Nonsteroidal Glucocorticoid Receptor Antagonists Research Articles

FX5, a non-steroidal glucocorticoid receptor antagonist, ameliorates diabetic cognitive impairment in mice

Diabetic cognitive impairment (DCI) is a common diabetic complication characterized by learning and memory deficits. In diabetic patients, hyperactivated hypothalamic-pituitary-adrenal (HPA) axis leads to abnormal increase of glucocorticoids (GCs), which causes the damage of hippocampal neurons and cognitive impairment. In this study we investigated the cognition-improving effects of a non-steroidal glucocorticoid receptor (GR) antagonist 5-chloro-N-[4-chloro-3-(trifluoromethyl) phenyl]thiophene-2-sulfonamide (FX5) in diabetic mice. Four weeks after T1DM or T2DM was induced, the mice were administered FX5 (20, 40 mg·kg−1·d−1, i.g.) for 8 weeks. Cognitive impairment was assessed in open field test, novel object recognition test, Y-maze test, and Morris water maze test. We showed that FX5 administration significantly ameliorated the cognitive impairments in both type 1 and 2 diabetic mice. Similar cognitive improvement was observed in diabetic mice following brain GR-specific knockdown by injecting AAV-si-GR. Moreover, AAV-si-GR injection occluded the cognition-improving effects of FX5, suggesting that FX5 functioning as a non-steroidal GR antagonist. In PA-treated primary neurons (as DCI model in vitro), we demonstrated that FX5 (2, 5, 10 μM) dose-dependently ameliorated synaptic impairment via upregulating GR/BDNF/TrkB/CREB pathway, protected against neuronal apoptosis through repressing GR/PI3K/AKT/GSK3β-mediated tauopathy and subsequent endoplasmic reticulum stress. In LPS-treated primary microglia, FX5 dose-dependently inhibited inflammation through GR/NF-κB/NLRP3/ASC/Caspase-1 pathway. These beneficial effects were also observed in the hippocampus of diabetic mice following FX5 administration. Collectively, we have elucidated the mechanisms underlying the beneficial effects of non-steroidal GR antagonist FX5 on DCI and highlighted the potential of FX5 in the treatment of the disease.

Ipriflavone as a non-steroidal glucocorticoid receptor antagonist ameliorates diabetic cognitive impairment in mice.

Diabetic cognitive impairment (DCI) is a common diabetic complication with hallmarks of loss of learning ability and disorders of memory and behavior. Glucocorticoid receptor (GR) dysfunction is a main reason for neuronal impairment in brain of diabetic patients. Here, we determined that ipriflavone (IP) a clinical anti‐osteoporosis drug functioned as a non‐steroidal GR antagonist and efficiently ameliorated learning and memory dysfunction in both type 1 and 2 diabetic mice. The underlying mechanism has been intensively investigated by assay against the diabetic mice with GR‐specific knockdown in the brain by injection of adeno‐associated virus (AAV)‐ePHP‐si‐GR. IP suppressed tau hyperphosphorylation through GR/PI3K/AKT/GSK3β pathway, alleviated neuronal inflammation through GR/NF‐κB/NLRP3/ASC/Caspase‐1 pathway, and protected against synaptic impairment through GR/CREB/BDNF pathway. To our knowledge, our work might be the first to expound the detailed mechanism underlying the amelioration of non‐steroidal GR antagonist on DCI‐like pathology in mice and report the potential of IP in treatment of DCI.

FX5 as a non-steroidal GR antagonist improved glucose homeostasis in type 2 diabetic mice via GR/HNF4α/miR-122-5p pathway

Type 2 diabetes mellitus (T2DM) is a chronic metabolic disease characterized by glucose metabolic disorders, and gluconeogenesis inhibiting is a promisingly therapeutic strategy for T2DM. Glucocorticoid receptor (GR) is tightly implicated in the regulation of gluconeogenesis, although the underlying mechanism remains obscure. Here, we discovered that small molecule, 5-chloro-N-[4-chloro-3-(trifluoromethyl)phenyl]thiophene-2-sulfonamide (FX5) as a new non-steroidal GR antagonist efficiently ameliorated glucose homeostasis in db/db and HFD/STZ-induced T2DM mice. The mechanism underlying the suppression of FX5 against gluconeogenesis was highly investigated. FX5 suppressed gluconeogenetic genes G6Pase and PEPCK in mouse primary hepatocytes and liver tissues of T2DM mice. Results of mammalian one-hybrid and transactivation as well as nuclear translocation assays totally evaluated the antagonistic features of FX5 against GR. Moreover, siRNA and overexpression related assays verified that FX5 alleviated gluconeogenesis either directly by antagonizing GR or indirectly through GR/HNF4α/miR122-5p signaling pathway. Our work has presented a new mode for GR antagonist in the regulation of gluconeogenesis, which is expected to highlight the potential of FX5 in the treatment of T2DM.

Abstract PD3-02: Second-generation selective glucocorticoid receptor modulators in triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) lacks expression of the estrogen receptor (ER), progesterone receptor (PR), and HER2. A subset of primary TNBCs (at least 30%) expresses moderate to strong glucocorticoid receptor (GR) in greater than 10% of invasive tumor cells; in addition, we have reported that increased GR signaling promotes TNBC cell survival. Furthermore, our laboratory recently observed that patients with early stage/high-GR-expressing TNBCs have a relatively poor prognosis. Therefore, GR is being explored as a target for improving outcome in TNBC. Indeed, mifepristone, a well-characterized non-selective steroidal GR/PR antagonist, has shown promise in reversing GR-mediated tumor cell-survival signaling in TNBC. In vivo, increased TNBC xenograft chemosensitivity was observed when mifepristone was added to chemotherapy treatment. Most recently, a Phase I clinical trial demonstrated the safety and potential efficacy of mifepristone added to nab-paclitaxel chemotherapy in Stage IV TNBC patients. Three highly selective non-steroidal GR antagonists (GRAs) have been investigated, CORT108297. CORT125134 (two aryl pyrazole-fused azadecalins) and CORT118335 (a pyrimidine dione). These new GRAs have 1) far less cross-reactivity than mifepristone with other nuclear receptor family members and 2) lower interaction profiles for drug metabolizing CYP enzymes. We hypothesized that selective GRAs would not only inhibit GR-induced pro-survival gene expression, but also increase TNBC chemosensitivity, and may eventually be a valuable adjunct therapy for treating chemotherapy-resistant TNBC. To understand how GR modulators interact with the GR ligand-binding domain (LBD), we are employing two approaches: 1) computational modeling of the GRA-GR LBD based on published crystal structures of the GR LBD with mifepristone (in collaboration with UIUC), and 2) crystal structure analysis of the GR LBD with GR modulators. Preliminary computational results indicate that CORT108297 docks into the LBD and antagonizes GR activity by indirectly disordering Helix 12. Experiments are ongoing using various buffer conditions to produce crystals of the GR LBD with CORT108297 and CORT118335 for x-ray crystallography. Secondly, we are functionally characterizing CORT108297, CORT125134, and CORT118335 for their ability to inhibit glucocorticoid induction of key GR-mediated target genes. Over the course of eight hours, we observed that the compounds exhibited temporal antagonism of the expression of GR target genes encoding anti-apoptotic proteins such as SGK-1, MCL-1, and MKP-1/DUSP1. Lastly, we are examining the selective GRAs' ability to enhance tumor cell cytotoxicity from paclitaxel in cell culture and in TNBC xenograft models. Preliminary results indicate that CORT125134 and CORT108297 significantly increase GR+ TNBC sensitivity to paclitaxel, and are well-tolerated in our in vivo models. Citation Format: West DC, Hosfield DJ, Mayne CG, Skor MN, Styke SC, Pierce CF, Kocherginsky M, Hunt H, Fleming GF, Szmulewitz RZ, Tajkhorshid E, Greene GL, Conzen SD. Second-generation selective glucocorticoid receptor modulators in triple-negative breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr PD3-02.

Indole Glucocorticoid Receptor Antagonists Active in a Model of Dyslipidemia Act via a Unique Association with an Agonist Binding Site.

To further elucidate the structural activity correlation of glucocorticoid receptor (GR) antagonism, the crystal structure of the GR ligand-binding domain (GR LBD) complex with a nonsteroidal antagonist, compound 8, was determined. This novel indole sulfonamide shows in vitro activity comparable to known GR antagonists such as mifepristone, and notably, this molecule lowers LDL (-74%) and raises HDL (+73%) in a hamster model of dyslipidemia. This is the first reported crystal structure of the GR LBD bound to a nonsteroidal antagonist, and this article provides additional elements for the design and pharmacology of clinically relevant nonsteroidal GR antagonists that may have greater selectivity and fewer side effects than their steroidal counterparts.

Synthesis and Biological Evaluation of Cyclopentaquinoline Derivatives as Nonsteroidal Glucocorticoid Receptor Antagonists.

The steroidal glucocorticoid antagonist mifepristone has been reported to improve the symptoms of depression. We report the discovery of 6-(3,5-dimethylisoxazol-4-yl)-2,2,4,4-tetramethyl-2,3,4,7,8,9-hexahydro-1H-cyclopenta[h]quinolin-3-one 3d (QCA-1093) as a novel nonsteroidal glucocorticoid receptor antagonist. The compound displayed potent in vitro activity, high selectivity over other steroid hormone receptors, and significant antidepressant-like activity in vivo.

Discovery of liver selective non-steroidal glucocorticoid receptor antagonist as novel antidiabetic agents

Series of benzyl-phenoxybenzyl amino-phenyl acid derivatives (8a–q) are reported as non-steroidal GR antagonist. Compound 8g showed excellent h-GR binding and potent antagonistic activity (in vitro). The lead compound 8g exhibited significant oral antidiabetic and antihyperlipidemic effects (in vivo), along with liver selectivity. These preliminary results confirm discovery of potent and liver selective passive GR antagonist for the treatment of T2DM.

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.

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.

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.

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.

Effects of acute and chronic insulin-induced hypoglycemia on type II glucocorticoid receptor (GR) gene expression in characterized CNS metabolic loci

The metabolic stressor, hypoglycemia, elicits integrated counterregulatory responses, including activation of the hypothalamic–pituitary–adrenal axis. Type II glucocorticoid receptors (GR) occur in multiple components of the central autonomic circuitry that regulates glucostasis, and antecedent GR stimulation is implicated in impaired glucagon and counterregulatory dysfunction during recurrent insulin-induced hypoglycemia (RIIH). To examine the hypothesis that this chronic stress may alter basal and/or hypoglycemic patterns of GR gene expression in a site-specific manner, real-time RT-PCR techniques were utilized to evaluate tissue GR mRNA levels in the microdissected lateral hypothalamic area (LHA) and paraventricular (PVH), dorsomedial (DMH), ventromedial (VMH), and arcuate (ARH) hypothalamic nuclei, before and after one or four injections, on as many days, of the intermediate-acting insulin formulation, Humulin NPH (NPH), while controls were treated with diluent alone. Rats injected with four doses of NPH were pretreated by intracerebroventricular (icv) administration of the selective nonsteroidal GR antagonist, CP-472555, or vehicle alone prior to insulin injections on days 1–3. The results show that acute hypoglycemia had no impact on GR mRNA levels in each structure evaluated. Basal GR gene expression was not altered by antecedent hypoglycemia, but tissue transcript levels were elevated in the DMH, PVH, VMH, and ARH during RIIH. Icv CP-472555 administration prior to antecedent hypoglycemia prevented RIIH-associated increases in GR mRNA in each of these sites. These data show that GR gene transcripts are increased in discrete CNS metabolic loci during RIIH, and that these local responses are attenuated by pharmacological blockade of central GR activation. The present studies demonstrate that hypoglycemic hypercorticosteronemia causes upregulated GR gene expression during RIIH, and implicate GR in mechanisms underlying this action.

I.c.v. administration of the nonsteroidal glucocorticoid receptor antagonist, CP-472555, prevents exacerbated hypoglycemia during repeated insulin administration

Hypoglycemia elicits an integrated array of CNS-mediated counterregulatory responses, including activation of the hypothalamic–pituitary–adrenal axis. The role of antecedent adrenocortical hypersecretion in impaired glucose counterregulation remains controversial. The present studies utilized the selective, nonsteroidal glucocorticoid receptor antagonist, CP-472555, as a pharmacological tool to investigate the hypothesis that hypoglycemic hypercorticosteronemia modulates CNS efferent autonomic and neuroendocrine motor responses to recurring insulin-induced hypoglycemia via glucocorticoid receptor-dependent mechanisms. Groups of adult male rats were injected s.c. with either one or four doses of the intermediate-acting insulin, Humulin neutral protamine Hagedorn (NPH), on as many days, while controls were injected with diluent alone. Animals injected with four doses of insulin were pretreated by i.c.v. administration of graded doses of the glucocorticoid receptor antagonist or vehicle alone prior to the first three doses of insulin. Repeated daily injection of NPH exacerbated hypoglycemia, attenuated patterns of glucagon and epinephrine secretion, and diminished neuronal transcriptional activation in discrete CNS metabolic loci, including the lateral hypothalamic area, dorsomedial hypothalamic nucleus, paraventricular hypothalamic nucleus, and nucleus of the solitary tract. While i.c.v. delivery of 25 or 100 ng doses of CP-472555 did not alter any of these parameters, animals treated with 500 ng exhibited circulating glucose, glucagon, and epinephrine levels that were similar to those in rats injected with one dose of insulin, as well as a reversal of recurring insulin-induced hypoglycemia-associated reductions in Fos immunolabeling in the lateral hypothalamic area, dorsomedial hypothalamic nucleus, and paraventricular hypothalamic nucleus. These results provide unique pharmacological evidence that antecedent activation of central glucocorticoid receptor is required for exacerbation of hypoglycemia during recurring insulin-induced hypoglycemia, and that these receptors mediate modulatory effects of hypoglycemic hypercorticosteronemia on autonomic efferent responses to recurring insulin-induced hypoglycemia. The data also suggest that neurons in central loci characterized here by antagonist-mediated overturn of recurring insulin-induced hypoglycemia-induced decreases in neuronal transcriptional activation may be direct or indirect substrates for this hormonal modulation action.

Synthesis and Biological Evaluation of Novel, Selective, Nonsteroidal Glucocorticoid Receptor Antagonists.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Synthesis and biological evaluation of novel, selective, nonsteroidal glucocorticoid receptor antagonists

We report the discovery of a novel class of glucocorticoid receptor (GR) antagonists based on the chromene molecular scaffold. The compounds exhibit good functional potency and an improved receptor selectivity profile for GR over other steroid receptors when compared to the classical steroidal GR-antagonist, RU-486.

Discovery of Potent, Non-Steroidal and Highly Selective Glucocorticoid Receptor Antagonists with Anti-Obesity Activity

To thwart phase II metabolism, Structure-Activity-Relationship (SAR) studies around the phenol of the potent glucocorticoid receptor (GR) antagonists CP-394531 and CP-409069 were examined. The discovery of the potent, selective, nonsteroidal GR antagonist (CP-472555) with anti-GR and anti-obesity activity in animal models is described.

Discovery of potent, nonsteroidal, and highly selective glucocorticoid receptor antagonists.

An approach to the computer-assisted, pharmacophore design of nonsteroidal templates for the glucocorticoid receptor (GR) that contained an element of pseudo-C2 symmetry was developed. The enatiomer of the initial design, 1Ra, and not the designed molecule, 1S, showed the desired ligand binding to the GR. The pseudo-C2 symmetry of the template allowed for rapid improvements in GR activity resulting in potent, selective, nonsteroidal GR antagonists, CP-394531 and CP-409069.