Glucocorticoid Receptor Ligand Research Articles

Insight into the molecular mechanisms of glucocorticoid receptor action promotes identification of novel ligands with an improved therapeutic index

Glucocorticoids are highly effective in the therapy of inflammatory and autoimmune disorders. Their beneficial action is restricted because of their adverse effects upon prolonged usage. Topical glucocorticoids that act locally have been developed to significantly reduce systemic side effects. Nonetheless, undesirable cutaneous effects such as skin atrophy persist from the use of topical glucocorticoids. There is therefore a high medical need for drugs as effective as glucocorticoids but with a reduced side-effect profile. Glucocorticoids function by binding to and activating the glucocorticoid receptor that positively or negatively regulates the expression of specific genes. Several experiments suggest that the negative regulation of gene expression by the glucocorticoid receptor accounts for its anti-inflammatory action. This occurs through direct or indirect binding of the receptor to transcription factors such as activator protein-1, nuclear factor-kappaB or interferon regulatory factor-3 that are already bound to their regulatory sites. The positive action of the receptor occurs through homodimer binding of the receptor to discrete nucleotide sequences and this possibly contributes to some of the adverse effects of the hormone. Glucocorticoid receptor ligands that promote the negative regulatory action of the receptor with reduced positive regulatory function should therefore show improved therapeutic potential. A complete separation of the positive from the negative regulatory activities of the receptor has so far not been possible because of the interdependent nature of the two regulatory processes. Nevertheless, considerable improvement in the therapeutic action of glucocorticoid receptor ligands is being achieved through the use of key molecular targets for screening novel glucocorticoid receptor ligands.

Cortisol Production Rate in Posttraumatic Stress Disorder

Several authors have reported the unsuspected finding of low cortisol levels (urinary, salivary, and serum) in patients with posttraumatic stress disorder (PTSD). Our objective was to assess concentrations of cortisol and its predominant metabolites, cortisol production rate (CPR), and glucocorticoid receptor (GR) binding characteristics in PTSD compared with normal subjects. Matched PTSD patients and control subjects had CPR determined by a stable isotope dilution technique after infusion of deuterated cortisol. Serum cortisol, urinary cortisol, and its metabolites were measured by gas chromatography/mass spectrometry. GR binding capacity (R(o)) and ligand binding affinity (K(d)) were measured in mononuclear leukocytes. All subjects were tested during a 40-h admission in an inpatient clinical research center. Ten patients with PTSD were matched by age and gender with 10 controls. Statistical comparison was conducted for various measures of cortisol in PTSD patients and normal subjects. No statistical difference was found in mean level or circadian pattern of cortisol secretion using serum or salivary immunoassay detection methods. Although in the normal range, urinary cortisol by immunoassay showed statistically lower values over a 24-h period in PTSD patients compared with controls. This finding was not confirmed by gas chromatography/mass spectrometry determination of cortisol or its metabolites. CPR was not statistically different between these groups. GR also showed no alteration in R(o) or K(d) between the groups. The data indicate that PTSD in the chronic and unprovoked state is not characterized by an acute biological stress response.

Discovery and SAR Study of Novel Dihydroquinoline Containing Glucocorticoid Receptor Ligands.

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Glucocorticoid therapy‐induced skin atrophy

Glucocorticoids (GCs) are highly effective for the topical treatment of inflammatory skin diseases. Their long-term use, however, is often accompanied by severe and partially irreversible adverse effects, with atrophy being the most prominent limitation. Progress in the understanding of GC-mediated molecular action as well as some advances in technologies to determine the atrophogenic potential of compounds has been made recently. It is likely that the detailed mechanisms of GC-induced skin atrophy will be discovered and in vitro models for the reliable prediction of atrophy will be established in the foreseeable future. This knowledge will not only facilitate safety profiling of established drugs but will also foster further drug discovery by improving compound characterization processes. New insights into GC modes of action will guide optimization strategies aiming at novel GC receptor ligands with improved effect/side effect profile.

Signaling pathway of ginsenoside-Rg1 leading to nitric oxide production in endothelial cells

We here provide definitive evidence that ginsenoside-Rg1, the pharmacologically active component of ginseng, is a functional ligand of the glucocorticoid receptor (GR) as determined by fluorescence polarization assay. Rg1 increased the phosphorylation of GR, phosphatidylinositol-3 kinase (PI3K), Akt/PKB and endothelial nitric oxide synthase (eNOS) leading to increase nitric oxide (NO) production in human umbilical vein endothelial cell. Rg1-induced eNOS phosphorylation and NO production were significantly reduced by RU486, LY294,002, or SH-6. Also, knockdown of GR completely eliminated the Rg1-induced NO production. This study revealed that Rg1 can indeed serve as an agonist ligand for GR and the activated GR can induce rapid NO production from eNOS via the non-transcriptional PI3K/Akt pathway.

Conformational changes of the glucocorticoid receptor ligand binding domain induced by ligand and cofactor binding, and the location of cofactor binding sites determined by hydrogen/deuterium exchange mass spectrometry

HXMS (hydrogen/deuterium exchange mass spectrometry) of the glucocorticoid receptor ligand-binding domain (GR LBD) complexed with the agonist dexamethasone and the antagonist RU-486 is described. Variations in the rates of exchange were observed in regions consistent with the published crystal structures of GR LBD complexed with RU-486 when compared with the GR dexamethasone complex. We also report the HXMS results for agonist-bound GR LBD with the coactivator transcriptional intermediary factor 2 (TIF2) and anatagonist-bound GR LBD with nuclear receptor corepressor (NCoR). Alterations in exchange rates observed for agonist-bound GR LBD with TIF2 present were consistent with the published crystal structural contacts for the complex. Alterations in exchange rates observed for antagonist-bound GR LBD with NCoR were a subset of those observed with TIF2 binding, suggesting a common or overlapping binding site for coactivator and corepressor.

Discovery and SAR study of novel dihydroquinoline containing glucocorticoid receptor ligands

We report the discovery of a novel class of glucocorticoid receptor (GR) ligands based on 1,2-dihydroquinoline molecular scaffold. The compounds exhibit good GR binding affinity and selectivity profile against other nuclear hormone receptors.

Ligand-specific glucocorticoid receptor activation in human platelets

Few studies have addressed the effects of classical anti-inflammatory glucocorticoids on platelet function. Here, we report for the first time that human platelets contain the glucocorticoid receptor (GR) as identified by a combination of biochemical and functional techniques. Ligand-binding studies revealed the presence of a high- and low-affinity binding site for [3H]-dexamethasone in platelets. The 2 GR ligands prednisolone and dexamethasone competed for [3H]-dexamethasone binding, as did the mineralocorticoid aldosterone. However, while prednisolone (1-10 microM) reduced adenosine diphosphate (ADP, 4 microM) and thromboxane A2 receptor agonist U46619 induced platelet aggregation (up to 75%), dexamethasone had no effect. The inhibition produced by prednisolone was reversed by preincubation with the GR antagonist mifepristone (10 microM; RU486), suggesting the functional importance of the ligand-receptor complex. In addition, prednisolone caused a marked (approximately 50%) reduction in thromboxane B2 levels, whereas dexamethasone was without effect. The apparently anomalous binding data were clarified by the fact that washed platelets (1) contained mineralocorticoid receptor and that (2) it was associated with GR. Taken together, our data suggest that platelet GR forms a heterodimeric complex with the mineralocorticoid receptor that is susceptible to differential activation by specific receptor ligands.

New and improved glucocorticoid receptor ligands

The therapeutic and prophylactic use of glucocorticoids is widespread due to their powerful anti-inflammatory, antiproliferative and immunomodulatory activity. However, long-term use of these drugs can result in severe dose-limiting side effects. One of the most critical and debilitating side effects is osteoporosis, which leads to increased risk of fractures. Glucocorticoids damage bone through several different mechanisms. The search for novel glucocorticoids that have reduced side effects in bone and other tissues is being driven by the identification of new mechanisms of action of the glucocorticoid receptor. This may facilitate the detection of new, safer therapies with efficacies equivalent to currently prescribed steroids.

Differential effects of selected natural compounds with anti-inflammatory activity on the glucocorticoid receptor and NF-κB in HeLa cells

Natural compounds have been used in the treatment of various diseases for centuries. Herein, we investigated the effects of structurally diverse alkaloids with anti-inflammatory activity (berberine, sanguinarine, chelerythrine, and colchicine) on two important anti-inflammatory and pro-inflammatory players, i.e. glucocorticoid receptor (GR) and nuclear factor kappa B (NF-κB), respectively. Sanguinarine and chelerythrine elicited nuclear translocation of the p65 subunit of NF-κB. The nuclear import of p65 was strongly augmented by these akaloids in non-stimulated cells as well as in cells challenged with tumor necrosis factor alpha (TNFα). Colchicine and berberine had no effect on p65 nuclear translocation regardless of the presence or absence of TNFα. Colchicine caused rapid degradation of the GR protein, whereas berberine had no effect on GR content or cellular localization. Sanguinarine and chelerythrine induced accumulation of GR in the nucleus with concomitant diminution of cytosolic GR. Analyses on the transcriptional activity of GR and NF-κB monitored by reporter assays using HeLa cells transiently transfected with glucocorticoid response element (pGRE-LUC) and/or NF-κB elements fused to luciferase gene (pNF-κB-luc) showed that none of the compounds tested had the capability to trigger GR and/or NF-κB transcriptional activities, respectively. The ligand binding assay showed that colchicine and berberine are not GR ligands whereas sanguinarine and chelerythrine significantly decreased binding of 3H-labelled dexamethasone to GR. In conclusion, structurally diverse natural antiflogistics displayed differential effects on GR and NF-κB in HeLa cells.

A concise asymmetric route for the synthesis of a novel class of glucocorticoid mimetics containing a trifluoromethyl-substituted alcohol

An asymmetric route was developed for the synthesis of a class of novel glucocorticoid receptor ligand derivatives 1. The key step of this synthesis involves a diastereoselective addition of chiral sulfoxide anion to a trifluoromethyl ketone precursor. The resulting diastereomers are readily separable and can be converted to the corresponding chiral epoxide and chiral alkyne intermediates ( 2 and 3). This sequence of reactions is suitable for large-scale preparation of these chiral intermediates and derivatives of 1. The absolute stereochemistry of the biologically active enantiomer of these GR ligands has also been determined.

Glucocorticoid Receptor Regulates ATP-Binding Cassette Transporter-A1 Expression and Apolipoprotein-Mediated Cholesterol Efflux From Macrophages

The ATP-binding cassette transporter-A1 (ABCA1) regulates cholesterol efflux from cells and is involved in high-density lipoprotein metabolism and atherogenesis. The objective of this study was to investigate the effect of dexamethasone (Dex) and other glucocorticoid receptor (GR) ligands on apolipoprotein AI-mediated cholesterol efflux from macrophages and ABCA1 expression in them. Dex, a GR agonist, decreased ABCA1 mRNA levels in a dose- and time-dependent fashion, and RU486, a GR antagonist, reversed the inhibitory effect of Dex. The effects of Dex and RU486 on ABCA1 protein levels and apolipoprotein AI-mediated cholesterol efflux from the macrophages were consistent with these changes in mRNA levels. Transfected RAW264.7, together with a human ABCA1 promoter-luciferase construct, inhibited transcriptional activity by Dex and overexpression of human GR. Transrepression by GR was not mediated by liver X receptor (LXR), because there were no differences in the effects of the GR ligands on promoter activity between a reporter construct with mutations at the LXR binding site and one without the mutations, and no changes were brought about in ABCG1 and ABCG4 expression by GR ligands. Our results showed that GR ligands affected ABCA1 expression and cholesterol efflux from macrophages, which are regulated by GR through a LXR-independent mechanism.

Trifluoromethyl group as a pharmacophore: Effect of replacing a CF 3 group on binding and agonist activity of a glucocorticoid receptor ligand

Compound 1, a potent glucocorticoid receptor ligand, contains a quaternary carbon bearing trifluoromethyl and hydroxyl groups. This paper describes the effect of replacing the trifluoromethyl group on binding and agonist activity of the GR ligand 1. The results illustrate that replacing the CF 3 group with a cyclohexylmethyl or benzyl group maintains the GR binding potency. These substitutions alter the functional behavior of the GR ligands from agonists to antagonists. Docking studies suggest that the benzyl analog 19 binds in a similar fashion as the GR antagonist, RU486. The central benzyl group of 19 and the C-11 dimethylaniline moiety of RU486 overlay. Binding of compound 19 is believed to force helix 12 to adopt an open conformation and this leads to the antagonist properties of the non-CF 3 ligands carrying a large group at the center of the molecule.

OP25. NEW GLUCOCORTICOIDS: SELECTIVE GLUCOCORTICOID RECEPTOR AGONISTS, NO-GLUCOCORTICOIDS AND LONG-CIRCULATING LIPOSOMAL GLUCOCORTICOIDS

Our understanding of the actions of glucocorticoids has greatly increased in the last few years. The various genomic and non-genomic mechanisms of glucocorticoid action provide interesting and sometimes very advanced starting points for the development of optimised glucocorticoids and glucocorticoid receptor ligands. SEGRAs, NO-glucocorticoids and long-circulating liposomal glucocorticods are examples to be mentioned in this regard. Selective glucocorticoid receptor agonists (SEGRAs): Currently glucocorticoid receptor ligands are being developed that cause predominantly transrepression but not transactivation. These drugs are called selective glucocorticoid receptor agonists (SEGRAs) and are thought to have an improved safety/efficacy profile. In vivo investigations and clinical trials will have to define whether SEGRAs will, as ‘improved glucocorticoids’, enter clinical medicine in the near future. NO-glucocorticoids: Recent experimental observations prompt the assessment of the clinical impact of another new class of glucocorticoid drugs, NO-glucocorticoids, which are able to release low levels of nitric oxide. They have been shown to be endowed with enhanced anti-inflammatory properties and reduced side effects. The prototype of these new steroids, 21-NO-prednisolone, is much more potent than prednisolone in models of acute and chronic inflammation but does not activate primary osteoclast activity (whereas prednisolone does). However, more studies are needed to confirm that NO-glucocorticoids will be effective as anti-inflammatory agents in clinical practice. Long-circulating liposomal glucocorticoids: The anti-inflammatory effectiveness of glucocorticoids can be improved by the additional benefits of the non-genomic actions of high glucocorticoid concentrations. On this basis, the successful use of long-circulating liposomal glucocorticoids has recently been reported which accumulate at sites of inflammation. This leads to very high glucocorticoid concentrations at e.g. the inflamed joint (but accompanied by low plasma concentrations with perhaps a lower rate of side effects) which is the key factor explaining the observed strong therapeutic effect. Conclusions: These new GCR-ligands and the administration of liposomes are very promising approaches that will hopefully soon be available in clinical practice to improve the benefit/risk ratio and well-being of patients being treated. Another intriguing issue is the study of membrane bound glucocorticoid receptors (mGCR). These surface receptors are suggested to mediate rapid nongenomic effects and have been found to be upregulated in active rheumatoid arthritis. The number of monocytes expressing mGCR is significantly correlated with disease activity. Therefore, drugs binding selectively to the mGCR may in future also prove to be of therapeutic value but the functions of mGCR must first be investigated in detail.

A Hierarchical Clustering Approach for Large Compound Libraries

A modified version of the k-means clustering algorithm was developed that is able to analyze large compound libraries. A distance threshold determined by plotting the sum of radii of leaf clusters was used as a termination criterion for the clustering process. Hierarchical trees were constructed that can be used to obtain an overview of the data distribution and inherent cluster structure. The approach is also applicable to ligand-based virtual screening with the aim to generate preferred screening collections or focused compound libraries. Retrospective analysis of two activity classes was performed: inhibitors of caspase 1 [interleukin 1 (IL1) cleaving enzyme, ICE] and glucocorticoid receptor ligands. The MDL Drug Data Report (MDDR) and Collection of Bioactive Reference Analogues (COBRA) databases served as the compound pool, for which binary trees were produced. Molecules were encoded by all Molecular Operating Environment 2D descriptors and topological pharmacophore atom types. Individual clusters were assessed for their purity and enrichment of actives belonging to the two ligand classes. Significant enrichment was observed in individual branches of the cluster tree. After clustering a combined database of MDDR, COBRA, and the SPECS catalog, it was possible to retrieve MDDR ICE inhibitors with new scaffolds using COBRA ICE inhibitors as seeds. A Java implementation of the clustering method is available via the Internet (http://www.modlab.de).

Novel ketal ligands for the glucocorticoid receptor: in vitro and in vivo activity

A novel series of selective ligands for the human glucocorticoid receptor is described. Structure–activity studies focused on variation of B-ring size, ketal ring size, and ketal substitution. These analogs were found to be potent and selective ligands for GR and have partial agonist profiles in functional assays for transactivation (TAT, GS) and transrepression (IL-6). Of these compounds, 27, 28, and 35 were evaluated further in a mouse LPS-induced TNF-α secretion model. Compound 28 had an ED 50 of 14.1 mg/kg compared with 0.5 mg/kg for prednisolone in the same assay.

Aktueller Stand zur Entwicklung neuer Glucocorticoidrezeptorliganden

Recent insights into the mechanisms of genomic and non-genomic glucocorticoid actions have stimulated the search for novel glucocorticoid receptor ligands. These efforts are driven by the need to improve the benefit-risk ratio of these important drugs. Glucocorticoids are very frequently and successfully used drugs which mediate important immunosuppressive and anti-inflammatory effects, but unfortunately they have pleiotropic effects causing a number of adverse reactions which limit their clinical use, especially at higher dosages and for longer periods. For this reason, novel glucocorticoid receptor ligands are being developed, among them selective glucocorticoid receptor agonists (SEGRAs). SEGRAs are drugs that predominantly induce transrepression effects (inhibition of protein synthesis), whereas the transactivation activity (induction of protein synthesis) is significantly reduced as compared with conventional glucocorticoid drugs. This makes sense since it became evident over the last few years that many adverse effects are predominantly caused by the transactivation mechanism, whereas anti-inflammatory effects are mostly mediated by transrepression mechanisms. Other interesting examples for exciting new developments are NO-glucocorticoids and long-circulating liposomal glucocorticoids. It is, however, true of all these developments that further in vivo and in vitro investigations and clinical trials will have to define in more detail their safety-efficacy profile in order to answer the questions whether these drugs as "improved glucocorticoids" will enter clinical medicine in the near future.

Synthesis and radiopharmacological evaluation of 2′-(4-fluorophenyl)-21-[ 18F]fluoro-20-oxo-11β,17α-dihydroxy-pregn-4-eno[3,2- c]pyrazole as potential glucocorticoid receptor ligand for positron emission tomography (PET)

The radiosynthesis and the radiopharmacological evaluation of pyrazolo steroid 2′-(4-fluorophenyl)-21-[ 18F]fluoro-20-oxo-11β,17α-dihydroxy-pregn-4-eno[3,2- c]pyrazole [ 18 F]-2 is described. The radiolabeling was accomplished in 3–4% decay-corrected radiochemical yield within 80 min at an specific radioactivity of 0.8–1.2 Ci/μmol. Biodistribution studies in male Wistar rats showed an initial brain uptake of 0.25 ± 0.03% ID/g after 5 min, which remained constant over 60 min. The radiopharmacological evaluation of compound [ 18 F]-2 was completed with autoradiography using rat brain sections and micro-PET imaging.

Ursodeoxycholic acid induced activation of the glucocorticoid receptor in primary rat hepatocytes

Ursodeoxycholic acid (UDCA), a hydrophilic bile acid, improves biochemical, immunopathological and histological parameters in chronic cholestatic liver diseases. The immunomodulatory properties of UDCA show interesting similarities with the effects of glucocorticoids. We investigated the activation of the glucocorticoid receptor by UDCA and the glucocorticoid receptor dependent gene expression in primary rat hepatocytes as well as binding of radiolabelled UDCA to the glucocorticoid receptor ligand binding site expressed in a glucocorticoid receptor fusion protein. Primary rat hepatocytes in culture were co-transfected with a luciferase reporter gene construct (GRE-luc) containing a glucocorticoid receptor responsive element (GRE) and a glucocorticoid receptor expression vector (6RGR) followed by stimulation with dexamethasone or UDCA. Luciferase activity was determined and specific binding of glucocorticoid receptor to the GRE was confirmed by an electrophoretic mobility shift assay (EMSA). The glucocorticoid receptor binding site was expressed in a GR-myc fusion protein and binding of radiolabelled UDCA to the fusion protein was determined. Incubation of co-transfected hepatocytes with 0.1-1.000 microM dexamethasone or 0.1-1.000 microM UDCA led to an 11.9- to 20.85-fold (dexamethasone) and 2.6- to 4.3-fold (UDC) increase of luciferase activity. Mobility shift assays using nuclear extracts from transfected and stimulated hepatocytes also showed a dose dependent increase of DNA binding after stimulation with UDCA. However, incubation of the GR-myc fusion protein with radiolabelled UDCA yielded no specific binding of UDCA to the glucocorticoid receptor binding site, whereas dexamethasone showed specific binding of the fusion protein. UDCA activates the intracellular glucocorticoid receptor in a dose-dependent manner. Direct binding of the glucocorticoid receptor by radiolabelled UDCA at the glucocorticoid receptor binding site could be excluded as the mechanism of activation. The mechanisms involved in UDCA-mediated glucocorticoid receptor activation and possible targeted glucocorticoid receptor activation due to partial UDCA tissue specificity warrant further elucidation.

Design and Evaluation of Novel Nonsteroidal Dissociating Glucocorticoid Receptor Ligands.

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