Non-steroidal Glucocorticoid Receptor Ligand Research Articles

Molecular View on the Dissociation Pathways and Transactivation Regulation Mechanism of Nonsteroidal GR Ligands.

As a major drug target for anti-inflammatory therapy, the glucocorticoid receptor (GR) regulates a wide range of physiological processes through transactivation (TA) or transrepression. GR TA is involved in many adverse effects of GR-targeting drugs, and therefore, the discovery of novel GR ligands with lower TA activity and longer residence time is quite urgent. Undoubtedly, understanding the ligand dissociation mechanisms and the structural basis of the TA regulation is crucial for the development of novel GR-targeting drugs. Here, we used random accelerated molecular dynamics (RAMD) and funnel metadynamics (FM) simulations to explore the dissociation mechanisms of 5 classic glucocorticoids and 6 nonsteroidal GR ligands. Multiple ligand dissociation pathways were discovered. The classic glucocorticoids exhibit a strong preference for Path I, and most nonsteroidal ligands tend to dissociate along mixed pathways. We also find that the distinct unbinding preferences for AZD2906 and AZD9567, two representative nonsteroidal ligands with similar scaffolds but different TA activities, are primarily determined by their different polar interactions with the surrounding residues. Notably, the binding of AZD9567 poses a substantial impact on the conformation of the GR homodimer interface, which provides a valuable clue to understand the mechanisms of the TA-related side effects induced by the adjustments of the homodimerization process. These findings are critical for the structure-based rational design of novel GR ligands with more potent anti-inflammatory potency and reduced side effects.

Bullous pemphigoid outcome is associated with CXCL10-induced matrix metalloproteinase 9 secretion from monocytes and neutrophils but not lymphocytes

The outcome of bullous pemphigoid (BP), the most frequent autoimmune skin-blistering disease, involves matrix metalloproteinase 9 (MMP-9), IL-17, and IL-23 release from infiltrated inflammatory cells. The chemokine CXCL10 has been associated with several autoimmune diseases, but its participation in BP pathophysiology still needs to be clarified. We sought to assess whether BP outcome was associated with different CXCL10 levels and to evaluate the contribution of CXCL10 to the described cytokine/protease inflammatory loop associated with disease outcome. Skin biopsy specimens (n=16), serum (n=114), blister fluid (n=23), and primary inflammatory cells from patients with BP were used to investigate CXCL10 expression and function. At baseline, both resident cells, such as keratinocytes and fibroblasts, and infiltrating immune cells expressed CXCL10 at lesional sites in skin of patients with BP. CXCL10 levels were higher in blister fluid (P<.0001) and serum (P<.005) from patients with BP than in serum from age- and sex-matched control subjects (n=34). Furthermore, CXCL10 serum levels increased at day 60 only in patients who relapsed within the first year of treatment (n=33, P<.005). Interestingly, CXCL10 expression could be upregulated by itself and IL-17 in inflammatory cells. Notably, neutrophils and monocytes from patients with BP, but not lymphocytes, responded to CXCL10 by increasing MMP-9 secretion through the activation of extracellular signal-regulated kinase 1/2, p38, phosphoinositide-3 kinase signaling pathways. Finally, CXCL10-increased MMP-9 secretion was inhibited by methylprednisolone and also by compound A, a novel nonsteroidal glucocorticoid receptor ligand. We showed that increased levels of inflammatory biomarkers in patients with BP, such as CXCL10, favor neutrophil- and monocyte-associated MMP-9 release and disease relapse and opened new therapeutic horizons in patients with this autoimmune disease.

Glucocorticoids Have Opposing Effects on Liver Fibrosis in Hepatic Stellate and Immune Cells.

Liver fibrosis is a reversible wound-healing process that is protective in the short term, but prolonged fibrotic responses lead to excessive accumulation of extracellular matrix components that suppresses hepatocyte regeneration, resulting in permanent liver damage. Upon liver damage, nonparenchymal cells including immune cells and hepatic stellate cells (HSCs) have crucial roles in the progression and regression of liver fibrosis. Here, we report differential roles of the glucocorticoid receptor (GR), acting in immune cells and HSCs, in liver fibrosis. In the carbon tetrachloride hepatotoxin-induced fibrosis model, both steroidal and nonsteroidal GR ligands suppressed expression of fibrotic genes and decreased extracellular matrix deposition but also inhibited immune cell infiltration and exacerbated liver injury. These counteracting effects of GR ligands were dissociated in mice with conditional GR knockout in immune cells (GR(LysM)) or HSC (GR(hGFAP)): the impacts of dexamethasone on immune cell infiltration and liver injury were totally blunted in GR(LysM) mice, whereas the suppression of fibrotic gene expression was diminished in GR(hGFAP) mice. The effect of GR activation in HSC was further confirmed in the LX-2 HSC cell line, in which antifibrotic effects were mediated by GR ligand inhibition of Sma and mad-related protein 3 (SMAD3) expression. We conclude that GR has differential roles in immune cells and HSCs to modulate liver injury and liver fibrosis. Specific activation of HSC-GR without alteration of GR activity in immune cells provides a potential therapeutic approach to treatment of hepatic fibrosis.

Selective Activator of the Glucocorticoid Receptor Compound A Dissociates Therapeutic and Atrophogenic Effects of Glucocorticoid Receptor Signaling in Skin.

Background:Glucocorticoids are effective anti-inflammatory drugs widely used in dermatology and for the treatment of blood cancer patients. Unfortunately, chronic treatment with glucocorticoids results in serious metabolic and atrophogenic adverse effects including skin atrophy. Glucocorticoids act via the glucocorticoid receptor (GR), a transcription factor that causes either gene transactivation (TA) or transrepression (TR). Compound A (CpdA), a novel non-steroidal GR ligand, does not promote GR dimerization and TA, retains anti-inflammatory potential but induces fewer metabolic side effects compared to classical glucocorticoids when used systemically. As topical effects of CpdA have not been well studied, this work goal was to compare the anti-inflammatory and side effects of topical CpdA and glucocorticoids and to assess their effect on GR TA and TR in keratinocytes.Methods:We used murine immortalized keratinocytes and F1 C57BlxDBA mice. Effect of glucocorticoid fluocinolone acetonide (FA) and CpdA on gene expression in keratinocytes in vitro and in vivo was evaluated by reverse transcription-PCR. The anti-inflammatory effects were assessed in the model of tumor promoter 12-O-tertradecanoyl-acetate (TPA)-induced dermatitis and in croton oil-induced ear edema test. Skin atrophy was assessed by analysis of epidermal thickness, keratinocyte proliferation, subcutaneous adipose hypoplasia, and dermal changes after chronic treatment with FA and CpdA.Results:In mouse keratinocytes in vitro and in vivo, CpdA did not activate GR-dependent genes but mimicked closely the inhibitory effect of glucocorticoid FA on the expression of inflammatory cytokines and matrix metalloproteinases. When applied topically, CpdA inhibited TPA-induced skin inflammation and hyperplasia. Unlike glucocorticoids, CpdA itself did not induce skin atrophy which correlated with lack of induction of atrophogene regulated in development and DNA damage response 1 (REDD1) causatively involved in skin and muscle steroid-induced atrophy.Conclusions:Overall, our results suggest that CpdA and its derivatives represent novel promising class of anti-inflammatory compounds with reduced topical side effects.

The discovery of potent and selective non-steroidal glucocorticoid receptor modulators, suitable for inhalation

We report the discovery of highly potent and selective non-steroidal glucocorticoid receptor modulators with PK properties suitable for inhalation. A high throughput screen of the AstraZeneca compound collection identified sulfonamide 3 as a potent non-steroidal glucocorticoid receptor ligand. Further optimization of this lead generated indazoles 30 and 48 that were progressed to characterization in in vivo models. X-ray crystallography was used to gain further insight into the binding mode of selected ligands.

A ligand-specific kinetic switch regulates glucocorticoid receptor trafficking and function.

The ubiquitously expressed glucocorticoid receptor (GR) is a major drug target for inflammatory disease, but issues of specificity and target tissue sensitivity remain. We now identify high potency, non-steroidal GR ligands, GSK47867A and GSK47869A, which induce a novel conformation of the GR ligand-binding domain (LBD) and augment the efficacy of cellular action. Despite their high potency, GSK47867A and GSK47869A both induce surprisingly slow GR nuclear translocation, followed by prolonged nuclear GR retention, and transcriptional activity following washout. We reveal that GSK47867A and GSK47869A specifically alter the GR LBD structure at the HSP90-binding site. The alteration in the HSP90-binding site was accompanied by resistance to HSP90 antagonism, with persisting transactivation seen after geldanamycin treatment. Taken together, our studies reveal a new mechanism governing GR intracellular trafficking regulated by ligand binding that relies on a specific surface charge patch within the LBD. This conformational change permits extended GR action, probably because of altered GR-HSP90 interaction. This chemical series may offer anti-inflammatory drugs with prolonged duration of action due to altered pharmacodynamics rather than altered pharmacokinetics.

Abstract 669: Anticancer effects of novel non steroidal glucocorticoid receptor ligand, Compound A, in T-cell leukemia lines

Abstract Glucocorticoids (GCs) are widely used in chemotherapy of hematologic malignancies, including T cell leukemia. The biological effects of GCs are mediated by the glucocorticoid receptor (GR), a well known transcription factor. Therapeutic effects of GCs are mainly mediated via GR transrepression that involves negative interaction between GR and other transcription factors. In contrast, side effects of glucocorticoids are mediated by GR transactivation that requires binding of GR homodimer to hormone-responsive elements in gene promoters. Selective GR activators (SEGRA) that shift GR functions towards transrepression have improved therapeutic index and hold a great potential for the GR-targeted chemotherapies. Recently we and others characterized a novel non-steroidal GR ligand, 2-(4-acetoxyphenyl)-2-chloro-N-methylethylammonium chloride also called Compound A (CpdA). CpdA prevents GR dimerization and transactivation, but strongly enhances GR transrepression. We also revealed that CpdA possesses anti-cancer activity: it inhibited growth and viability of prostate cancer cells in GR-dependent fashion. Here we analyzed CpdA effects in K562 and CEM T-cell leukemia lines. We found that both cell lines express functional GR. CpdA at 10-6-10-5M concentration range exerted cytostatic and apoptotic effects in both cell lines. CEM cells were more sensitive to CpdA than K562 that correlated with the higher amount of GR protein in these cells. We found that in contrast to GCs, CpdA did not induce GR transactivation evaluated by RT-PCR of known GR-target genes such as FKBP51. At the same time, Luciferase assay showed that CpdA efficiently activated transrepression of NF-kB and AP-1 factors. Currently, GCs are used for the treatment of blood cancers in combined chemotherapy with different anti-cancer drugs including proteasome inhibitor Bortezomib (BZ). It is known that proteasome inhibitors increase GR stability. Thus, we expected that BZ will maximize ligand properties of CpdA as GR modulator and will increase its anti-cancer effects. We found that BZ induced caspase-dependent apoptosis in both T-cell leukemia cell lines. The effect of BZ on GR expression and function and on apoptosis induced by CpdA is under study. In addition, we have performed Ames test and Topoisomerase I assay as first steps towards the evaluation of toxicological profile of CpdA. We have not revealed mutagenic effect of CpdA either in frameshift or in base-substitution indicator strains at the wide range of concentrations (10-9-10-4M). CpdA also did not inhibit eukaryotic topoisomerase I activity. Overall, our data suggest that novel GR modulator CpdA has a high chemotherapeutic potential for the treatment of T-cell leukemia. The detailed mechanisms of its anti-cancer effects in transformed T-cells are under investigation. Work is supported by grants: RFBR 10-04-00979, RO1CA118890, ACS IL 160185, UICC ICRETT-09-137. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 669. doi:10.1158/1538-7445.AM2011-669

Abstract 214: Anticancer effect of Compound A, a novel modulator of the glucocorticoid receptor, in acute leukemia cells

Abstract Glucocorticoids (GCs) are widely used in chemotherapy of blood cancers, including childhood acute leukemia. The biological effects of GCs are mediated by the glucocorticoid receptor (GR), a well known transcription factor. Therapeutic effects of GCs are mainly mediated via GR transrepression that involves negative interaction between GR and other transcription factors. In contrast, many adverse metabolic effects of glucocorticoids are mediated by GR transactivation that requires GR binding to hormone-responsive elements in gene promoters. Thus, selective GR activators (SEGRA) that modify GR, shift GR functions towards transrepression resulting in improved therapeutic index, hold a great potential for the GR-targeted chemotherapies. Recently we and others characterized novel non-steroidal GR ligand, 2-(4-acetoxyphenyl)-2-chloro-N-methylethylammonium chloride or Compound A (CpdA), and showed that CpdA prevents GR dimerization and transactivation, but strongly enhances GR transrepression. We also revealed that CpdA possesses anti-cancer properties: it inhibited growth and viability of prostate cancer cells in GR-dependent fashion. Here we report that CpdA at concentrations 10−6-10−5M exerted cytostatic (evaluated by MTT test) and cytotoxic (evaluated by FACS analysis of DNA fragmentation as sub-G1 peak in propidium iodide-labeled cells) effects in K564, NB-4 and CEM leukemia cell lines. We found that these cell lines express wild type nonmutated GR. Consequently, these cell lines present a good model system for the further comparative analysis of CpdA and GCs effects. Leukemia cell lines were sensitive to apoptotic effect of both GR ligands: Dexamethasone (Dex) and CpdA. Dex induced apoptosis at the concentrations 10−7 to 10−5M; and CpdA affected cell viability at concentrations 5×10−6−10−5M. As a first step towards the evaluation of toxicological profile of this promising anti-cancer compound, we assessed its mutagenicity using Ames test. CpdA was not mutagenic either in frameshift or in base-substitution indicator strains, TA98 and TA100, respectively, at the wide range of concentrations (10−9-10−4M). Overall, our data suggest that novel non-mutagenic GR modulator has a high chemotherapeutic potential. The detailed mechanisms of its anti-cancer effects in leukemia cell lines is under investigation. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 214.

Therapeutic and Adverse Effects of a Non-Steroidal Glucocorticoid Receptor Ligand in a Mouse Model of Multiple Sclerosis

BackgroundDissociating glucocorticoid receptor (GR) ligands hold great promise for treating inflammatory disorders since it is assumed that they exert beneficial activities mediated by transrepression but avoid adverse effects of GR action requiring transactivation. Here we challenged this paradigm by investigating 2-(4-acetoxyphenyl)-2-chloro-N-methyl-ethylammonium chloride (CpdA), a dissociating non-steroidal GR ligand, in the context of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS).Methodology/Principal FindingsCpdA inhibited pro-inflammatory mediators in myelin-specific T cells and fibroblasts in a GR-dependent manner while gene activation was abolished. However, it also induced massive apoptosis in various cell types even in the absence of the GR by engaging a Bcl-2- and caspase-dependent pathway. 1H NMR spectroscopy corroborated these findings by revealing that CpdA dissolved in buffered solutions rapidly decomposes into aziridine intermediates known to act as alkylating pro-apoptotic agents. Importantly, the dichotomy of CpdA action also became evident in vivo. Administration of high-dose CpdA to mice was lethal while treatment of EAE with low to intermediate amounts of CpdA dissolved in water significantly ameliorated the disease. The beneficial effect of CpdA required expression of the GR in T cells and was achieved by down regulating LFA-1 and CD44 on peripheral Th cells and by repressing IL-17 production.Conclusions/SignificanceCpdA has significant therapeutic potential although adverse effects severely compromise its application in vivo. Hence, non-steroidal GR ligands require careful analysis prior to their translation into new therapeutic concepts.

Glucocorticoid Receptor in Health and Disease

Glucocorticoid Receptor in Health and DiseaseGlucocorticoid hormones are essential for life, have a vital place in the treatment of inflammatory and autoimmune diseases and are increasingly implicated in the pathogenesis of a number of common disorders. Their action is mediated by an intracellular receptor protein, the glucocorticoid receptor (GR), functioning as a ligand-inducible transcription factor. Multiple synthetic glucocorticoids are used as potent antiinflammatory and immunosuppressive agents, but their therapeutic usefulness is limited by a wide range and severity of side-effects. One of the most important pharmaceutical goals has been to design steroidal and non-steroidal GR ligands with profound therapeutic efficacy and reduced unwanted effects. The therapeutic benefit of glucocorticoid agonists is frequently compromised by resistance to glucocorticoids, which may depend on: access of the hormones to target cells, steroid metabolism, expression level and isoform composition of the GR protein, mutations and polymorphisms in the GR gene and association of the receptor with chaperone proteins. The major breakthrough into the critical role of glucocorticoid signaling in the maintenance of homeostasis and pathogenesis of diseases, as well as into the molecular mechanisms underlying the therapeutic usefulness of antiinflammatory drugs acting through the GR is expected to result from the current progress in large-scale gene expression profiling technologies and computational biology.

Combining 3D-QSAR, docking, molecular dynamics and MM/PBSA methods to predict binding modes for nonsteroidal selective modulator to glucocorticoid receptor

AL-438 is a selective and dissociated GR (glucocorticoid receptor) agonist. In this letter, the binding mode of AL-438 to GR is predicted by using multiple computational methods including 3D-QSAR, molecular docking and molecular dynamics simulation. This provides a guideline for rational design of novel and dissociated nonsteroidal GR ligand.

Discovery of nonsteroidal glucocorticoid receptor ligands based on 6-indole-1,2,3,4-tetrahydroquinolines

A series of nonsteroidal glucocorticoid receptor (GR) ligands based on a 6-indole-1,2,3,4-tetrahydroquinoline scaffold are reported. Structure–activity relationship (SAR) of the pendent indole group identified compound 20 exhibiting good GR binding affinity ( K i = 1.5 nM) and 100- to 1000-fold selectivity over MR, PR, and AR while showing activity in an E-selectin repression assay.

12th Workshop of the Central European Division e.V. of the International Isotope Society

AbstractV Snieckus [Queen's University, Canada]—The Directed Ortho Metalation—Cross Coupling Symbiosis in Aromatic Synthesis.N Geach, GP Randall, CJ Winfield [Scynexis, UK]—Isotopically Labelled 4‐Formylimidazole.A Studer [Marburg University, Germany]—New Concepts in Tin‐Free Radical Chemistry.AC Spivey, C Noban [Imperial College London, UK]. T Jones [Sheffield University, UK], GJ Ellames, A Kohler [Sanofi‐Synthélabo, UK], H Wadsworth [Amersham Biosciences, UK]—Opportunities for Isotope Labelling via Solid Phase Synthesis with Organogermanium Linkers.J Rieke‐Zapp [Aventis Pharma, Germany]—Optimization of Reaction Conditions on mg‐Scale by Use of Screening Devices.S Stone‐Elander [Karolinska Institute, Sweden]—Rerouting Radiochemistry with Microwaves.J Goehl, B Perry, D Leuck, J Windels, M Marx [EaglePicher Pharmaceutical, USA]—GMP and Radiolabelled Syntheses of Capsaicin.J Krüger, B Manmontri, G Fels [Paderborn University, Germany]—H/D‐Exchange in Aromatic Systems Using Transition Metal Catalysis.F Wüst, T Kniess, R Bergmann [Research Centre Rossendorf, Germany]—Synthesis of a 11C‐Labelled Nonsteroidal Glucocorticoid Receptor Ligand as Potential Radiotracer for Imaging Brain Glucocorticoid Receptors with Positron Emission Tomography (PET).P Mäding, F Füchtner [Research Centre Rossendorf, Germany], CS Hilger [Schering AG, Germany]. M Halks‐Miller, R Horuk [Berlix Biosciences, USA]—18F‐Labelling of a Potent Nonpeptide CCR1 Antagonist for the Diagnosis of the Alzheimer's Disease.L Matei, C Postolache, V Fugaru, E Condac, A Dinischiotu, M Costache [Bukarest University, Romania]—Use of [1,2‐3H] Testosterone in 5α‐ Reductase Enzymatic Activity Dosing in Dermal Fibroblast Cultures from Polycystic Ovarian Patients.L Matei, C Postolache, V Fugaru [Bukarest University, Romania]—Synthesis and Stability Studies of Acyclovir Labelled with Tritium.J Atzrodt, V Derdau [Aventis Pharma, Germany]—Studies on Directed Ortho‐Metalation (DoM) as Convenient Tool for the Synthesis of Labeled Compounds.

Trans-Activation and repression properties of the novel nonsteroid glucocorticoid receptor ligand 2,5-dihydro-9-hydroxy-10-methoxy-2,2,4-trimethyl-5-(1-methylcyclohexen-3-y1)-1H-[1]benzopyrano[3,4-f]quinoline (A276575) and its four stereoisomers.

Glucocorticoids are potent anti-inflammatory and immunosuppressant agents. However, they also produce serious side effects that limit their usage. It has been proposed that anti-inflammatory properties of glucocorticoids are caused mostly by repression of activator protein 1- and nuclear factor kappabeta-stimulated synthesis of inflammatory mediators, whereas most of their adverse effects are associated with trans-activation of genes involved with metabolic processes. Our laboratories have sought to discover novel glucocorticoid receptor (GR) ligands that have high repression but low trans-activation activities. We describe here cellular properties of 2,5-dihydro-9-hydroxy-10-methoxy-2,2,4-trimethyl-5-(1-methylcyclohexen-3-y1)-1H-[1]benzopyrano[3,4-f]quinoline (A276575) and its four enantiomers. Similar to dexamethasone, A276575 exhibited high affinity for GR and potently repressed interleukin (IL) 1beta-stimulated IL-6 production in human skin fibroblasts, prostaglandin (PG) E(2) production in A549 human lung epithelial cells, and concanavalin A-induced monocyte proliferation. In contrast to dexamethasone, A276575 caused smaller induction of aromatase activity in human skin fibroblasts and antagonized dexamethasone-induced activation of an mouse mammary tumor virus-glucocorticoid-response element (GRE) reporter gene construct. Among the four enantiomers of A276575, the two (-)-enantiomers showed 10- to 30-fold higher affinities for GR than their respective (+)-enantiomers. Both (-)-Syn and (-)-Anti enantiomers of A276575 were potent inhibitors of IL-1beta-stimulated PGE2 production in A549 lung epithelial cells; unexpectedly, however, only the (-)-Anti enantiomer inhibited regulated on T-cell activation, normal T-cell expressed and secreted (RANTES) production in A549 cells. In summary, A276575 is a novel, nonsteroidal GR ligand that possesses high repression activities against inflammatory mediator production but has lower GRE trans-activation activities than traditional steroids. Differential repression of RANTES and PGE2 production in a cell by the two (-)-enantiomers of A276575 illustrates the complexity of repression by GR.