Non-steroidal Glucocorticoid Receptor Research Articles

Tetrahydroquinoline glucocorticoid receptor agonists: Discovery of a 3-hydroxyl for improving receptor selectivity

We have previously disclosed a series of glucocorticoid receptor (GR) ligands derived from 6-indole-1,2,3,4-tetrahydroquinolines through structure–activity relationship (SAR) of the pendent C6-indole ring. In parallel with this effort, we now report SAR of the tetrahydroquinoline A-ring that identified the importance of a C3 hydroxyl in improving GR selectivity within a series of non-steroidal GR agonists.

Nonsteroidal Dissociated Glucocorticoid Agonists Containing Azaindoles as Steroid A-Ring Mimetics

Syntheses and structure-activity relationships (SAR) of nonsteroidal glucocorticoid receptor (GR) agonists are described. These compounds contain azaindole moieties as A-ring mimetics and display various degrees of in vitro dissociation between gene transrepression and transactivation. Collagen induced arthritis studies in mouse have demonstrated that in vitro dissociated compounds (R)-16 and (R)-37 have steroid-like anti-inflammatory properties with improved metabolic side effect profiles, such as a reduced increase in body fat and serum insulin levels, compared to steroids.

Correlation of Skin Blanching and Percutaneous Absorption for Glucocorticoid Receptor Agonists by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry Imaging and Liquid Extraction Surface Analysis with Nanoelectrospray Ionization Mass Spectrometry

Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) and liquid extraction surface analysis (LESA) with nanoelectrospray ionization mass spectrometry (nESI-MS) have both been successfully employed to determine the degree of percutaneous absorption of three novel nonsteroid glucocorticoid receptor (GR) agonists in porcine ear sections. Historically, the ability of a glucocorticoid to elicit a skin blanching response when applied at low dose in ethanol solution to the forearms of healthy human volunteers has been a reliable predictor of their topical anti-inflammatory activity. While all three nonsteroidal GR agonists under investigation caused a skin blanching effect, the responses did not correlate with in vitro GR agonist potencies and different time courses were also observed for the skin blanching responses. MALDI MSI and LESA with nESI-MS were used to investigate and understand these different responses. The findings of the investigation was that the depth of porcine skin penetration correlates to the degree of skin blanching obtained for the same three compounds in human volunteers.

Structure Guided Design of 5-Arylindazole Glucocorticoid Receptor Agonists and Antagonists

Glucocorticoid receptor (GR) agonists have been used for more than half a century as the most effective treatment of acute and chronic inflammatory conditions despite serious side effects that accompany their extended use that include glucose intolerance, muscle wasting, skin thinning, and osteoporosis. As a starting point for the identification of GR ligands with an improved therapeutic index, we wished to discover selective nonsteroidal GR agonists and antagonists with simplified structure compared to known GR ligands to serve as starting points for the optimization of dissociated GR modulators. To do so, we selected multiple chemical series by structure guided docking studies and evaluated GR agonist activity. From these efforts we identified 5-arylindazole compounds that showed moderate binding to the glucocorticoid receptor (GR) with clear opportunities for further development. Structure guided optimization was used to design arrays that led to potent GR agonists and antagonists. Several in vitro and in vivo experiments were utilized to demonstrate that GR agonist 23a (GSK9027) had a profile similar to that of a classical steroidal GR agonist.

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.

Characterization of a novel non-steroidal glucocorticoid receptor antagonist

Selective antagonists of the glucocorticoid receptor (GR) are desirable for the treatment of hypercortisolemia associated with Cushing’s syndrome, psychic depression, obesity, diabetes, neurodegenerative diseases, and glaucoma. NC3327, a non-steroidal small molecule with potent binding affinity to GR ( K i = 13.2 nM), was identified in a high-throughput screening effort. As a full GR antagonist, NC3327 greatly inhibits the dexamethasone (Dex) induction of marker genes involved in hepatic gluconeogenesis, but has a minimal effect on matrix metalloproteinase 9 (MMP-9), a GR responsive pro-inflammatory gene. Interestingly, the compound recruits neither coactivators nor corepressors to the GR complex but competes with glucocorticoids for the interaction between GR and a coactivator peptide. Moreover, NC3327 does not trigger GR nuclear translocation, but significantly blocks Dex-induced GR transportation to the nucleus, and thus appears to be a ‘competitive’ GR antagonist. Therefore, the non-steroidal compound, NC3327, may represent a new class of GR antagonists as potential therapeutics for a variety of cortisol-related endocrine disorders.

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.

Rational Ligand-Based Virtual Screening and Structure–Activity Relationship Studies in the Ligand-Binding Domain of the Glucocorticoid Receptor-α

The interest in developing synthetic glucocorticoids (GCs) arises from the utility of endogenous steroids as potent anti-inflammatory and immunosuppressant agents. The first GCs to be discovered, such as cortisol or dexamethasone, still represent the main treatment for conditions of the inflammatory process, despite the fact that they carry a significant risk of side effects. Hence, there is a continuing need to find drugs that preserve the immune effects of GCs without the side effects, such as those on metabolism (diabetes), bone tissue (osteoporosis), muscles (myopathy), eyes and skin. In this review, we focus on the recent use of ligand-based computational approaches in glucocorticoid receptor (GR) drug-design efforts for the determination of novel GR ligands. We examine a number of ligand-based (similarity searches, pharmacophore screens and quantitative structure-activity relationships) approaches that have been implemented in recent years. A recent virtual high-throughput screening similarity search was successful in developing a novel series of nonsteroidal GR antagonists. Additionally, there has been considerable success in ligand-based structure-analysis relationship generation and lead optimization studies for the GR. Future trends toward integrated GR ligand design incorporating ligand- and structure-based methodologies are inevitable.

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.

Glucocorticoid Receptor Antagonists

This review covers recent progress in the discovery of selective glucocorticoid receptor (GR) antagonists. Potential therapeutic applications of selective GR antagonists are described including the pharmacological rationale and, in some cases, clinical evidence that underlies these proposed uses. Disease areas that are discussed are Cushing's syndrome, psychotic depression, diabetes, obesity, Alzheimer's disease, neuropathic pain, drug abuse, and glaucoma. Methods for evaluating GR antagonist properties (binding, functional, and in vivo assays) are briefly covered. Early research on steroidal ligands which led to the identification of the non-selective GR antagonist RU-486 (mifepristone) and the GR-selective steroid RU-43044 is reviewed as is subsequent work on related steroidal compounds. Structure activity relationships (SAR) of nonsteroidal GR antagonists from the following structural classes are presented: octahydrophenanthrenes, spirocyclic dihydropyridines, triphenylmethanes and diaryl ethers, chromenes, dibenzyl anilines, dihydroisoquinolines, pyrimidinediones, azadecalins, and aryl pyrazolo azadecalins.

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.

Dissociated non-steroidal glucocorticoid receptor modulators: an update on new compounds

Background: Glucocorticoids are the most potent anti-inflammatory drugs available and allow successful treatment of several chronic inflammatory and autoimmune diseases. The discovery of the molecular mechanisms of the glucocorticoid receptor (GR), and the recognition that activation and repression of gene expression could be addressed separately, opened the possibility of improving safety profiles by identifying ligands that predominantly induce repression over activation. Recent progress in the development of dissociated and other non-steroidal GR modulators is discussed here. Objective: To summarize the data and progress of current compounds reported in GR programs carried out by pharmaceutical companies. Methods: The review is primarily of patents and publications disclosed over the last two years. Results/conclusion: Although the mechanistic drivers for development of safer GR ligands have expanded from the basic transrepression/transactivation hypothesis, the concept appears to have led to the identification of some drug candidates that show dissociated in vitro profiles and improved in vivo therapeutic indices.

Synthesis and Characterization of Nonsteroidal Glucocorticoid Receptor Modulators for Multiple Myeloma

Structure-activity relationship studies centered around 3'-substituted (Z)-5-(2'-(thienylmethylidene))1,2-dihydro-9-hydroxy-10-methoxy-2,2,4-trimethyl-5H-chromeno[3,4-f]quinolines are described. A series of highly potent and efficacious selective glucocorticoid receptor modulators were identified with in vitro activity comparable to dexamethasone. In vivo evaluation of these compounds utilizing a 28 day mouse tumor xenograft model demonstrated efficacy equal to dexamethasone in the reduction of tumor volume.

Identification of dissociated non-steroidal glucocorticoid receptor agonists

A new series of ligands for the glucocorticoid receptor (GR) is described. SAR development was guided by docking 3 into the GR active site and optimizing an unsubstituted phenyl ring for key interactions found in the steroid A-ring binding pocket. To identify compounds with an improved side effect profile over marketed steroids the functional activity of compounds was evaluated in cell based assays for transactivation (aromatase) and transrepression (IL-6). Through this effort, 36 has been identified as a partial agonist with a dissociated profile in these cell based assays.

Non-steroidal glucocorticoid agonists—The discovery of aryl pyrazoles as A-ring mimetics

Starting from an established series of non-steroidal glucocorticoid receptor (GR) agonists, a large array was designed where a metabolically labile benzoxazinone moiety was replaced. Initial hits bound to GR but lacked agonist activity. Following two further iterations, potent GR agonists were discovered with 20D1E1 having NFκB agonism pIC 50 8.8 (103%). Other analogues such as 23D1E1 display a dissociated profile (NFκB pIC 50 8.1 (103%), MMTV pEC 50 7.02 (36%)). The tetrahydronaphthalene moiety can also be replaced with substituted aryls such as 24E1 and 25E1.

Discovery and optimization of novel, non-steroidal glucocorticoid receptor modulators

A virtual screening approach comprising a 3-D similarity search based on known GR modulators was used to identify a novel series of non-steroidal glucocorticoid receptor (GR) antagonists. Optimization of the initial hit to provide potent compounds which exhibit good selectivity against other steroidal nuclear hormone receptors is described.

5( Z)-Benzylidene-1,2-dihydro-9-hydroxy-10-methoxy-2,2,4-trimethyl-5 H-1-aza-6-oxa-chrysenes as non-steroidal glucocorticoid receptor modulators

A series of 5-benzylidene-1,2-dihydro-2,2,4-trimethyl-5 H-1-aza-6-oxa-chrysenes was synthesized and profiled for their ability to act as selective glucocorticoid receptor modulators (SGRMs). The synthesis and structure–activity relationships for this series of compounds are presented.

Non-steroidal glucocorticoid receptor antagonists: the race to replace RU-486 for anti-glucocorticoid therapy

The endogenous glucocorticoid, cortisol, elevates blood glucose and suppresses the immune system. Glucocorticoid (GC) levels rapidly increase in response to physiologic and mental stress, thereby allowing stress adaptation. Unfortunately, the GC response can be excessive, especially under stressful conditions for the organism. The resulting hypercortisolemia is associated with a cluster of symptoms called Cushing’s syndrome, a serious and potentially fatal illness involving hyperglycemia, hypertension, osteoporosis, muscle atrophy and fat maldistribution, as well as psychoses and immunosuppresion. Several disease states, such as diabetes and Cushing’s, would benefit from blocking the actions of endogenous cortisol. The only glucocorticoid receptor (GR) antagonist available in the clinic is the steroid mifepristone (RU-486), whose primary potency is antigestagenic, making its utility as a GR antagonist limited. This manuscript reviews the current patent literature on selective non-steroidal GR antagonists.

Dissociated Nonsteroidal Glucocorticoid Receptor Modulators; Discovery of the Agonist Trigger in a Tetrahydronaphthalene−Benzoxazine Series

The tetrahydronaphthalene-benzoxazine glucocorticoid receptor (GR) partial agonist 4b was optimized to produce potent full agonists of GR. Aromatic ring substitution of the tetrahydronaphthalene leads to weak GR antagonists. Discovery of an "agonist trigger" substituent on the saturated ring of the tetrahydronaphthalene leads to increased potency and efficacious GR agonism. These compounds are efficacy selective in an NFkB GR agonist assay (representing transrepression effects) over an MMTV GR agonist assay (representing transactivation effects). 52 and 60 have NFkB pIC(50) = 8.92 (105%) and 8.69 (92%) and MMTV pEC(50) = 8.20 (47%) and 7.75 (39%), respectively. The impact of the trigger substituent on agonism is modeled within GR and discussed. 36, 52, and 60 have anti-inflammatory activity in a mouse model of inflammation after topical dosing with 52 and 60, having an effect similar to that of dexamethasone. The original lead was discovered by a manual agreement docking method, and automation of this method is also described.