Glucocorticoid Receptor Transactivation Research Articles

Intermittent glucocorticoid treatment improves muscle metabolism via the PGC1α/Lipin1 axis in an aging-related sarcopenia model.

Sarcopenia burdens the older population through loss of muscle energy and mass, yet treatments to functionally rescue both parameters are lacking. The glucocorticoid prednisone remodels muscle metabolism on the basis of frequency of intake, but its mechanisms in sarcopenia are unknown. We found that once-weekly intermittent prednisone administration rescued muscle quality in aged 24-month-old mice to a level comparable to that seen in young 4-month-old mice. We discovered an age- and sex-independent glucocorticoid receptor transactivation program in muscle encompassing peroxisome proliferator-activated receptor γ coactivator 1 α (PGC1α) and its cofactor Lipin1. Treatment coordinately improved mitochondrial abundance through isoform 1 and muscle mass through isoform 4 of the myocyte-specific PGC1α, which was required for the treatment-driven increase in carbon shuttling from glucose oxidation to amino acid biogenesis. We also probed myocyte-specific Lipin1 as a nonredundant factor coaxing PGC1α upregulation to the stimulation of both oxidative and anabolic effects. Our study unveils an aging-resistant druggable program in myocytes for the coordinated rescue of energy and mass in sarcopenia.

Comparison of the Glucocorticoid Receptor Binding and Agonist Activities of Typical Glucocorticoids: Insights into Their Endocrine Disrupting Effects.

Over the past decades, the synthetic glucocorticoids (GCs) have been widely used in clinical practice and animal husbandry. Given the health hazard of these toxic residues in food, it is necessary to explore the detailed interaction mechanisms of typical GCs and their main target glucocorticoid receptor (GR). Hence, this work compared the GR binding and agonist activities of typical GCs. Fluorescence polarization assay showed that these GCs were potent ligands of GR. Their GR binding affinities were in the order of methylprednisolone>betamethasone≈prednisolone>dexamethasone, with IC50 values of 1.67, 2.94, 2.95, and 5.58 nM. Additionally, the limits of detection of dexamethasone, betamethasone, prednisolone, and methylprednisolone were 0.32, 0.14, 0.19, and 0.09 μg/kg in fluorescence polarization assay. Reporter gene assay showed that these GCs induced GR transactivation in a dose-dependent manner, confirming their GR agonist activities. Among which, dexamethasone at the concentration of 100 nM produced a maximal induction of more than 11-fold over the blank control. Molecular docking and molecular dynamics simulations suggested that hydrogen-bonding and hydrophobic interactions played an important role in stabilizing the GC-GR-LBD complexes. In summary, this work might help to understand the GR-mediated endocrine disrupting effects of typical GCs.

PFO5DoDA disrupts hepatic homeostasis primarily through glucocorticoid signaling inhibition

Legacy per- and polyfluoroalkyl substances (PFASs) are a worldwide health concern due to their potential bioaccumulation and toxicity in humans. A variety of perfluoroether carboxylic acids (PFECAs) have been developed as next-generation replacements of legacy PFASs. However, information regarding their possible environmental and human health risks is limited. In the present study, we explored the effects of PFECAs on mice based on long-term exposure to environmentally relevant doses of perfluoro-3,5,7,9,11-pentaoxadodecanoic acid (PFO5DoDA). Results showed that PFECAs exposure suppressed many cellular stress signals and resulted in hepatomegaly. PFO5DoDA acted as an agonist of the peroxisome proliferator-activated receptor (PPAR) in vitro and modulated PPAR-dependent gene expression in the liver. Importantly, PFECAs had an inhibitory effect on the glucocorticoid receptor (GR), which may contribute to the extensive suppression of stress signals. Of note, the GR suppression induced by PFECAs was not reported by legacy perfluorooctanoic acid (PFOA). PFO5DoDA-induced changes in both GR and PPAR signals remodeled hepatic metabolic profiles, including decreased fatty acids and amino acids and increased β-oxidation. Mechanistically, PFO5DoDA inhibited GR transactivation by degradation of GR proteins. Our results emphasize the potential risk of PFECAs to human health, which were introduced to ease concerns regarding legacy PFASs.

The long winding road to the safer glucocorticoid receptor (GR) targeting therapies.

Glucocorticoids (Gcs) are widely used to treat inflammatory diseases and hematological malignancies, and despite the introduction of novel anti-inflammatory and anti-cancer biologics, the use of inexpensive and effective Gcs is expected to grow. Unfortunately, chronic treatment with Gcs results in multiple atrophic and metabolic side effects. Thus, the search for safer glucocorticoid receptor (GR)-targeted therapies that preserve therapeutic potential of Gcs but result in fewer adverse effects remains highly relevant. Development of selective GR agonists/modulators (SEGRAM) with reduced side effects, based on the concept of dissociation of GR transactivation and transrepression functions, resulted in limited success, and currently focus has shifted towards partial GR agonists. Additional approach is the identification and inhibition of genes associated with Gcs specific side effects. Others and we recently identified GR target genes REDD1 and FKBP51 as key mediators of Gcs-induced atrophy, and selected and validated candidate molecules for REDD1 blockage including PI3K/Akt/mTOR inhibitors. In this review, we summarized classic and contemporary approaches to safer GR-mediated therapies including unique concept of Gcs combination with REDD1 inhibitors. We discussed protective effects of REDD1 inhibitors against Gcs–induced atrophy in skin and bone and underlined the translational potential of this combination for further development of safer and effective Gcs-based therapies.

TAZ inhibits glucocorticoid receptor and coordinates hepatic glucose homeostasis in normal physiological states.

The elucidation of the mechanisms whereby the liver maintains glucose homeostasis is crucial for the understanding of physiological and pathological states. Here, we show a novel role of hepatic transcriptional co-activator with PDZ-binding motif (TAZ) in the inhibition of glucocorticoid receptor (GR). TAZ is abundantly expressed in pericentral hepatocytes and its expression is markedly reduced by fasting. TAZ interacts via its WW domain with the ligand-binding domain of GR to limit the binding of GR to the GR response element in gluconeogenic gene promoters. Therefore, liver-specific TAZ knockout mice show increases in glucose production and blood glucose concentration. Conversely, the overexpression of TAZ in mouse liver reduces the binding of GR to gluconeogenic gene promoters and glucose production. Thus, our findings demonstrate that hepatic TAZ inhibits GR transactivation of gluconeogenic genes and coordinates gluconeogenesis in response to physiological fasting and feeding.

GR-mediated anti-inflammation of α-boswellic acid: Insights from in vitro and in silico studies

Although multiple bioactivities of α-boswellic acid have been reported, the molecular mechanism of its anti-inflammatory action is not yet clear. Hence, glucocorticoid receptor (GR)-mediated anti-inflammation of α-boswellic acid was investigated in this work. Fluorescence polarization assay suggested that α-boswellic acid bound to GR with IC50 value of 658.00 ± 0.21 μM. Upon binding to α-boswellic acid, GR translocated from cytoplasm into nucleus of HeLa cells, facilitating sequential transcriptional regulation of GR-related genes. Luciferase reporter assay suggested that α-boswellic acid lacked GR transcriptional activity, indicating its potential as a dissociative GR ligand. Interestingly, α-boswellic acid selectively modulated the anti-inflammatory gene CBG (marker for GR transrepression), while leaving the “side-effect” gene TAT (marker for GR transactivation) unaffected in HepG2 cells. Furthermore, α-boswellic acid inhibited lipopolysaccharide-stimulated cytokines production in U937 macrophages, confirming its anti-inflammation property in vitro. Molecular docking showed that both hydrogen-bonding and hydrophobic interactions helped to stabilize α-boswellic acid-GR binding. Their binding stability was further confirmed in a 70-ns dynamics simulation. In summary, α-boswellic acid could bind to and translocate GR but did not induce glucocorticoid response element-mediated transcription. Since α-boswellic acid showed the dissociated characteristic that separated transrepression from transactivation, it might be a selective GR modulator against inflammatory disorders.

In vitro assessment of corticosteroid effects of eight chiral herbicides

Information regarding the enantioselective endocrine disruption of chiral herbicides is scarce. This study assessed the disrupting effects of eight typical chiral herbicides on corticosteroids (including glucocorticoids and mineralocorticoids). Enantioselectivity of eight chiral herbicides were evaluated for their agonistic/antagonistic effects on glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) with CHOK1 cell line using reporter gene assay. Their influence on the production of corticosteroids were further investigated in H295R cell line using enzyme-linked immunosorbent assay (ELISA). None of the racemates or enantiomers of eight chiral herbicides exhibited GR or MR agonistic activity at non-cytotoxic concentrations. However, rac-propisochlor and S-imazamox antagonized cortisol-induced transactivation of GR by 21.79% and 38.73% at the concentration of 1.0 × 10−7 M and 1.0 × 10−6 M, respectively, and R-napropamide remarkably attenuated aldosterone-induced MR transactivation by 68.78% at 1.0 × 10−6 M. The secretion of cortisol was significantly restrained after treated with 1.0 × 10−6 M rac-propisochlor and rac-/R-napropamide at the concentration of 1.0 × 10−6 M by 26.49%, 30.10% and 35.27%, respectively, while this glucocorticoid was remarkably induced by 1.0 × 10−5 M rac-diclofop-methyl and its two enantiomers at the concentration of 1.0 × 10−5 M by 75.60%, 100.1% and 68.78%, respectively. Exposure to rac-propisochlor (1.0 × 10−6 M), S-diclofop-methyl (1.0 × 10−5 M) or rac-/S-/R- acetochlor (1.0 × 10−6 M) and rac-/S-/R-lactofen (1.0 × 10−6 M) inhibited the secretion of aldosterone by approximately 40%. Our findings suggested that chiral herbicides disrupted corticosteroid homeostasis in an enantioselective way. Therefore, more comprehensive screening is required to better understand the ecological and health risks of chiral pesticides.

Abstract 3450: Impact of BRCA1 on glucocorticoid signaling in fallopian tube epithelial cells

Abstract Background: High-grade serous ovarian cancer (HGSC), the most predominant ovarian cancer histotype, originates in the fallopian tube epithelium (FTE). Women with germline BRCA1 mutations are at high risk for this cancer subtype. Our previous work indicates that FTE cells from BRCA1 mutation carriers (mtBRCA1) have increased EGFR and NFκB signaling compared to FTE from control patients, and that glucocorticoid receptor (GR) signaling may be impaired in BRCA1-deficient cells. Glucocorticoids are potent anti-inflammatory steroids that inhibit NFκB signaling. Studies using breast cancer cells report that BRCA1 increases microRNA miR-146 expression, which targets transcripts for EGFR and activators of NFκB. In this study, we examined the impact of BRCA1 deficiency on GR expression and signaling, and on miR-146 expression. Methods: Primary FTE cells were derived from mtBRCA1 carriers or control patients. Immortalized human FTE OE6/7 cells were engineered to overexpress BRCA1. UWB1.289 and UWB1.289+BRCA1 HGSC cells were obtained from ATCC. Levels of GRα, GRβ (splice variant isoform that acts as a dominant-negative of GRα), inflammatory cytokines, and GR-regulated genes were quantified using qPCR and western blotting. miR146 was quantified using miR PCR assays. Results: FTE and HGSC cells express both GR isoforms. GR mRNA levels in primary cultured FTE cells from mtBRCA1 carriers were similar to FTE cells from mtBRCA2 and control patients, although mtBRCA1 FTE cells had increased GRα:GRβ transcript ratios. In contrast, BRCA1 overexpression in OE6/7 cells and UWB1.1 289 cells increased the GRα:GRβ transcript ratio. Total GR protein levels were similar in UWB1.289 and UWB1.289+BRCA1 cells; however, BRCA1 expression associated with increased dexamethasone (DEX)-induced GR transactivation. GR target genes SGK1 and GILZ were upregulated by DEX similarly regardless of BRCA1 expression. TNFα was decreased in OE6/7 and UWB1.1 289 cells expressing higher levels of BRCA1 and was further decreased by DEX. miR-146 levels were elevated, rather than suppressed, in BRCA1-deficient cells, and correlated with decreased expression of targeted NFκB activators. Conclusions: These data indicate GR signaling is affected by BRCA1 and that both GRα and GRβ are expressed in FTE cells. BRCA1 expression alters relative isoform expression ratios raising the possibility of altered response to glucocorticoids that could impact NFκB signaling. Furthermore, these studies indicate that mechanisms other than decreased miRNA-146 underlie increased NFκB and EGFR signaling in mtBRCA1 FTE cells. Citation Format: Kasra Khalaj, Alexandra Kollara, Julia Hollingsworth, Vladimir Djedovic, Theodore J. Brown. Impact of BRCA1 on glucocorticoid signaling in fallopian tube epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3450.

Identification of 20(R, S)-protopanaxadiol and 20(R, S)-protopanaxatriol for potential selective modulation of glucocorticoid receptor

Although glucocorticoids (GCs) are widely used as anti-inflammatory drugs, they are often accompanied by adverse effects, which are mainly due to the transactivation of glucocorticoid receptor (GR) target genes. In order to screen novel plant-derived GR ligands (phytocorticoids) capable of separating transrepression from transactivation, this work focuses on the estimation of 20(R, S)-protopanaxadiol [PPD(R, S)] and 20(R, S)-protopanaxatriol [PPT(R, S)] for their dissociated characteristics. The reporter gene assay shows that ginsenosides cannot enhance glucocorticoid-responsive element-driven genes. The cytotoxicity assay shows that PPT(S), PPT(R), and PPD(S) can inhibit cell proliferation while PPD(R) does not suppress cell growth at available concentration. Further analysis of transactivation and transrepression activities indicates that PPD(R) can repress the transcription of GR target transrepressed gene without activating the expression of the GR target transactivated gene. Results of molecular docking suggest that PPD(R) yields more hydrogen bond interactions and a lower binding energy than its counterparts, resulting in tighter binding between PPD(R) and GR. In addition, PPD(R) achieves stability in the pocket after 2 ns, thereby facilitating exerting its regulatory role of GR target genes. By contrast, other ginsenosides fluctuate drastically during the simulations. In conclusion, PPD(R) may serve as a potential selective GR modulator (SEGRM).

Caveolin1 interacts with the glucocorticoid receptor in the lung but is dispensable for its anti-inflammatory actions in lung inflammation and Trichuris Muris infection

Glucocorticoids (Gcs) are widely prescribed anti-inflammatory compounds, which act through the glucocorticoid receptor (GR). Using an unbiased proteomics screen in lung tissue, we identified the membrane protein caveolin -1 (Cav1) as a direct interaction partner of the GR. In Cav1 knockout mice GR transactivates anti-inflammatory genes, including Dusp1, more than in controls. We therefore determined the role of Cav1 in modulating Gc action in two models of pulmonary inflammation. We first tested innate responses in lung. Loss of Cav1 impaired the inflammatory response to nebulized LPS, increasing cytokine/chemokine secretion from lung, but impairing neutrophil infiltration. Despite these changes to the inflammatory response, there was no Cav1 effect on anti-inflammatory capacity of Gcs. We also tested GR/Cav1 crosstalk in a model of allergic airway inflammation. Cav1 had a very mild effect on the inflammatory response, but no effect on the Gc response – with comparable immune cell infiltrate (macrophage, eosinophils, neutrophils), pathological score and PAS positive cells observed between both genotypes. Pursuing the Th2 adaptive immune response further we demonstrate that Cav1 knockout mice retained their ability to expel the intestinal nematode parasite T.muris, which requires adaptive Th2 immune response for elimination. Therefore, Cav1 regulates innate immune responses in the lung, but does not have an effect on Th2-mediated adaptive immunity in lung or gut. Although we demonstrate that Cav1 regulates GR transactivation of anti-inflammatory genes, this does not translate to an effect on suppression of inflammation in vivo.

Glucocorticoid Receptor Transactivation Is Required for Glucocorticoid-Induced Ocular Hypertension and Glaucoma.

PurposeGlucocorticoid (GC)–induced ocular hypertension (GC-OHT) is a serious side effect of prolonged GC therapy that can lead to glaucoma and permanent vision loss. GCs cause a plethora of changes in the trabecular meshwork (TM), an ocular tissue that regulates intraocular pressure (IOP). GCs act through the glucocorticoid receptor (GR), and the GR regulates transcription both through transactivation and transrepression. Many of the anti-inflammatory properties of GCs are mediated by GR transrepression, while GR transactivation largely accounts for GC metabolic effects and side effects of GC therapy. There is no evidence showing which of the two mechanisms plays a role in GC-OHT.MethodsGRdim transgenic mice (which have active transrepression and impaired transactivation) and wild-type (WT) C57BL/6J mice received weekly periocular dexamethasone acetate (DEX-Ac) injections. IOP, outflow facilities, and biochemical changes to the TM were determined.ResultsGRdim mice did not develop GC-OHT after continued DEX treatment, while WT mice had significantly increased IOP and decreased outflow facilities. Both TM tissue in eyes of DEX-treated GRdim mice and cultured TM cells isolated from GRdim mice had reduced or no change in the expression of fibronectin, myocilin, collagen type I, and α-smooth muscle actin (α-SMA). GRdim mouse TM (MTM) cells also had a significant reduction in DEX-induced cytoskeletal changes, which was clearly seen in WT MTM cells.ConclusionsWe provide the first evidence for the role of GR transactivation in regulating GC-mediated gene expression in the TM and in the development of GC-OHT. This discovery suggests a novel therapeutic approach for treating ocular inflammation without causing GC-OHT and glaucoma.

SUN-019 The Selective Progesterone Receptor Modulator Ulipristal Acetate Blocks Glucocorticoid Receptor Transactivation

Progesterone is a crucial regulator of the female reproductive system, but also contributes to the pathogenesis of certain gynecological diseases. As such, selective progesterone receptor modulators (SPRMs) have shown potential for the treatment of such diseases. The SPRM Ulipristal acetate (UPA) offers a much-needed therapeutic option for the clinical management of uterine fibroids. Although UPA initially passed safety evaluations in Europe, post-marketing analysis identified cases of liver injury and hepatic failure, leading to temporary restrictions on UPA use. The structural analysis predicted low glucocorticoid receptor (GR) antagonist properties of UPA, however the anti-glucocorticoid activity of Ulipristal has been demonstrated on reporter constructs in HeLa cells. To determine whether UPA can alter the activity of endogenous GR in various relevant cell types, immortalized human uterine fibroid cells (UtLM) and hepatocytes (HepG2) were treated with the synthetic glucocorticoid dexamethasone and/or UPA. Co-treatment of 100 nM UPA with dexamethasone reduced glucocorticoid-induced expression of PER1, FKBP5, and GILZ in HepG2 and UtLM cells by 30-55%. At 1μM, UPA completely blocked glucocorticoid regulation of these genes in both cell lines. As a comparison, the extent of glucocorticoid inhibition by UPA was comparable to the known PR and GR antagonist RU 486. To discover whether the anti-glucocorticoid activity of UPA was mediated by PR, we knocked down PR expression by siRNA and found that the inhibitory activity of UPA does not require PR, suggesting a direct effect on GR. To determine the mechanism by which UPA inhibits GR activity, we evaluated hallmarks of GR transactivation. As hormone-dependent phosphorylation at S211 is associated with transcriptionally active GR, we evaluated p211 status in response to dexamethasone treatment with and without UPA. In the presence of UPA, dexamethasone-mediated phosphorylation at 211 was significantly diminished. Furthermore, dexamethasone-dependent nuclear translocation of GR was reduced by approximately 70% in both cell lines. Reduced activation of GR resulted in diminished GR recruitment to known regulatory regions in the GILZ promoter in the presence of UPA. We next evaluated the effect of UPA on glucocorticoid-mediated transcription in primary cells. Co-treatment of dexamethasone with UPA blunted glucocorticoid responsiveness in primary human uterine fibroid tissue cultured ex vivo. To determine whether UPA could inhibit endogenous glucocorticoid signaling, mice were treated with vehicle or 2 mg/kg UPA and various tissues were isolated for gene expression analysis. UPA inhibited basal expression of Per1, Fkbp5, and Gilz in the uterus and liver, and lung but not the spleen. These studies suggest that UPA selectively regulates GR activity, which is an important consideration for its use as a long-term therapeutic agent.

A Model of Glucocorticoid Receptor Interaction With Coregulators Predicts Transcriptional Regulation of Target Genes.

Regulatory factors that control gene transcription in multicellular organisms are assembled in multicomponent complexes by combinatorial interactions. In this context, nuclear receptors provide well-characterized and physiologically relevant systems to study ligand-induced transcription resulting from the integration of cellular and genomic information in a cell- and gene-specific manner. Here, we developed a mathematical model describing the interactions between the glucocorticoid receptor (GR) and other components of a multifactorial regulatory complex controlling the transcription of GR-target genes, such as coregulator peptides. We support the validity of the model in relation to gene-specific GR transactivation with gene transcription data from A549 cells and in vitro real time quantification of coregulator-GR interactions. The model accurately describes and helps to interpret ligand-specific and gene-specific transcriptional regulation by the GR. The comprehensive character of the model allows future insight into the function and relative contribution of the molecular species proposed in ligand- and gene-specific transcriptional regulation.

Profiling withanolide A for therapeutic targets in neurodegenerative diseases.

To identify new potential therapeutic targets for neurodegenerative diseases, we initiated activity-based protein profiling studies with withanolide A (WitA), a known neuritogenic constituent of Withania somnifera root with unknown mechanism of action. Molecular probes were designed and synthesized, and led to the discovery of the glucocorticoid receptor (GR) as potential target. Molecular modeling calculations using the VirtualToxLab predicted a weak binding affinity of WitA for GR. Neurite outgrowth experiments in human neuroblastoma SH-SY5Y cells further supported a glucocorticoid-dependent mechanism, finding that WitA was able to reverse the outgrowth inhibition mediated by dexamethasone (Dex). However, further GR binding and transactivation assays found no direct interference of WitA. Further molecular modeling analysis suggested that WitA, although forming several contacts with residues in the GR binding pocket, is lacking key stabilizing interactions as observed for Dex. Taken together, the data suggest that WitA-dependent induction of neurite outgrowth is not through a direct effect on GR, but might be mediated through a closely related pathway. Further experiments should evaluate a possible role of GR modulators and/or related signaling pathways such as ERK, Akt, NF-κB, TRα, or Hsp90 as potential targets in the WitA-mediated neuromodulatory effects.

Potential Dissociative Glucocorticoid Receptor Activity for Protopanaxadiol and Protopanaxatriol.

Glucocorticoids are steroid hormones that regulate inflammation, growth, metabolism, and apoptosis via their cognate receptor, the glucocorticoid receptor (GR). GR, acting mainly as a transcription factor, activates or represses the expression of a large number of target genes, among them, many genes of anti-inflammatory and pro-inflammatory molecules, respectively. Transrepression activity of glucocorticoids also accounts for their anti-inflammatory activity, rendering them the most widely prescribed drug in medicine. However, chronic and high-dose use of glucocorticoids is accompanied with many undesirable side effects, attributed predominantly to GR transactivation activity. Thus, there is a high need for selective GR agonist, capable of dissociating transrepression from transactivation activity. Protopanaxadiol and protopanaxatriol are triterpenoids that share structural and functional similarities with glucocorticoids. The molecular mechanism of their actions is unclear. In this study applying induced-fit docking analysis, luciferase assay, immunofluorescence, and Western blot analysis, we showed that protopanaxadiol and more effectively protopanaxatriol are capable of binding to GR to activate its nuclear translocation, and to suppress the nuclear factor-kappa beta activity in GR-positive HeLa and HEK293 cells, but not in GR-low level COS-7 cells. Interestingly, no transactivation activity was observed, whereas suppression of the dexamethasone-induced transactivation of GR and induction of apoptosis in HeLa and HepG2 cells were observed. Thus, our results indicate that protopanaxadiol and protopanaxatriol could be considered as potent and selective GR agonist.

Glucocorticoid Receptor Transactivation is Required for Glucocorticoid-Induced Ocular Hypertension and Glaucoma: A Deleterious Effect of Chronic Glucocorticoid Therapy

Glucocorticoid (GC)-induced ocular hypertension (OHT) is a serious side-effect of prolonged GC therapy that can lead to glaucoma and permanent vision loss. GCs cause a plethora of changes in the trabecular meshwork (TM), an ocular tissue that regulates intraocular pressure (IOP). GCs act through the glucocorticoid receptor (GR), and the GR regulates transcription both through transactivation and transrepression. Many of the anti-inflammatory properties of GCs are mediated by GR transrepression, while GR transactivation largely accounts for GC metabolic effects and side effects of GC therapy. There is no evidence showing which of the two mechanisms play role in GC- OHT. Methods: GRdim transgenic mice (which have active transrepression and impaired transactivation) and wild type (WT) C57BL/6J mice received weekly periocular DEX-Ac injections. IOP, outflow facilities, and biochemical changes to the TM were determined. Results: GRdim mice did not develop GC-OHT DEX treatment, while WT mice had significantly increased IOP and decreased outflow facilities. Both TM tissue in eyes of DEX-treated GRdim mice and cultured TM cells isolated from GRdim mice had reduced or no change in the expression of fibronectin, myocilin, collagen type I, and α-SMA. GRdim MTM cells also had a significant reduction in DEX-induced cytoskeletal changes, which was clearly seen in WT MTM cells. Conclusion: We provide the first evidence for the role of GR transactivation in regulating GC-mediated gene expression in the TM and in the development of GC- OHT. This discovery suggests a novel therapeutic approach for treating ocular inflammation that will not cause the serious side effect of GC-OHT and glaucoma. Funding: NIH/NEI EY016242. Declaration of Interest: AFC is a consultant for Goodmans LLP, LayerBio, Kala Pharmacuticals and has received research support from Unity Biotechnology and Lung Therapeutics. However, none of these are related to the study presented. The other authors declare that they have no competing interests. Ethical Approval: All experimental protocols were approved by University of North Texas Health Science Center Institutional Animal Care and Use Committee regulations.

PO-260 Anti-lymphoma activity of novel selective glucocorticoid receptor agonists (SEGRAs) in vitro and in vivo

IntroductionGlucocorticoids (GCs) are widely used in blood cancer treatment; although, they cause metabolic disorders. Biological response to GCs is mediated by glucocorticoid receptor (GR) regulating gene expression via transactivation (TA), which requires GR binding to GC-responsive elements in gene promoters, and transrepression (TR), negative interaction between GR and transcription factors. TR mediates anticancer effects of GR, while side effects are associated with GR TA. Selective GR agonists (SEGRAs) that preferentially activate GR TR could be a better option for cancer treatment. One of well characterised SEGRAs is 2-(4-acetoxyphenyl)−2-chloro-N-methylethylammonium-chloride, or CpdA, isolated from Namibian shrub Salsola tuberculatiformis. CpdA demonstrated anticancer activity in vitro and in vivo. We extended SEGRA list by synthesis of CpdA enantiomers and its chemical derivatives.Material and methodsSynthesis of (S) and (R)-CpdA was based on Sharpless asymmetric dihydroxylation. Chemical analogues of CpdA, CpdA01-08, were designed by appending of bulky substituent into benzene ring and to nitrogen atom or alkylation of carbon atom adjacent to chlorine atom. All experiments in vitro were carried out on Granta (lymphoma) and CEM (leukaemia) cells. Cells were treated with Dex, CpdA, (R) and (S)-CpdA, CpdA01-08. Effects on cell growth were evaluated by cell counting and flow cytometry. Affinity to GR was measured by competitive binding assay. Gene expression was measured by qPCR and Western blotting. GR and NF-kB activity was evaluated using Luciferase reporter analysis. Anticancer effect in vivo was determined using the model of murine lymphoma P388.Results and discussionsThe most cytotoxic compounds among 10 newly synthesised, CpdA03 and CpdA05, demonstrated the highest affinity to GR. They induced GR TR but not TA and proved their SEGRA properties. Effect of CpdA enantiomers on cell growth and survival was not significantly different from Dex and CpdA. CpdA03, cytotoxic SEGRA with the highest affinity to GR, comparable with DEX and CpdA, was tested in vivo for evaluation its anti-lymphoma activity, and demonstrated 3-fold decrease of tumour size in comparison with 2–2,5-fold decrease after Dex or CpdA treatment.ConclusionThe design of synthesis and evaluation of anticancer properties of new SEGRA are provided. According to our data one novel SEGRA, CpdA03, is perspective for further investigation as anti-lymphoma drug with reduced side effects.Study is funded by Russian Science Foundation No. 17-75-20124.

Roles of 14-3-3β and γ in regulation of the glucocorticoid receptor transcriptional activation and hepatic gluconeogenesis

The glucocorticoid receptor (GR) is a ligand-dependent transcription factor that mediates the effects of glucocorticoids, and plays a crucial role in cell growth, development, inflammation, and gluconeogenesis. The 14-3-3 proteins bind to target proteins via phosphorylation, and influence many cellular events by altering their subcellular localization or by acting as chaperones. However, the mechanisms by which 14-3-3 proteins regulate GR transactivation and their involvement in gluconeogenesis remain uncharacterized. We found that 14-3-3β and γ increased GR transcriptional activity and the promoter activities of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase in the presence of glucocorticoids. Inhibition of the endogenous 14-3-3β and γ decreased dexamethasone- and cAMP-stimulated PEPCK expression. Further, both 14-3-3β and γ increased glucose production in response to glucocorticoids. Our findings suggest that 14-3-3β and γ function as positive regulators of GR transactivation and glucocorticoid-mediated hepatic gluconeogenesis.

Imidazo[2,1-b]benzothiazol Derivatives as Potential Allosteric Inhibitors of the Glucocorticoid Receptor.

Glucocorticoid receptor (GCR) transactivation reporter gene assays were used as an initial high-throughput screening on a diversified library of 1200 compounds for their evaluation as GCR antagonists. A class of imidazo[2,1-b]benzothiazole and imidazo[2,1-b]benzoimidazole derivatives were identified for their ability to modulate GCR transactivation and anti-inflammatory transrepression effects utilizing GCR and NF-κB specific reporter gene assays. Modeling studies on the crystallographic structure of the GCR ligand binding domain provided three new analogues bearing the tetrahydroimidazo[2,1-b]benzothiazole scaffold able to antagonize the GCR in the presence of dexamethasone (DEX) and also defined their putative binding into the GCR structure. Both mRNA level measures of GCR itself and its target gene GILZ, on cells treated with the new analogues, showed a GCR transactivation inhibition, thus suggesting a potential allosteric inhibition of the GCR.

Selective Glucocorticoid Receptor Properties of GSK866 Analogs with Cysteine Reactive Warheads.

Synthetic glucocorticoids (GC) are the mainstay therapy for treatment of acute and chronic inflammatory disorders. Due to the high adverse effects associated with long-term use, GC pharmacology has focused since the nineties on more selective GC ligand-binding strategies, classified as selective glucocorticoid receptor (GR) agonists (SEGRAs) or selective glucocorticoid receptor modulators (SEGRMs). In the current study, GSK866 analogs with electrophilic covalent-binding warheads were developed with potential SEGRA properties to improve their clinical safety profile for long-lasting topical skin disease applications. Since the off-rate of a covalently binding drug is negligible compared to that of a non-covalent drug, its therapeutic effects can be prolonged and typically, smaller doses of the drug are necessary to reach the same level of therapeutic efficacy, thereby potentially reducing systemic side effects. Different analogs of SEGRA GSK866 coupled to cysteine reactive warheads were characterized for GR potency and selectivity in various biochemical and cellular assays. GR- and NFκB-dependent reporter gene studies show favorable anti-inflammatory properties with reduced GR transactivation of two non-steroidal GSK866 analogs UAMC-1217 and UAMC-1218, whereas UAMC-1158 and UAMC-1159 compounds failed to modulate cellular GR activity. These results were further supported by GR immuno-localization and S211 phospho-GR western analysis, illustrating significant GR phosphoactivation and nuclear translocation upon treatment of GSK866, UAMC-1217, or UAMC-1218, but not in case of UAMC-1158 or UAMC-1159. Furthermore, mass spectrometry analysis of tryptic peptides of recombinant GR ligand-binding domain (LBD) bound to UAMC-1217 or UAMC-1218 confirmed covalent cysteine-dependent GR binding. Finally, molecular dynamics simulations, as well as glucocorticoid receptor ligand-binding domain (GR-LBD) coregulator interaction profiling of the GR-LBD bound to GSK866 or its covalently binding analogs UAMC-1217 or UAMC-1218 revealed subtle conformational differences that might underlie their SEGRA properties. Altogether, GSK866 analogs UAMC-1217 and UAMC-1218 hold promise as a novel class of covalent-binding SEGRA ligands for the treatment of topical inflammatory skin disorders.