Glucocorticoid Receptor Ligand Research Articles

LGD-5552, an Antiinflammatory Glucocorticoid Receptor Ligand with Reduced Side Effects, in Vivo

Treatment of inflammation is often accomplished through the use of glucocorticoids. However, their use is limited by side effects. We have examined the activity of a novel glucocorticoid receptor ligand that binds the receptor efficiently and strongly represses inflammatory gene expression. This compound has potent antiinflammatory activity in vivo and represses the transcription of the inflammatory cytokine monocyte chemoattractant protein-1 and induces the antiinflammatory cytokine IL-10. The compound demonstrates differential gene regulation, compared with commonly prescribed glucocorticoids, effectively inducing some genes and repressing others in a manner different from the glucocorticoid prednisolone. The separation between the antiinflammatory effects of LGD-5552 and the side effects commonly associated with glucocorticoid treatment suggest that this molecule differs significantly from prednisolone and other steroids and may provide a safer therapeutic window for inflammatory conditions now commonly treated with steroidal glucocorticoids.

Effect of Glucocorticoid Receptor Ligand Dexamethasone on the Expression of Organic Cation Transporter in Rat Liver

Since rat organic cation transporter 1 (Oct1, Slc22a1) is expressed mainly in the liver and mediates drug transport, its activity may determine the hepatic handling of cationic drugs. Here, we studied the regulation mechanism of the expression of rat Oct1, focusing on the nuclear receptors. Various nuclear receptors are considered to regulate expressions of many genes for membrane transporters and enzymes that are involved in the drug absorption and disposition. Previously, we demonstrated that some ligands of nuclear receptors affected the transcriptional regulation of rat Oct1 when examined in the primary cultured rat hepatocytes. In the present study, dexamethasone, a ligand of glucocorticoid receptor, down-regulated the expression of rat Oct1. In addition, the transport activity of rat Oct1, evaluated by the uptake of substrates of rat Oct1, was decreased by treatment of dexamethasone in comparison with untreated rat hepatocytes, showing a good agreement with the change in mRNA level. In conclusion, these observations suggested that the expression of rat Oct1 gene and the apparent organic cation uptake activity of rat hepatocytes are down-regulated by dexamethasone presumably via a glucocorticoid receptor.

Induction of Metabolism and Transport in Human Intestine: Validation of Precision-Cut Slices as a Tool to Study Induction of Drug Metabolism in Human Intestine in Vitro

Induction of drug enzyme activity in the intestine can strongly determine plasma levels of drugs. It is therefore important to predict drug-drug interactions in human intestine in vitro. We evaluated the applicability of human intestinal precision-cut slices for induction studies in vitro. Morphological examination and intracellular ATP levels indicated tissue integrity up to 24 h of incubation, whereas in proximal jejunum slices, the metabolic rate toward most substrates remained at 40 to 50% of initial values. In colon slices, the cytochrome P450 conversions were below the detection limit, but conjugation rates remained relatively constant during incubation. The inducibility of drug-metabolizing enzymes and P-glycoprotein was evaluated using prototypical inducers for five induction pathways. beta-Naphthoflavone (aryl hydrocarbon receptor ligand) induced CYP1A1 (132-fold in colon and 362-fold in proximal jejunum) and UDP glucuronosyltransferase (UGT) 1A6 mRNA (9.8-fold in colon and 3.2-fold in proximal jejunum). In proximal jejunum, rifampicin (RIF) [pregnane X receptor (PXR) ligand] induced CYP3A4 (5.2-fold), CYP2B6 (2-fold), UGT1A6 (2.2-fold), and multidrug resistance-1 (MDR1)/ABCB1 mRNA (2.7-fold), whereas 6beta-hydroxytestosterone formation (CYP3A4) increased 2-fold. In colon, RIF induced UGT1A6 32-fold and MDR1 2.2-fold. Dexamethasone (glucocorticoid receptor and PXR ligand) induced CYP3A4 mRNA (3.5-fold) and activity (5-fold) in proximal jejunum. Phenobarbital (constitutive androstane receptor activator) induced CYP3A4 (4.1-fold, only in jejunum), CYP2B6 (4.9-fold in colon and 2.3-fold in proximal jejunum), and MDR1/ABCB1 mRNA and CYP3A4 activity (2-fold only proximal jejunum). Quercetin (nuclear factor-E2-related factor 2 activator) induced UGT1A6 mRNA (6.7-fold in colon and 2.2-fold in proximal jejunum). In conclusion, this study shows that human intestinal precision-cut slices are useful to study induction of drug-metabolizing enzymes and transporters in the human intestine.

Novel insights into mechanisms of glucocorticoid action and the development of new glucocorticoid receptor ligands

Glucocorticoids (GCs) are potent anti-inflammatory and immunosuppressant agents. Unfortunately, they also produce serious side effects that limit their usage. This discrepancy is the driving force for the intensive search for novel GC receptor ligands with a better benefit–risk ratio as compared to conventional GCs. A better understanding of the molecular mode of GC action might result in the identification of novel drug targets. Genomic GC effects are mediated by transrepression or transactivation, the latter being largely responsible for GC side effects. We here discuss novel GC receptor ligands, such as selective glucocorticoid receptor agonists (SEGRAs), which might optimize genomic GC effects as they preferentially induce transrepression with little or no transactivating activity. In addition to genomic GC effects, GCs also produce rapid genomic-independent activities, termed nongenomic, and we here review the possible implications of a recently reported mechanism underlying nongenomic GC-induced immunosuppression in T cells. It was shown that the synthetic GC dexamethasone targets membrane-bound GC receptors leading to impaired T cell receptor signaling. As a consequence, membrane-linked GC receptors might be a potential candidate target for GC therapy. The ultimate goal is to convert these molecular insights into new GC receptor modulators with an improved therapeutic index.

The ginsenoside metabolite compound K, a novel agonist of glucocorticoid receptor, induces tolerance to endotoxin‐induced lethal shock

Compound K (C-K), a protopanaxadiol ginsenoside metabolite, was previously shown to have immunomodulatory effects. Here, we describe a novel therapeutic role for C-K in the treatment of lethal sepsis through the modulation of Toll-like receptor (TLR) 4-associated signalling via glucocorticoid receptor (GR) binding. In mononuclear phagocytes, C-K significantly repressed the activation of TLR4/lipopolysaccharide (LPS)-induced NF-κB and mitogen-activated protein kinases (MAPKs), as well as the secretion of pro-inflammatory cytokines. However C-K did not affect the TLR3-mediated expression of interferon-β or the nuclear translocation of IRF-3. C-K competed with the synthetic glucocorticoid dexamethasone for binding to GR and activated glucocorticoid responsive element (GRE)-containing reporter plasmids in a dose-dependent manner. In addition, the blockade of GR with either the GR antagonist RU486 or a siRNA against GR substantially reversed the anti-inflammatory effects of C-K. Furthermore, TLR4-dependent repression of inflammatory response genes by C-K was mediated through the disruption of p65/interferon regulatory factor complexes. Importantly, pre- or post-treatment with C-K significantly rescued mice from Gram-negative bacterial LPS-induced lethal shock by lowering their systemic inflammatory cytokine levels and by reversing the lethal sequelae of sepsis. Collectively, these results demonstrate that C-K, as a functional ligand of GR, regulates distinct TLR4-mediated inflammatory responses, and suggest a novel therapy for Gram-negative septic shock.

Antiinflammatory glucocorticoid receptor ligand with reduced side effects exhibits an altered protein–protein interaction profile

Glucocorticoids are commonly used antiinflammatory agents whose use is limited by side effects. We have developed a series of glucocorticoid receptor (GR) ligands that retain the strong antiinflammatory activity of conventional glucocorticoids with reduced side effects. We present a compound, LGD5552, that binds the receptor efficiently and strongly represses inflammatory gene expression. LGD5552 bound to GR activates gene expression somewhat differently than glucocorticoids. It activates some genes with an efficacy similar to that of the glucocorticoids. However, other glucocorticoid-activated genes are not regulated by LGD5552. These differences may be because of the more efficient binding of corepressor in the presence of LGD5552, compared with glucocorticoid agonists. This class of nonsteroidal, GR-dependent antiinflammatory drugs may offer a safer alternative to steroidal glucocorticoids in the treatment of inflammatory disease.

Selective Glucocorticoid Receptor Ligands

Glucocorticoids are four-ring steroid compounds that regulate a wide range of physiological systems ranging from embryonic respiratory development, immune function and responses to acute or chronic stress. Glucocorticoids are taken up by many target cells where they bind and activate cytoplasmic glucocorticoid receptors (GRs), which then dimerize, translocate to the nucleus and function as ligand-dependent transcriptional regulators. Synthetic glucocorticoids such as dexamethasone and prednisolone have for decades been the cornerstone for the clinical treatment of inflammatory diseases, such as rheumatoid arthritis and asthma, yet prolonged use have undesirable side-effects such as persistent immune suppression, metabolic imbalance, obesity, diabetes, and osteoporosis. Detailed understanding of the cell signaling mechanisms of GR action has led to the development of novel selective glucocorticoid receptor ligands that appear to offer more efficient treatments for a number of diseases while eliciting fewer side-effects. Additionally, in cell-based and animal model systems a number of compounds such as the methane sulphonamides and a novel compound A-348441 have shown promise as GR antagonists. Other classes of ligands such as the benzopyranoquinolines and the arylpyrazoles have further been shown to selectively influence the transcriptional regulatory properties of GRs on different target gene in various cellular contexts. These selective GR modulators are believed to initiate transcriptional co-regulator recruitment that in turn promotes specific gene responses relevant to the more efficient and specific treatment of inflammatory conditions and metabolic diseases such as type-2 diabetes.

Optical probes to identify the glucocorticoid receptor ligands in living cells

Glucocorticoids act through glucocorticoid receptor (GR) and are used for the treatment of several diseases. Ligand-induced recruitment of coregulator protein(s), coactivator/corepressor, to GR is an initial step in transcriptional activation/inhibition of GR. We describe herein genetically encoded fluorescent probes for screening of glucocorticoids, natural and synthetic, in single living cells. The GR ligand binding domain was connected to the GR interacting peptide sequence from coactivator or corepressor protein via a flexible linker sequence. This fusion protein was sandwiched between cyan and yellow fluorescent proteins (CFP and YFP, respectively) to complete the construct of the probe. This construct functions as an optical probe for imaging ligand-induced interaction between the glucocorticoid receptor and the coregulator protein (GLUCOCOR) in live cells. The interaction between GR LBD and coregulator peptide within GLUCOCOR brings CFP in close proximity of YFP to induce fluorescence resonance energy transfer from CFP to YFP. The GLUCOCORs can identify functionally active GR ligands, rapidly and conveniently, in a high-throughput screen; and are capable of distinguishing GR agonists, antagonists, and selective GR modulators in intact living cells. Therefore, the present method may play a significant role in developing new glucocorticoids for clinical use.

Separating Transrepression and Transactivation: A Distressing Divorce for the Glucocorticoid Receptor?

Glucocorticoids (corticosteroids) are highly effective in combating inflammation in the context of a variety of diseases. However, clinical utility can be compromised by the development of side effects, many of which are attributed to the ability of the glucocorticoid receptor (GR) to induce the transcription of, or transactivate, certain genes. By contrast, the anti-inflammatory effects of glucocorticoids are due largely to their ability to reduce the expression of pro-inflammatory genes. This effect has been predominantly attributed to the repression of key inflammatory transcription factors, including AP-1 and NF-kappaB, and is termed transrepression. The ability to functionally separate these transcriptional functions of GR has prompted a search for dissociated GR ligands that can differentially induce transrepression but not transactivation. In this review, we present evidence that post-transcriptional mechanisms of action are highly important to the anti-inflammatory actions of glucocorticoids. Furthermore, we present the case that mechanistically distinct forms of glucocorticoid-inducible gene expression are critical to the development of anti-inflammatory effects by repressing inflammatory signaling pathways and inflammatory gene expression at multiple levels. Considerable care is therefore required to avoid loss of anti-inflammatory effectiveness in the development of novel transactivation-defective ligands of GR.

Signal Sequences for Targeting of Gene Therapy Products to Subcellular Compartments: The Role Of CRM1 in Nucleocytoplasmic Shuttling of the Protein Switch

The purpose of this study was to understand the mechanism of nuclear export of the protein switch, used for controlled intracellular delivery of gene products, by studying the involvement of classical export receptor CRM1. Transient transfections of protein switch constructs, isolated nuclear export and import signals were carried out. Effect of leptomycin B (inhibitor of export receptor) and geldanamycin (inhibitor of Hsp90) on localization of these constructs was studied using fluorescence microscopy. Putative nuclear export signals in the glucocorticoid and progesterone receptor ligand binding domains were identified and studied. It was observed that treatment with leptomycin B caused nuclear accumulation of the protein switch constructs. However, geldanamycin did not have any pronounced effect on the localization. The isolated nuclear export signal from glucocorticoid receptor localized mostly in the cytoplasm, while its mutated version was present everywhere. The localization controlled protein switch constructs are exported out of the nucleus by the classical CRM1 receptors. The ligand binding domain of these protein switch constructs plays an important role in maintaining these constructs in the cytoplasm in the absence of ligand, as well the re-export back to the cytoplasm from the nucleus after ligand washout.

A novel quantitative flow cytometric method for measuring glucocorticoid receptor (GR) in cell lines: Correlation with the biochemical determination of GR

Currently, a time consuming biochemical method is used for GR quantification. Here we compare the biochemical approach with a newly developed flow cytometric method of measuring GR in cell lines, which is less time consuming and does not requires the use of radioactive materials. The biochemical assay is easy to apply but the cells need to be grown in media free of endogenous glucocorticoids, in order to prevent them from interfering with radiolabelled hormone binding to the receptor. The presence of endogenous GR ligands is known to reduce receptor levels and to often produce false negative results. The immunofluorescent method is free of such limitations, as it depends entirely on detecting the receptor using a highly specific monoclonal antibody. Additionally, the biochemical assay cannot measure heterogeneity in individual cells, in contrast the flow cytometric one enables the enumeration of the receptor on a per cell basis, allowing exact description of differences in receptor levels amongst intact cells. Our results demonstrate that the flow cytometric method is of similar accuracy but of higher precision compared to the biochemical one. Also, the data we obtained using the immunofluorescent method correlated well with the biochemical one ( R 2 = 0.9712). In conclusion, flow cytometric method requires small cell numbers, is more accurate and lesser time consuming than the biochemical one. Thus, it could be useful for the quantification of GR in lymphocyte subpopulations, in lymphoproliferative disorders and in tumor cells from cancer patients, in order to design more efficient clinical treatment protocols.

Non-genomic effects of ginsenoside-Re in endothelial cells via glucocorticoid receptor

We demonstrated that ginsenoside-Re (Re), a pharmacological active component of ginseng, is a functional ligand of glucocorticoid receptor (GR) using competitive ligand-binding assay (IC 50 = 156.6 nM; K d = 49.7 nM) and reporter gene assay. Treatment with Re (1 μM) raises intracellular Ca 2+ ([Ca 2+] i) and nitric oxide (NO) levels in human umbilical vein endothelial cells as measured using fura-2 and 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate, respectively. Western blot analysis shows that Re increased phosphorylation of endothelial nitric oxide synthase. These effects were abolished by GR antagonist RU486, siRNA targeting GR, non-selective cation channel blocker 2-aminoethyldiphenylborate, or in the absence of extracellular Ca 2+, indicating Re is indeed an agonistic ligand for the GR and the activated GR induces rapid Ca 2+ influx and NO production in endothelial cells.

Development of a Preparative-Scale Asymmetric Synthesis of (R)-p-Tolyl Methyl Sulfoxide for Use in a One-Pot Synthesis of a Drug Intermediate Containing a Trifluoromethyl-Substituted Alcohol Functionality

A one-pot process for the synthesis of (S)-1,1,1-trifluoro-4-(5-fluoro-2-methoxy-phenyl)-4-methyl-2-((R)-toluene-4-sulfinylmethyl)pentan-2-ol (3) is described, which was a key intermediate for the preparation of a class of novel glucocorticoid receptor ligands. The chemistry features the preparative-scale synthesis of (R)-p-tolyl methyl sulfoxide [(R)-pTMSO] from (2R,4S,5R)-4-methyl-5-phenyl-3-(tolene-4-sulfonyl)oxathiazolidine-2-oxide (5) and the synthesis of 3 from 5 without isolation of (R)-pTMSO during the process.

Agonist versus antagonist induce distinct thermodynamic modes of co-factor binding to the glucocorticoid receptor

The glucocorticoid receptor (GR) is involved in the transcriptional regulation of genes associated with inflammation, glucose homeostasis, and bone turnover through the association with ligands, such as corticosteroids. GR-mediated gene transcription is regulated or fine-tuned via the recruitment of co-factors including coactivators and corepressors. Current therapeutic approaches to targeting GR aim to retain the beneficial anti-inflammatory activity of the corticosteroids while eliminating negative side effects. Towards achieving this goal the experiments discussed here reveal a mechanism of co-factor binding in the presence of either bound agonist or antagonist. The GR ligand binding domain (GR-LBD(F602S)), in the presence of agonist or antagonist, utilizes different modes of binding for coactivator versus corepressor. Coactivator binding to the co-effector binding pocket of GR-LBD(F602S) is driven both by favorable enthalpic and entropic interactions whereas corepressor binding to the same pocket is entropically driven. These data support the hypothesis that ligand-induced conformational changes dictate co-factor binding and subsequent trans-activation or trans-repression.

Modelling the glucocorticoid receptor and producing therapeutic agents with anti‐inflammatory effects but reduced side‐effects

Glucocorticoid hormones exert a wide spectrum of metabolic and immunological effects. They are synthesized from a cholesterol precursor and are structurally related to the other steroid hormones, progesterone, aldosterone and oestrogen. They act through the glucocorticoid receptor (GR), a member of the nuclear receptor superfamily. The GR is an intracellular receptor; the hydrophobic ligand accesses its receptor by diffusion across the plasma membrane. The ligand-activated GR translocates to the nucleus to regulate expression of its target genes. The GR, in common with the rest of the receptor family, can be functionally divided into an N-terminal transcription activation domain, a central DNA binding domain and a C-terminal ligand binding domain, which also includes a second transactivation domain. Although synthetic glucocorticoids are the most potent anti-inflammatory agents known, their use is limited owing to the range and severity of their side-effects. The structure of the ligand binding domain of the glucocorticoid receptor has now been solved, and a series of studies has shown that even subtle changes to the ligand structure alter the final conformation of the ligand-receptor complex, with consequences for further protein recruitment and for the function of the receptor. This, coupled with the successful development of selective oestrogen receptor agonists, has led to concerted efforts to find selective GR ligands, with preserved beneficial anti-inflammatory activity, but reduced side-effect profile. Current efforts have identified several useful tool compounds, and further molecules are in development in several pharmaceutical companies.

Unliganded and hormone-bound glucocorticoid receptors interact with distinct hydrophobic sites in the Hsp90 C-terminal domain

Unlike most chaperones, heat-shock protein 90 (Hsp90) interacts with a select group of "client proteins" that regulate essential biological processes. Little is known about how Hsp90 recognizes and binds these proteins. The glucocorticoid receptor (GR) is a well characterized Hsp90 client protein, whose hormone binding, nuclear-cytoplasmic trafficking, and transcriptional activity are regulated by Hsp90. Here, we provide evidence that unliganded and hormone-bound GR interact with two distinct, solvent-exposed hydrophobic sites in the Hsp90 C-terminal domain that contain the sequences "MxxIM" (HM10) and "L/MxxIL" (HM9). Our results indicate that binding of Hsp90 HM10 to unliganded GR stabilizes the unliganded ligand-binding pocket of GR indirectly by promoting an intramolecular interaction between the C-terminal alpha-helix (H12) and a solvent-exposed hydrophobic groove in the GR ligand binding domain. In the presence of hormone, Hsp90 appears to bind the hydrophobic groove of GR directly by mimicking the interactions of GR with transcriptional coactivators. The identified interactions provide insights into the mechanisms that enable Hsp90 to regulate the activity of both unliganded and hormone-bound GR and to sharpen the cellular response to hormone.

α-Methyltryptamine sulfonamide derivatives as novel glucocorticoid receptor ligands

α-Methyltryptamine sulfonamides were identified as human glucocorticoid receptor (hGR) ligands in an ultra high throughput screening (UHTS) campaign. Described will be the hit-to-lead activities, including parallel and single point analog synthesis to map the scaffold. Ligands were identified that exhibited 30 nM binding to hGR. The SAR and selectivity of these compounds will be discussed.

Patterns of exposure to glucocorticoid receptor ligand

Hormones are characteristically secreted in distinct pulses. Corticosterone (in rodents) and cortisol (in human) show profound circadian and ultradian rhythmicity which results in rapid changes in ligand concentration in the circulating blood. The pattern of ultradian glucocorticoid levels shows genetic modulation, can change according to the physiological and pathological status of the animal and can be programmed by neonatal events. Since the tissue response to changes in circulating glucocorticoid levels can be very rapid, changes in ultradian rhythm could allow tissue-specific regulation of glucocorticoid signalling without major changes in mean plasma hormone levels.

Analysis of the Dissociated Steroid RU24858 Does Not Exclude a Role for Inducible Genes in the Anti-Inflammatory Actions of Glucocorticoids

Although repression of inflammatory gene expression makes glucocorticoids powerful anti-inflammatory agents, side effects limit usage and drive the search for improved glucocorticoid receptor (GR) ligands. In A549 pulmonary cells, dexamethasone and the prototypical dissociated ligand RU24858 (Mol Endocrinol 11:1245-1255, 1997) repress interleukin (IL)-1beta-induced expression of cyclooxygenase (COX)-2 and IL-8. Although RU24858 is a weaker GR ligand, both glucocorticoids showed similar efficacies on transrepression of nuclear factor kappaB (NF-kappaB)-dependent transcription, whereas RU24858 yielded less than 12% of the response to dexamethasone on a classic glucocorticoid response element (GRE) reporter (transactivation). Modest NF-kappaB-dependent transrepression ( approximately 40%), along with analysis of IL-8 transcription rate and the accumulation of unspliced nuclear RNA, indicates that transrepression does not fully account for the repression of genes such as IL-8. This was confirmed by the finding that mRNA degradation is increased by both dexamethasone and RU24858. Analysis of IL-1beta-induced steady-state mRNA levels for IL-8 and COX-2 show that dexamethasone- and RU24858-dependent repression of these genes is attenuated by inhibitors of transcription and protein synthesis. Because similar effects were observed with respect to COX-2 and IL-8 protein expression, we conclude that glucocorticoid-dependent gene expression is necessary for repression by both glucocorticoids. Despite RU24858 being defective at classic GRE-dependent transactivation, both dexamethasone and RU24858 induced the expression of potentially anti-inflammatory genes and metabolic genes, suggesting the importance of nontraditional glucocorticoid-dependent gene expression. Thus, classic transactivation- and transrepressionbased screens for anti-inflammatory "dissociated" GR ligands may be flawed because they may not reflect the effects on real glucocorticoid-inducible genes.

Reduction of glucocorticoid receptor ligand binding by the 11-β hydroxysteroid dehydrogenase type 2 inhibitor, Thiram

Endogenous and synthetic glucocorticoids (GCs), such as cortisol and dexamethasone (Dex), modulate airway inflammation, regulate the production of surfactant by lung epithelial cells, and influence fetal lung maturation. The 11-β hydroxysteroid dehydrogenase type 2 (HSD2) enzyme catalyzes the oxidation of bioactive cortisol and Dex to their 11-keto metabolites. Thiram (tetramethylthiuram disulfide) specifically inhibits HSD2 activity by oxidizing cysteine residues located in the cofactor binding domain of the enzyme. During studies performed to define a potential role for HSD2 in modulating GC action in human lung epithelial cells, we observed that exposure of intact human lung epithelial cells (NCI-H441) to 50 μM Thiram significantly attenuated the down-stream effects of Dex (100 nM) on the expression of two GC-sensitive genes, pulmonary surfactant proteins A and B. This observation appeared to be inconsistent with simple inhibition of HSD2 activity. Although Thiram inhibited HSD2 oxidase activity in a dose-dependent manner without affecting HSD2 protein expression, Thiram also reduced specific binding of [ 3H]-Dex to the glucocorticoid receptor (GR). Pre-treatment of cells with 1 mM dithiothreitol (DTT), a thiol-reducing agent, completely blocked the inhibitory effect of Thiram on ligand binding. These results are suggestive that Thiram may alter the ligand-binding domain of the GR by oxidizing critical thiol-containing amino acid residues. Taken collectively, these data demonstrate that attenuated down-stream GC signaling, via decreased binding of ligand to the GR, is a novel cellular effect of Thiram exposure in human lung epithelial cells.