL-1 beta versus TNF alpha-induced NF-kappa B activation. These data suggest that the C-terminal domain may link IRAK-1 to downstream signalling components common to both the IL-1 and TNF pathways. Furthermore, we have demonstrated that endogenous IRAK-1 becomes phosphorylated upon IL-1 beta treatment and can be detected along with NF-kappa B essential modulator (NEMO) and I kappa B kinase beta (IKK beta) in high-molecular-mass complexes of 600-800 kDa. Moreover, IRAK-1 could be detected in NEMO immunoprecipitates from IL-1 beta-stimulated cells. We conclude that IRAK-1 mediates IL-1 beta signal transduction through a ligand-dependent association of IRAK-1 with the IKK complex.

Modified peptide antagonists of interleukin 5 exhibit extended in vivo persistence but restricted species specificity.

AF18748 is disulphide-linked homodimeric peptide with 19 amino acids in each chain that antagonises the action of the eosinophil-specific cytokine, interleukin 5 (IL-5). We have generated a set of N-terminally truncated peptides derived from AF18748 and demonstrated that the first five amino acids of the peptide do not contribute to receptor binding activity. The shortened peptide blocked IL-5-dependent adhesion of eosinophils with an IC(50)of 350 pM, and had no effect on stimulation by IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), tumour necrosis factor (TNF)-alpha or fMet-Leu-Phe. The peptides were rapidly broken down in mouse plasma through cleavage of a single chain of the dimer. However, this breakdown did not correlate with loss of biological activity, indicating that the asymmetric peptide fragment retains full receptor binding capacity. The activity of AF18748 disappeared rapidly from the blood following intravenous injection into mice. Coupling of polyethylene glycol to the N-terminus of AF18748 resulted in a moderate loss in biological potency (IC(50)30 nM), but the resulting conjugate persisted in the circulation for more than 8 h after injection. Despite its high potency at the human IL-5 receptor, AF18748 was unable to antagonise the activity of IL-5 on murine B13 cells, or on canine eosinophils, indicating that the peptide is highly specific for the human IL-5 receptor.

Cytokine-specific transcriptional regulation through an IL-5Ralpha interacting protein.

Cytokine receptors consist of multiple subunits, which are often shared between different receptors, resulting in the functional redundancy sometimes observed between cytokines. The interleukin 5 (IL-5) receptor consists of an IL-5-specific alpha-subunit (IL-5Ralpha) and a signal-transducing beta-subunit (betac) shared with the IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors. In this study, we sought to find a role for the cytoplasmic domain of IL-5Ralpha. We show that syntenin, a protein containing PSD-95/Discs large/zO-1 (PDZ) domains, associates with the cytoplasmic tail of the IL-5Ralpha. Syntenin was found to directly associate with the transcription factor Sox4. Association of syntenin with IL-5Ralpha was required for IL-5-mediated activation of Sox4. These studies identify a mechanism of transcriptional activation by cytokine-specific receptor subunits.

Cell receptors and cell signalling.

All cells in a multicellular organism are constantly exposed to a variety of extracellular signals that they need to interpret and translate into an appropriate response to their environment. These signals can be soluble factors generated locally (for example, synaptic transmission) or distantly (for example, hormones and growth factors), ligands on the surface of other cells, or the extracellular matrix itself. To achieve this, cells maintain a diversity of receptors on their surface that respond specifically to individual stimuli. These receptors fall into families, based primarily on the way in which they generate the intracellular signals that give rise to the particular functional responses. Moreover, the activity of a given receptor can be modulated by other signalling pathways in a variety of ways, generating the flexibility required of such a complex system. This review aims to describe the function of the major classes of receptor, including G protein coupled receptors, receptor tyrosine kinases, ligand gated ion channels, integrins, and cytokine receptors, and to demonstrate the "crosstalk" that exists between these systems.

Effects of the indolocarbazole 3744W on the tyrosine kinase activity of the cytoplasmic domain of the platelet-derived growth factor beta-receptor.

The cytoplasmic domain of the platelet-derived growth factor (PDGF) beta-receptor was expressed in insect cells by using a baculovirus system. The resulting protein was a constitutively active tyrosine kinase that could phosphorylate both protein and peptide substrates. A recently identified potent and selective inhibitor of intact PDGF receptor autophosphorylation, 3744W, inhibited the autophosphorylation of the cytoplasmic domain both in vitro (IC50 1.8+/-0.12 microM) and within intact insect cells (IC50 2.0 microM). However, under identical assay conditions, 3744W did not inhibit the phosphorylation of the synthetic polymeric peptide poly(Glu4Tyr1) even at concentrations as high as 100 microM. These results suggest that, although 3744W inhibits PDGF receptor autophosphorylation directly, it can discriminate between phosphate acceptor substrates.

Indolocarbazoles: potent and selective inhibitors of platelet-derived growth factor receptor autophosphorylation.

A quantitative assay for measuring the autophosphorylation of platelet-derived growth factor (PDGF) receptors in intact vascular smooth muscle cells has been developed and used to screen for novel tyrosine kinase (TK) inhibitors. Several novel inhibitors of PDGF receptor autophosphorylation have been identified from the indolocarbazole series, including the 3,9 dimethoxy derivative, 3744W (IC50 = 14.5+/-2 nM). Tested against a panel of tyrosine and serine/threonine kinases, 3744W is at least 1,000 fold selective for the PDGF receptor tyrosine kinase and was found to inhibit autophosphorylation of both the alpha and beta isoforms of the PDGF receptor in human smooth muscle cells. PDGF-BB-stimulated DNA synthesis in quiescent cultures of human smooth muscle cells was blocked in a concentration-dependent manner by 3744W, IC50 = 10 nM. Binding studies showed that 3744W did not block the binding of PDGF-BB to cell surface receptors on human airway smooth muscle cells. Furthermore, inhibition of bone marrow stem cell proliferation by 3744W was only observed at concentrations 100-1,000 times greater than those needed to block PDGF-driven DNA synthesis in human smooth muscle cells. 3744W represents a novel, potent and selective inhibitor of PDGF receptor autophosphorylation and a powerful biochemical probe for investigating PDGF-dependent responses in vitro.

An interleukin 5 mutant distinguishes between two functional responses in human eosinophils.

Interleukin 5 (IL-5) is the key cytokine involved in regulating the production and many of the specialized functions of mature eosinophils including priming, adhesion, and survival. We have generated a point mutant of human IL-5, IL-5 (E12K), which is devoid of agonist activity in both a TF-1 cell proliferation assay and a human eosinophil adhesion assay. However, IL-5 (E12K) is a potent and specific antagonist of both these IL-5-dependent functional responses. In both receptor binding and cross-linking studies the wild-type and IL-5 (E12K) mutant exhibit virtually identical properties. This mutant protein was unable to stimulate tyrosine phosphorylation in human eosinophils, and blocked the phosphorylation stimulated by IL-5. In contrast, IL-5 (E12K) is a full agonist in a human eosinophil survival assay, although with reduced potency compared to the wild-type protein. This IL-5 mutant enables us to clearly distinguish between two IL-5-dependent functional responses and reveals distinct mechanisms of receptor/cellular activation.

Tyrosine phosphorylation is involved in receptor coupling to phospholipase D but not phospholipase C in the human neutrophil.

The tyrosine kinase inhibitors ST271, ST638 and erbstatin inhibited phospholipase D (PLD) activity in human neutrophils stimulated by fMet-Leu-Phe, platelet-activating factor and leukotriene B4. These compounds did not inhibit phorbol ester-stimulated PLD, indicating that they do not inhibit PLD per se, but probably act at a site between the receptor and the phospholipase. In contrast, the protein kinase C inhibitor Ro-31-8220 inhibited phorbol 12,13-dibutyrate- but not fMet-Leu-Phe-stimulated PLD activity, arguing against the involvement of protein kinase C in the receptor-mediated activation of PLD. ST271 did not inhibit Ins(1,4,5)P3 generation, but did inhibit protein tyrosine phosphorylation stimulated by fMet-Leu-Phe. The phosphotyrosine phosphatase inhibitor pervanadate increased tyrosine phosphorylation and stimulated PLD. These results suggest that tyrosine kinase activity is involved in receptor coupling to PLD but not to PtdIns(4,5)P2-specific phospholipase C in the human neutrophil.