Glucocorticoid Receptor Ligand Binding Research Articles

The Hippocampal Response to Acute Corticosterone Elevation Is Altered in a Mouse Model for Angelman Syndrome.

Angelman Syndrome (AS) is a severe neurodevelopmental disorder, caused by the neuronal absence of the ubiquitin protein ligase E3A (UBE3A). UBE3A promotes ubiquitin-mediated protein degradation and functions as a transcriptional coregulator of nuclear hormone receptors, including the glucocorticoid receptor (GR). Previous studies showed anxiety-like behavior and hippocampal-dependent memory disturbances in AS mouse models. Hippocampal GR is an important regulator of the stress response and memory formation, and we therefore investigated whether the absence of UBE3A in AS mice disrupted GR signaling in the hippocampus. We first established a strong cortisol-dependent interaction between the GR ligand binding domain and a UBE3A nuclear receptor box in a high-throughput interaction screen. In vivo, we found that UBE3A-deficient AS mice displayed significantly more variation in circulating corticosterone levels throughout the day compared to wildtypes (WT), with low to undetectable levels of corticosterone at the trough of the circadian cycle. Additionally, we observed an enhanced transcriptomic response in the AS hippocampus following acute corticosterone treatment. Surprisingly, chronic corticosterone treatment showed less contrast between AS and WT mice in the hippocampus and liver transcriptomic responses. This suggests that UBE3A limits the acute stimulation of GR signaling, likely as a member of the GR transcriptional complex. Altogether, these data indicate that AS mice are more sensitive to acute glucocorticoid exposure in the brain compared to WT mice. This suggests that stress responsiveness is altered in AS which could lead to anxiety symptoms.

Structure of Hsp90-p23-GR reveals the Hsp90 client-remodelling mechanism.

Hsp90 is a conserved and essential molecular chaperone responsible for the folding and activation of hundreds of 'client' proteins1-3. The glucocorticoid receptor (GR) is a model client that constantly depends on Hsp90 for activity4-9. GR ligand binding was previously shown to nr inhibited by Hsp70 and restored by Hsp90, aided by the co-chaperone p2310. However, a molecular understanding of the chaperone-mediated remodelling that occurs between the inactive Hsp70-Hsp90 'client-loading complex' and an activated Hsp90-p23 'client-maturation complex' is lacking for any client, including GR. Here we present a cryo-electron microscopy (cryo-EM) structure of the human GR-maturation complex (GR-Hsp90-p23), revealing that the GR ligand-binding domain is restored to a folded, ligand-bound conformation, while being simultaneously threaded through the Hsp90 lumen. In addition, p23 directly stabilizes native GR using a C-terminal helix, resulting in enhanced ligand binding. This structure of a client bound to Hsp90 in a native conformation contrasts sharply with the unfolded kinase-Hsp90 structure11. Thus, aided by direct co-chaperone-client interactions, Hsp90 can directly dictate client-specific folding outcomes. Together with the GR-loading complex structure12, we present the molecular mechanism of chaperone-mediated GR remodelling, establishing the first, to our knowledge,complete chaperone cycle for anyHsp90 client.

Discovery of Novel GR Ligands toward Druggable GR Antagonist Conformations Identified by MD Simulations and Markov State Model Analysis.

Binding of different ligands to glucocorticoid receptor (GR) may induce different conformational changes and even trigger completely opposite biological functions. To understand the allosteric communication within the GR ligand binding domain, the folding pathway of helix 12 (H12) induced by the binding of the agonist dexamethasone (DEX), antagonist RU486, and modulator AZD9567 are explored by molecular dynamics simulations and Markov state model analysis. The ligands can regulate the volume of the activation function‐2 through the residues Phe737 and Gln738. Without ligand or with agonist binding, H12 swings from inward to outward to visit different folding positions. However, the binding of RU486 or AZD9567 perturbs the structural state, and the passive antagonist state appears more stable. Structure‐based virtual screening and in vitro bioassays are used to discover novel GR ligands that bias the conformation equilibria toward the passive antagonist state. HP‐19 exhibits the best anti‐inflammatory activity (IC50 = 0.041 ± 0.011 µm) in nuclear factor‐kappa B signaling pathway, which is comparable to that of DEX. HP‐19 also does not induce adverse effect‐related transactivation functions of GR. The novel ligands discovered here may serve as promising starting points for the development of GR modulators.

Glucocorticoid Receptor Condensates Link DNA-Dependent Receptor Dimerization and Transcriptional Transactivation

The glucocorticoid receptor (GR) is a ligand-regulated transcription factor (TF) that controls the tissue- and gene-specific transactivation and transrepression of thousands of target genes. Distinct GR DNA binding sequences with activating or repressive activities have been identified, but how they modulate transcription in opposite ways is not known. We show that GR forms phase-separated condensates that specifically concentrate known co-regulators via their intrinsically disordered regions (IDRs) in vitro. A combination of dynamic, multivalent (between IDRs) and specific, stable interactions (between LxxLL motifs and the GR ligand binding domain) control the degree of recruitment. Importantly, GR DNA-binding directs the selective partitioning of co-regulators within GR condensates such that activating DNAs cause enhanced recruitment of co-activators. Our work shows that condensation controls GR function by modulating co-regulator recruitment and provides a mechanism for the up- and down-regulation of GR target genes controlled by distinct DNA recognition elements.

The role of pro-opiomelanocortin in the ACTH\u2013cortisol dissociation of sepsis

BackgroundSepsis is typically hallmarked by high plasma (free) cortisol and suppressed cortisol breakdown, while plasma adrenocorticotropic hormone (ACTH) is not increased, referred to as ‘ACTH–cortisol dissociation.’ We hypothesized that sepsis acutely activates the hypothalamus to generate, via corticotropin-releasing hormone (CRH) and vasopressin (AVP), ACTH-induced hypercortisolemia. Thereafter, via increased availability of free cortisol, of which breakdown is reduced, feedback inhibition at the pituitary level interferes with normal processing of pro-opiomelanocortin (POMC) into ACTH, explaining the ACTH–cortisol dissociation. We further hypothesized that, in this constellation, POMC leaches into the circulation and can contribute to adrenocortical steroidogenesis.MethodsIn two human studies of acute (ICU admission to day 7, N = 71) and prolonged (from ICU day 7 until recovery; N = 65) sepsis-induced critical illness, POMC plasma concentrations were quantified in relation to plasma ACTH and cortisol. In a mouse study of acute (1 day), subacute (3 and 5 days) and prolonged (7 days) fluid-resuscitated, antibiotic-treated sepsis (N = 123), we further documented alterations in hypothalamic CRH and AVP, plasma and pituitary POMC and its glucocorticoid-receptor-regulated processing into ACTH, as well as adrenal cortex integrity and steroidogenesis markers.ResultsThe two human studies revealed several-fold elevated plasma concentrations of the ACTH precursor POMC from the acute to the prolonged phase of sepsis and upon recovery (all p < 0.0001), coinciding with the known ACTH–cortisol dissociation. Elevated plasma POMC and ACTH–corticosterone dissociation were confirmed in the mouse model. In mice, sepsis acutely increased hypothalamic mRNA of CRH (p = 0.04) and AVP (p = 0.03) which subsequently normalized. From 3 days onward, pituitary expression of CRH receptor and AVP receptor was increased. From acute throughout prolonged sepsis, pituitary POMC mRNA was always elevated (all p < 0.05). In contrast, markers of POMC processing into ACTH and of ACTH secretion, negatively regulated by glucocorticoid receptor ligand binding, were suppressed at all time points (all p ≤ 0.05). Distorted adrenocortical structure (p < 0.05) and lipid depletion (p < 0.05) were present, while most markers of adrenocortical steroidogenic activity were increased at all time points (all p < 0.05).ConclusionTogether, these findings suggest that increased circulating POMC, through CRH/AVP-driven POMC expression and impaired processing into ACTH, could represent a new piece in the puzzling ACTH–cortisol dissociation.

Unravelling Allosteric Cross-Talk between Co-Activator Peptide and Ligand Binding Site in Glucocorticoid Receptor

Glucocorticoid receptor (GR) is a nuclear receptor that controls critical biological processes by regulating the transcription of specific genes. There is a known interconnection between the ligand and co-regulator binding sites of the GR ligand binding domain that is crucial for the functional differentiation of the downstream gene transcription. However, the molecular mechanisms underlying such an allosteric control remain elusive. The integration of molecular dynamics simulations, bioinformatic analysis and biophysical measurements led us to detect a network of dynamically interconnected evolutionary conserved residues that mediates the allosteric signal propagation between the co-regulator docking site and the ligand binding pocket. Co-regulator peptides alter the functionality of this network, explaining why the allosteric response depends on the peptide sequence. These findings provide useful insights for the comprehension of GR allosteric regulation that should help the design of novel drugs.

Deciphering the structural basis for glucocorticoid resistance caused by missense mutations in the ligand binding domain of glucocorticoid receptor

The glucocorticoid resistance hereditary condition may emerge from the occurrence of point mutations in the glucocorticoid receptor (GR), which could impair its functionality. Because the main feature of such pathology is the resistance of the hypothalamic-pituitary-adrenal axis to the hormone cortisol, we used the GR ligand binding domain three-dimensional structure to perform computational analysis for eight variants known to cause this clinical condition (I559 N, V571A, D641V, G679S, F737L, I747 M, L753F and L773P), aiming to understand, on the atom scale, how they cause glucocorticoid resistance. We observed that the mutations generated a reduced affinity to cortisol and they alter some loop conformations, which could be a consequence from changes in protein motion, which in turn could result from the reduced stability of mutant GR structures. Therefore, the analyzed mutations compromise the GR ligand binding domain structure and cortisol binding, which could characterize the glucocorticoid resistance phenotype.

Structural and Dynamics of Glucocorticoid Receptor Ligand Binding Domain in Complex with PGC1α and Synthetic Glucocorticoids

Synthetic glucocorticoids are important pharmaceutical drugs that are known to bind to the ligand binding domain (LBD) of glucocorticoid receptor (GR), a member of the nuclear receptor superfamily. These binding will induce conformational changes within GR for the subsequent DNA binding and different co‐regulators recruitments, ultimately lead to activate or repress target gene expression. One of the crucial co‐regulators is peroxisome proliferator‐activated gamma coactivator 1‐α (PGC1α), known to regulates energy metabolism by direct interacting with GR to modulate gene expression. However, the mechanisms through which PGC1α changes GR conformation to drive transcription are unknown. Here, an ancestral variant of GR (GR2) was utilized as a tool to produce stable protein for biochemical and structural studies. We report the first high resolution X‐ray crystal structures of GR2 LBD in complexes with PGC1α and dexamethasone and hydrocortisone. These structural analyses reveal how distinct steroid drugs bind to GR with different affinities via unique hydrogen bonding and hydrophobic interactions. Hydrogen‐deuterium exchange mass spectrometry showed different effects on GR2 LBD allosteric communication after glucocorticoids binding. Molecular dynamics simulations and network analysis identified the key residues involved in the intramolecular communication between ligand binding site and activation function surface, which is the PGC1α binding site.Support or Funding InformationThis work was supported by W.M. Keck Foundation Medical Research Grant to E. A. O. X. L. was supported by an American Heart Association postdoctoral fellowship [17POST33660110].This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Discovery of New non-steroidal selective glucocorticoid receptor agonists

Glucocorticoids (GCs) are widely used as potent anti-inflammatory drugs; however, GC therapy is often accompanied by adverse side effects. The anti-inflammatory action of GCs is exerted through the glucocorticoid receptor (GR) in part by antagonizing the pro-inflammatory nuclear factor k B (NF-kB) whereas the majority of side effects are assumed to be mediated by transactivation of GR target genes. We set out to identify novel non-steroidal selective GR agonists (SEGRA) favoring transrepression of NF-kB target genes over transactivation of genes associated with undesirable effects. Our virtual screening protocol was driven by a pharmacophore model based on a pyrrolidinone amide analogue (named as ‘compound 12′ in Biggadike et al 2009, PNAS USA 106, 18,114) bound to the extended binding pocket of the GR ligand binding domain (GR-LBD). Ambinter library (7.8 million compounds) was queried by our validated pharmacophore hypothesis and the prioritized compounds were biologically evaluated using a series of well-established screening assays. Two structurally similar hits (1 and 13) were identified that bind to GR, induce its translocation to the nucleus, do not mediate transactivation of GR target genes whereas partially repress a number of pro-inflammatory NF-kB target genes, in a GR-dependent manner. Explanatory molecular dynamics (MD) calculations could detail the per-residue interactions accounting for the binding of 1 and 13 to the extended binding pocket of GR. The discovered 1,3-benzothiazole analogs introduce a new class of genuine SEGRA paving the way for hit-to-lead optimization.

Polygodial, a sesquiterpene isolated from Drimys brasiliensis (Winteraceae), triggers glucocorticoid-like effects on pancreatic β-cells

Despite its common use, the synthetic glucocorticoid dexamethasone can cause several adverse effects, such as diabetes and insulin-related metabolic impairment. Thus, research on molecules that could provide the same anti-inflammatory response with milder side effects is constant. In this work the anti-inflammatory activity of the natural sesquiterpene polygodial, extracted from the endemic Brazilian plant Drimys brasiliensis Miers (Winteraceae), was investigated. Employing a pancreatic β-cell model (INS 1E), the effect of polygodial on signaling pathways is similar to that caused by dexamethasone – both increased MKP1 and decreased ERK1/2 expression in a dose-response and time-dependent manner. Relating to such finding, nuclear translocation of the glucocorticoid receptor was also discovered to be induced by the sesquiterpene. Molecular modeling results indicated that polygodial was capable of docking to the glucocorticoid receptor, but presented preference for the Arg611 binding site rather than Thr739 when set to bind freely inside the pocket. At last, fragmentation of DNA was verified as consequence of sesquiterpene-induced cell death. Altogether, our results suggest that, like dexamethasone, polygodial interacts the glucocorticoid receptor ligand binding domain but create fewer ligand-protein interactions at the site, yielding a weaker effector response. Such property provides an advantage when regarding the adverse effects resulting from stronger affinity ligands of the glucocorticoid receptor, such as in the case of the current standard dexamethasone-based treatment. This aspect, also, turns polygodial an interesting hit compound to the development of new drugs based on its backbone structure providing less harmful anti-inflammatory treatments.

Investigation on critical structural motifs of ligands for triggering glucocorticoid receptor nuclear migration through molecular docking simulations

The glucocorticoid receptor (GR), a transcription factor regulating gene expression in a ligand-dependent fashion, is known for flexibility in adapting various ligands with their structures ranging from steroid to non-steroid. However, in our previous study, GR shows a stringent discrimination against a set of steroid ligands with highly similar structures for triggering its nuclear migration. In order to resolve this puzzle, we employed molecular docking simulations to investigate the origin of this structural discrimination. By analyzing the docking orientations and the related ligand–GR interaction patterns, we found that the hydrophilicity mismatch between the docking ligand and the GR ligand-binding site is the main cause combined with the steric hindrance and structural rigidness of these steroid ligands. Furthermore, we utilized this knowledge to rationalize how the structure–binding interaction of non-steroid ligands triggers GR nuclear migration with their structures available in Protein Data Bank.

Association of the glucocorticoid receptor D641V variant with steroid-resistant asthma: a case-control study.

Several mutations of the glucocorticoid receptor (GR) gene cause malfunction of the protein, resulting in steroid resistance. In diseases other than asthma, the GR variants I559N, D641V, and V729I have been linked to steroid resistance. The aim of this study was to evaluate the link of these GR variants in steroid-resistant (SR) asthma in the Chinese Han population. GR polymorphisms were determined in 64 SR asthma patients, 217 steroid-sensitive (SS) asthma patients and 221 healthy control (CTR) individuals. The analysis of the GR variants was performed using PCR-sequence specific primers according to the European Molecular Biology Laboratory database (NC_000005.8). In addition, ligand binding and serum cortisol levels were determined. Compared with SS asthma patients and CTRs, a significant lower frequency of the GR D641V variant AA genotype (P=0.003, 0.014, respectively) and the A allele (P=0.001, 0.009, respectively) was found in SR asthma patients. Furthermore, the equilibrium dissociation constant (Kd) of GR ligand binding in SR asthma patients with the GR D641V variant AA genotype was significantly lower compared with the AT or the TT genotype carriers (P=0.006, 0.016, respectively). There was no significant difference between the I559N and V729I GR variants on comparing SR asthma patients with SS asthma patients or CTRs. This study suggests that the D641V variant of the GR is probably associated with SR asthma in the Chinese Han population.

Development of a Nonradioactive Steroid Receptor Ligand Binding Assay Using Fluorescence Polarization for Studying Hsp90‐Mediated Chaperone Protein Activity

While glucocorticoid receptor (GR) ligand binding has historically been measured using radiolabeled agonists, such as [3H]dexamethasone, new methods have sought to use fluorescently conjugated ligands, such as dexamethasone‐fluorescein (Dex‐Fl), as more sustainable alternatives. The molecular chaperone protein hsp90 has been shown to be essential for facilitating GR ligand binding, and structurally unrelated hsp90 inhibitors are currently in various phases of clinical investigation. The purpose of our study was to develop a fluorescence polarization (FP)‐based assay for measuring GR binding activity suitable for use in undergraduate research environments, and then apply this method to evaluating the extent to which specific agonists and antagonists of the GR and hsp90 chaperone machinery affect ligand binding. We constructed a versatile FP assay with comparable results to the previously used radiation‐based method. Interestingly, GR agonists dexamethasone and cortisol produced equivalent Dex‐Fl competition to the GR antagonist RU486, and did so in a concentration‐dependent manner. Data from hsp90 and hsp70 inhibitors showed mixed results, with hsp90 antagonists radicicol, NVP‐AUY922, and BIIB012 having limited effects, while the hsp70 antagonist novobiocin showed a potentiation of FP‐detectable steroid binding. Using the described FP method to evaluate steroid binding can be applied to broader studies examining clinically relevant GR and hsp90‐based drug therapies.

Triterpenes from Alisma orientalis act as androgen receptor agonists, progesterone receptor antagonists, and glucocorticoid receptor antagonists

Alisma orientalis, a well-known traditional medicine, exerts numerous pharmacological effects including anti-diabetes, anti-hepatitis, and anti-diuretics but its bioactivity is not fully clear. Androgen receptor (AR), progesterone receptor (PR), and glucocorticoid receptor (GR) are three members of nuclear receptor superfamily that has been widely targeted for developing treatments for essential diseases including prostate cancer and breast cancer. In this study, two triterpenes, alisol M 23-acetate and alisol A 23-acetate from Alisma orientalis were determined whether they may act as androgen receptor (AR), progesterone receptor (PR), or glucocorticoid receptor (GR) modulators. Indeed, in the transient transfection reporter assays, alisol M 23-acetate and alisol A 23-acetate transactivated AR in dose-dependent manner, while they transrepressed the transactivation effects exerted by agonist-activated PR and GR. Through molecular modeling docking studies, they were shown to respectively interact with AR, PR, or GR ligand binding pocket fairly well. All these results indicate that alisol M 23-acetate and alisol A 23-acetate from Alisma orientalis might possess therapeutic effects through their modulation of AR, PR, and GR pathways.

Glucocorticoid Receptor Modulators Informed by Crystallography Lead to a New Rationale for Receptor Selectivity, Function, and Implications for Structure-Based Design

The structural basis of the pharmacology enabling the use of glucocorticoids as reliable treatments for inflammation and autoimmune diseases has been augmented with a new group of glucocorticoid receptor (GR) ligands. Compound 10, the archetype of a new family of dibenzoxepane and dibenzosuberane sulfonamides, is a potent anti-inflammatory agent with selectivity for the GR versus other steroid receptors and a differentiated gene expression profile versus clinical glucocorticoids (lower GR transactivation with comparable transrepression). A stereospecific synthesis of this chiral molecule provides the unique topology needed for biological activity and structural biology. In vivo activity of 10 in acute and chronic models of inflammation is equivalent to prednisolone. The crystal structure of compound 10 within the GR ligand binding domain (LBD) unveils a novel binding conformation distinct from the classic model adopted by cognate ligands. The overall conformation of the GR LBD/10 complex provides a new basis for binding, selectivity, and anti-inflammatory activity and a path for further insights into structure-based ligand design.

Unbinding Pathways from the Glucocorticoid Receptor Shed Light on the Reduced Sensitivity of Glucocorticoid Ligands to a Naturally Occurring, Clinically Relevant Mutant Receptor

Glucocorticoids are endogenous steroid hormones that regulate essential biological functions, including metabolism, growth, and apoptosis. Glucocorticoids represent the most effective anti-inflammatory agents for the treatment of several inflammatory conditions. However, the clinical use of such drugs is hampered by severe side effects. Therefore, the development of novel glucocorticoid receptor (GR) modulators with an increased therapeutic index is impelling. Herein, using steered molecular dynamics (SMD) simulations, we provide a detailed picture of the unbinding process of three clinically relevant GR modulators from GR ligand binding domains. The SMD protocol described here can be used to prioritize the synthesis of structural analogues on the basis of their potentials of mean force and calculated unbinding energies. Moreover, our results are instrumental in explaining at atomic resolution the weakened ability of dexamethasone to activate the naturally occurring mutant I747M GR, which is implicated in rare familial glucocorticoid resistance, clinically characterized by glucocorticoid insensitivity.

Alternate glucocorticoid receptor ligand binding structures influence outcomes in an in vivo tissue regeneration model

Since their characterization, glucocorticoids (GCs), the most commonly prescribed immunomodulatory drugs, have undergone numerous structural modifications designed to enhance their activity. In vivo assessment of these corticosteroid analogs is essential to understand the difference in molecular signaling of the ligands that share the corticosteroid backbone. Our research identified a novel function of GCs as modulators of tissue regeneration and demonstrated that GCs activate the glucocorticoid receptor (GR) to inhibit early stages of tissue regeneration in zebrafish (Danio rerio). We utilized this phenomenon to assess the effect of different GC analogs on tissue regeneration and identified that some GCs such as beclomethasone dipropionate (BDP) possess inhibitory properties, while others, such as dexamethasone and hydrocortisone have no effect on regeneration. We performed in silico molecular docking and dynamic studies and demonstrated that type and size of substitution at the C17 position of the cortisol backbone confer a unique stable conformation to GR on ligand binding that is critical for inhibitory activity. In the field of tissue regeneration, our study is one of the first Structure Activity Relationship (SAR) investigations performed in vertebrates demonstrating that the in vivo tissue regeneration model is a powerful tool to probe structure function relationships, to understand regenerative biology, and to assist in rational drug design.

Biochemical Reconstitution of Steroid Receptor•Hsp90 Protein Complexes and Reactivation of Ligand Binding

Hsp90 is an essential and highly abundant molecular chaperone protein that has been found to regulate more than 150 eukaryotic signaling proteins, including transcription factors (e.g. nuclear receptors, p53) and protein kinases (e.g. Src, Raf, Akt kinase) involved in cell cycling, tumorigenesis, apoptosis, and multiple eukaryotic signaling pathways (1,2). Of these many 'client' proteins for hsp90, the assembly of steroid receptor•hsp90 complexes is the best defined (Figure 1). We present here an adaptable glucocorticoid receptor (GR) immunoprecipitation assay and in vitro GR•hsp90 reconstitution method that may be readily used to probe eukaryotic hsp90 functional activity, hsp90-mediated steroid receptor ligand binding, and molecular chaperone cofactor requirements. For example, this assay can be used to test hsp90 cofactor requirements and the effects of adding exogenous compounds to the reconstitution process. The GR has been a particularly useful system for studying hsp90 because the receptor must be bound to hsp90 to have an open ligand binding cleft that is accessible to steroid (3). Endogenous, unliganded GR is present in the cytoplasm of mammalian cells noncovalently bound to hsp90. As found in the endogenous GR•hsp90 heterocomplex, the GR ligand binding cleft is open and capable of binding steroid. If hsp90 dissociates from the GR or if its function is inhibited, the receptor is unable to bind steroid and requires reconstitution of the GR•hsp90 heterocomplex before steroid binding activity is restored (4) . GR can be immunoprecipitated from cell cytosol using a monoclonal antibody, and proteins such as hsp90 complexed to the GR can be assayed by western blot. Steroid binding activity of the immunoprecipitated GR can be determined by incubating the immunopellet with [(3)H]steroid. Previous experiments have shown hsp90-mediated opening of the GR ligand binding cleft requires hsp70, a second molecular chaperone also essential for eukaryotic cell viability. Biochemical activity of hsp90 and hsp70 are catalyzed by co-chaperone proteins Hop, hsp40, and p23 (5). A multiprotein chaperone machinery containing hsp90, hsp70, Hop, and hsp40 are endogenously present in eukaryotic cell cytoplasm, and reticulocyte lysate provides a chaperone-rich protein source (6). In the method presented, GR is immunoadsorbed from cell cytosol and stripped of the endogenous hsp90/hsp70 chaperone machinery using mild salt conditions. The salt-stripped GR is then incubated with reticulocyte lysate, ATP, and K(+), which results in the reconstitution of the GR•hsp90 heterocomplex and reactivation of steroid binding activity (7). This method can be utilized to test the effects of various chaperone cofactors, novel proteins, and experimental hsp90 or GR inhibitors in order to determine their functional significance on hsp90-mediated steroid binding (8-11).

Biochemical Reconstitution of Steroid Receptor&amp;#x2022;Hsp90 Protein Complexes and Reactivation of Ligand Binding

Hsp90 is an essential and highly abundant molecular chaperone protein that has been found to regulate more than 150 eukaryotic signaling proteins, including transcription factors (e.g. nuclear receptors, p53) and protein kinases (e.g. Src, Raf, Akt kinase) involved in cell cycling, tumorigenesis, apoptosis, and multiple eukaryotic signaling pathways 1,2. Of these many 'client' proteins for hsp90, the assembly of steroid receptor•hsp90 complexes is the best defined (Figure 1). We present here an adaptable glucocorticoid receptor (GR) immunoprecipitation assay and in vitro GR•hsp90 reconstitution method that may be readily used to probe eukaryotic hsp90 functional activity, hsp90-mediated steroid receptor ligand binding, and molecular chaperone cofactor requirements. For example, this assay can be used to test hsp90 cofactor requirements and the effects of adding exogenous compounds to the reconstitution process. The GR has been a particularly useful system for studying hsp90 because the receptor must be bound to hsp90 to have an open ligand binding cleft that is accessible to steroid 3. Endogenous, unliganded GR is present in the cytoplasm of mammalian cells noncovalently bound to hsp90. As found in the endogenous GR•hsp90 heterocomplex, the GR ligand binding cleft is open and capable of binding steroid. If hsp90 dissociates from the GR or if its function is inhibited, the receptor is unable to bind steroid and requires reconstitution of the GR•hsp90 heterocomplex before steroid binding activity is restored 4 . GR can be immunoprecipitated from cell cytosol using a monoclonal antibody, and proteins such as hsp90 complexed to the GR can be assayed by western blot. Steroid binding activity of the immunoprecipitated GR can be determined by incubating the immunopellet with [3H]steroid. Previous experiments have shown hsp90-mediated opening of the GR ligand binding cleft requires hsp70, a second molecular chaperone also essential for eukaryotic cell viability. Biochemical activity of hsp90 and hsp70 are catalyzed by co-chaperone proteins Hop, hsp40, and p23 5. A multiprotein chaperone machinery containing hsp90, hsp70, Hop, and hsp40 are endogenously present in eukaryotic cell cytoplasm, and reticulocyte lysate provides a chaperone-rich protein source 6. In the method presented, GR is immunoadsorbed from cell cytosol and stripped of the endogenous hsp90/hsp70 chaperone machinery using mild salt conditions. The salt-stripped GR is then incubated with reticulocyte lysate, ATP, and K+, which results in the reconstitution of the GR•hsp90 heterocomplex and reactivation of steroid binding activity 7. This method can be utilized to test the effects of various chaperone cofactors, novel proteins, and experimental hsp90 or GR inhibitors in order to determine their functional significance on hsp90-mediated steroid binding 8-11.

Ursodeoxycholic Acid Amides As Novel Glucocorticoid Receptor Modulators

Ursodeoxycholic acid (UDCA) is used for the treatment of hepatic inflammatory diseases. Recent studies have shown that UDCA's biological effects are partly glucocorticoid receptor (GR) mediated. UDCA derivatives were synthesized and screened for ability to induce GR translocation in a high content analysis assay using the esophageal cancer SKGT-4 cell line. UDCA derivatives induced GR translocation in a time dependent manner with equal efficacy to that of dexamethasone (Dex) and with greatly increased potency relative to UDCA. The cyclopropylamide 1a suppressed TNF-α induced NF-κB activity and it induced GRE transactivation. 1a was unable to displace Dex from the GR ligand binding domain (LBD) in a competition experiment but was capable of coactivator recruitment in a time-resolved fluorescence energy transfer assay (TR-FRET). This represents a novel mechanism of action for a GR modulator. These derivatives could result in a new class of GR modulators.