Identification and characterization of lipopolysaccharide binding protein (LBP) as an estrogen receptor α specific serum biomarker.

Estrogen Receptor α (ERα) and Estrogen Receptor ß (ERß) are steroid nuclear receptors that transduce estrogen signaling to control diverse physiological processes linked to reproduction, bone remodeling, behavior, immune response and endocrine-related diseases. In order to differentiate between ERα and ERß mediated effects in vivo, ER subtype selective biomarkers are essential. We utilized ERα knockout (AERKO) and ERß knockout (BERKO) mouse liver RNA and genome wide profiling to identify novel ERα selective serum biomarker candidates. Results from the gene array experiments were validated using real-time RT-PCR and subsequent ELISA's to demonstrate changes in serum proteins. Here we present data that Lipopolysacharide Binding Protein (LBP) is a novel liver-derived ERα selective biomarker that can be measured in serum.

Antagonist-induced, activation function-2-independent estrogen receptor alpha phosphorylation.

Estrogen receptor alpha (ERalpha) serine 118 (Ser118) phosphorylation modulates activation function-1 (AF1) function. Correct positioning of helix 12 promotes agonist-dependent recruitment of cyclin-dependent kinase-7 to catalyze this event. In this study we show robust cyclin-dependent kinase-7-independent, AF2 antagonist-induced Ser118 phosphorylation. Estradiol (E2) and ICI-182,780 (ICI-780) induce Ser118 phosphorylation of wild-type ERalpha and either of two helix 12 mutants, suggesting AF2-independent action, probably via shedding of 90-kDa heat shock protein. With E2 treatment, the predominantly nuclear, phosphorylated ERalpha in COS-1 cells is detergent soluble. Although levels of ICI-780-induced phosphorylation are profound, Ser118-phosphorylated ERalpha is aggregated over the nucleus or in the cytoplasm, fractionating with the cell debris and making detection in cleared lysates improbable. Selective ER modulators (SERMs) elicit a mixed response with phosphorylated ERalpha in both detergent-soluble and -insoluble compartments. Apparent ligand-induced loss of ERalpha protein from cleared lysates is thus due to ligand-induced redistribution into the pellet, not degradation. The COS-1 response to ICI-780 can be mimicked in MCF-7 cells treated with a proteasome inhibitor to block authentic ligand-induced degradation. With SERMs and antagonists, the magnitude of Ser118-phosphorylated receptor redistribution into the insoluble fraction of COS-1 cells correlates with the magnitude of authentic ERalpha degradation in MCF-7 cells. A strong inverse correlation with ligand-induced uterotropism in vivo (P < 0.0001) and direct correlation with AF2-independent transrepression of the matrix metalloprotease-1 promoter in endometrial cells in vitro are seen. These data suggest that ligand-induced Ser118 phosphorylation of ERalpha can be AF2 independent. Furthermore, they identify translocation of Ser118-phosphorylated ERalpha out of the nucleus, leading to cytoplasmic aggregation, as an antagonist pathway that may precede receptor degradation.

Antidiabetic activity of a highly potent and selective nonpeptide somatostatin receptor subtype-2 agonist.

Somatostatin inhibits both glucagon and insulin secretion. Glucagon significantly contributes to hyperglycemia in type 2 diabetes. Despite its function in the inhibition of glucagon secretion, somatostatin fails to reduce hyperglycemia in type 2 diabetes, due to a parallel suppression of insulin secretion. Five pharmacologically distinct somatostatin receptor subtypes (sst(1)-sst(5)) mediate the effects of somatostatin on a cellular level. Pancreatic A cells express sst(2), whereas B cells express sst(5). In this study, we describe a novel approach to the treatment of type 2 diabetes using a highly sst(2)-selective, nonpeptide agonist (compound 1). Compound 1 effectively inhibited glucagon secretion from pancreatic islets isolated from wild-type mice, whereas glucagon secretion from sst(2)-deficient islets was not suppressed. Compound 1 did not influence nonfasted insulin concentration. In sst(2)-deficient mice, compound 1 did not have any effects on glucagon or glucose levels, confirming its sst(2) selectivity. In animal models of type 2 diabetes in the nonfasted state, circulating glucagon and glucose levels were decreased after treatment with compound 1. In the fasting state, compound 1 lowered blood glucose by approximately 25%. In summary, small-molecule sst(2)-selective agonists that suppress glucagon secretion offer a novel approach toward the development of orally bioavailable drugs for treatment of type 2 diabetes.

Differential hormonal regulation of tryptophan hydroxylase-2 mRNA in the murine dorsal raphe nucleus.

BACKGROUND: Recently a novel tryptophan hydroxylase isoform (TPH2) was identified and shown to be highly expressed in the central nervous system (CNS). Hormonal effects on TPH2 mRNA expression in the rodent dorsal raphe nucleus (DRN) are unknown. METHODS: In situ hybridization histochemistry and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) were used to assess the effects of dexamethasone or estradiol on TPH2 mRNA levels in the DRN of C57/Bl6 mice. RESULTS: Dexamethasone reduced TPH2 mRNA levels in the DRN of both ovx female and intact male mice. Reduction of TPH2 mRNA in the DRN was blocked by co-administration of mifepristone. Estradiol had no detectable effect on TPH2 mRNA levels in the DRN. CONCLUSIONS: TPH2 mRNA is regulated by glucocorticoids but not estradiol in the mouse DRN. Glucocorticoid-mediated reduction of TPH2 message may have relevance to the etiology of major depression, psychotic major depression in particular, where elevated glucocorticoids are one hallmark of the disease.

Estrogen receptor-beta regulates tryptophan hydroxylase-1 expression in the murine midbrain raphe.

BACKGROUND: Distinct expression patterns of estrogen receptor (ER)-alpha and ER-beta are displayed in the murine central nervous system. ER-beta is the predominant form of the receptor expressed in the murine midbrain dorsal raphe nucleus (DRN). Tryptophan hydroxylase (TPH) is abundantly expressed in the serotonergic neurons of the DRN and is regulated by estrogen in both the monkey and the guinea pig. METHODS: In this study we used immunocytochemistry to show that ER-beta and TPH are colocalized in the serotonergic cells of the murine DRN. We utilized the ER-alpha and ER-beta gene deletion mouse models and in situ hybridization to demonstrate that ER-beta is responsible for regulating TPH1 mRNA expression. RESULTS: Estrogen increased TPH1 mRNA expression in the DRN of wild type and ER-alpha knockout mice (alpha-ERKO) but not ER-beta knockouts (beta-ERKO). CONCLUSIONS: These data indicate that ER-beta is responsible for mediating estrogen regulated TPH1 expression in the murine DRN.

17 Beta-estradiol-induced antidepressant-like effect in the forced swim test is absent in estrogen receptor-beta knockout (BERKO) mice.

RATIONALE: The decrease in levels of estrogens (ER) that occurs in menopause has been correlated with depressive disorders, probably due to ER direct and/or indirect effects in the brain, where these hormones act through both genomic (i.e. interaction as transcription factors with nuclear receptors ER-alpha and ER-beta) and non-genomic (i.e. binding with cell-membrane receptors) mechanisms. With respect to mood related disorders the interaction between ER-beta and the serotonin (5-HT) system is highly relevant. 17beta-Estradiol (E2) induces expression of the enzyme implicated in 5-HT synthesis - tryptophan hydroxylase (TPH), and this effect is mediated through ER-beta located in 5-HT cell bodies of the dorsal raphe nucleus (DRN). OBJECTIVE: The present studies tested the hypothesis that E2 induces antidepressant-like effects in female ovariectomized (OVX) mice, and that expression of ER-beta is mandatory for such effects. METHODS: The Forced Swim Test (FST) was used in three experiments to assess (a) dose response effect of E2 in outbred and inbred mouse strains, (b) length of treatment necessary for effect, (c) and role of ER-beta receptors. RESULTS: E2 (100 or 200 microg/kg), as well as the antidepressant desipramine (DMI), significantly reduced total duration of immobility in the FST in mice from different strains. Four consecutive daily doses (200 microg/kg) were required for such effect, which was absent in mice lacking the gene coding for ER-beta (BERKO mice). CONCLUSION: These data suggest that E2-induced antidepressant-like effects in mice are mediated through activation of ER-beta. They offer preliminary support to the hypothesis that specific compounds acting at ER-beta may influence mood in postmenopausal women.

Immunolocalization of estrogen receptor beta in the mouse brain: comparison with estrogen receptor alpha.

Estrogen receptor alpha (ER alpha) and ER beta are members of the steroid nuclear receptor family that modulate gene transcription in an estrogen-dependent manner. ER mRNA and protein have been detected both peripherally and in the central nervous system, with most data having come from the rat. Here we report the development of an ER beta-selective antibody that cross-reacts with mouse, rat, and human ER beta protein and its use to determine the distribution of ER beta in the murine brain. Further, a previously characterized polyclonal antibody to ER alpha was used to compare the distribution of the two receptors in the first comprehensive description of ER distribution specifically in the mouse brain. ER beta immunoreactivity (ir) was primarily localized to cell nuclei within select regions of the brain, including the olfactory bulb, cerebral cortex, septum, preoptic area, bed nucleus of the stria terminalis, amygdala, paraventricular hypothalamic nucleus, thalamus, ventral tegmental area, substantia nigra, dorsal raphe, locus coeruleus, and cerebellum. Extranuclear-ir was detected in several areas, including fibers of the olfactory bulb, CA3 stratum lucidum, and CA1 stratum radiatum of the hippocampus and cerebellum. Although both receptors were generally expressed in a similar distribution through the brain, nuclear ER alpha-ir was the predominant subtype in the hippocampus, preoptic area, and most of the hypothalamus, whereas it was sparse or absent from the cerebral cortex and cerebellum. Collectively, these findings demonstrate the region-selective expression of ER beta and ER alpha in the adult ovariectomized mouse brain. These data provide an anatomical framework for understanding the mechanisms by which estrogen regulates specific neural systems in the mouse.

Effects of heterocyclic aromatic substituents on binding affinities at two distinct sites of somatostatin receptors. Correlation with the electrostatic potential of the substituents.

In our continuing program exploring glucose-based peptidomimetics of somatostatin (SRIF-14), we sought to improve the water solubility of our glycosides. This led to insights into the nature of the ligand binding sites at the SRIF receptor. Replacement of the C4 benzyl substituent in glucoside (+)-2 with pyridinylmethyl or pyrazin-2-ylmethyl congeners increased water solubility and enhanced affinity for the human SRIF subtype receptor 4 (sst4). We attribute this effect to hydrogen bond formation. The pyridin-3-ylmethyl substituent at C4, when combined with the imidazol-4-ylmethyl group at C2, generated (-)-19, which has the highest affinity of a glucose-based peptidomimetic at a human SRIF receptor to date (K(i) 53 +/- 23 nM, n = 6 at sst4). The C4 heterocyclic congeners of glucosides bearing a 1-methoxy substituent rather than an indole side chain at the anomeric carbon, such as (+)-16, also provided information about the Trp(8) binding pocket. We correlated the SARs at both the C4 and the Trp(8) binding pockets with calculations of the electrostatic potentials of the diverse C4 aromatic substituents using Spartan 3-21G(*) MO analysis. These calculations provide an approximate analysis of a molecule's ability to interact within a receptor binding site. Our binding studies show that benzene and indole rings, but not pyridinylmethyl nor pyrazin-2-ylmethyl rings, can bind the hydrophobic Trp(8) binding pocket of sst4. The Spartan 3-21G(*) MO analysis reveals significant negative electrostatic potential in the region of the pi-clouds for the benzene and indole rings but not for the pyridinylmethyl or pyrazin-2-ylmethyl congeners. Our data further demonstrate that the replacement of benzene or indole side chains by heterocyclic aromatic rings typified by pyridine and pyrazine not only enhances water solubility and hydrogen bonding capacity as expected, but can also profoundly diminish the ability of the pi-cloud of the aromatic substituent to interact with side chains of an aromatic binding pocket such as that for Trp(8) of SRIF-14. Conversely, these calculations accommodate the experimental findings that pyrazin-2-ylmethyl and pyridinylmethyl substituents at C4- of C1-indole-substituted glycosides afford higher affinities at sst4 than the C4-benzyl group of (+)-2. This result is consistent with the high electron density in the plane of the heterocycle depicted in Figure 6 which can accept hydrogen bonds from the C4 binding pocket of the receptor. Unexpectedly, we found that the 2-fluoropyridin-5-ylmethyl analogue (+)-14 more closely resembles the binding affinity of (+)-8 than that of (+)-2, thus suggesting that (+)-14 represents a rare example of a carbon linked fluorine atom acting as a hydrogen bond acceptor. We attribute this result to the ability of the proton to bind the nitrogen and fluorine atoms simultaneously in a bifurcated arrangement. At the NK1 receptor of substance P (SP), the free hydroxyl at C4 optimizes affinity.

Estrogen receptor ligands. II. Discovery of benzoxathiins as potent, selective estrogen receptor alpha modulators.

The discovery and synthesis of dihydrobenzoxathiins as potent, ERalpha subtype selective ligands are described. The most active analogue, 4-D, was found to be 50-fold selective in a competitive binding assay and 100-fold selective in a transactivation assay in HEK-293 cells. The alpha selectivity was postulated to lie in the interaction of the sulfur atom of the benzoxathiin ring with the two discriminating residues in the binding pocket of the receptor isoforms.

A beta-lactamase-dependent Gal4-estrogen receptor beta transactivation assay for the ultra-high throughput screening of estrogen receptor beta agonists in a 3456-well format.

Estrogen action is mediated via two estrogen receptor (ER) subtypes, ERalpha and ERbeta. Selective ER modulators with balanced high affinity for ERalpha and ERbeta have been developed as therapeutics for the treatment of a variety of diseases, including hormone-responsive breast cancer and osteoporosis. Recent data based primarily on the evaluation of ER-knockout mice have revealed that ERalpha and ERbeta may regulate separate and distinct biological processes. The identification of ERbeta specific ligands could further enhance our understanding of ERbeta biology. In addition, compounds targeting ERbeta may prove useful as therapeutic agents with activity profiles distinguishable from that of estradiol. To discover novel selective ligands for ERbeta, we developed and characterized a cell-based Gal4-ERbeta beta-lactamase reporter gene assay (GERTA) in CHO cells for the ligand-induced activation of the human ERbeta. The sensitivity and selectivity of this assay were found to be comparable to those of an ER ligand-binding assay. The assay was optimized for screening in an ultra high throughput 3456-well nanoplate format and was successfully used to screen a large compound collection for ERbeta agonists. Compounds identified in a primary screen were tested in an in vitro ligand-binding assay to characterize further the selectivity and potency for ERbeta.

Androstene-3,5-dienes as ER-beta selective SERMs.

A series of androstene-3,5-diene derivatives were prepared. Despite lacking the C-3 hydroxyl previously believed necessary for ER activity, some of the analogs retained surprising affinity for ER-beta. For example, diene 4 retained excellent selectivity and potency as an ER-beta agonist and was more selective for ER-beta over the androgen receptor (AR).

Estrogen receptor ligands. Part 16: 2-Aryl indoles as highly subtype selective ligands for ERalpha.

A novel class of indole ligands for estrogen receptor alpha have been discovered which exhibit potent affinity and high selectivity. Substitution of the bazedoxifene skeleton to the linker present in the HTS lead 1a provided 22b which was found to be 130-fold alpha-selective and acted as an antagonist of estradiol activity in uterine tissue and MCF-7 cancer cells.

Bridged androstenediol analogs as ER-beta selective SERMs.

A series of bridged androstenediol derivatives was prepared. The bridged compounds exhibited reduced ER-beta selectivity relative to uncyclized analogs.

Tetrahydrofluorenones with conformationally restricted side chains as selective estrogen receptor beta ligands.

A series of 2-9a bridged tetrahydrofluorenone derivatives were prepared which exhibited significant binding affinity for ERbeta and were highly selective.

6H-Benzo[c]chromen-6-one derivatives as selective ERbeta agonists.

A series of 6H-benzo[c]chromen-6-one and 6H-benzo[c]chromene derivatives were prepared, and the affinity and selectivity for ERalpha and ERbeta was measured. Many of the analogs were found to be potent and selective ERbeta agonists. Bis hydroxyl at positions 3 and 8 is essential for activity in a HTRF coactivator recruitment assay. Additional modifications at both phenyl rings led to compounds with ERbeta<10nM potency and >100-fold selectivity over ERalpha.

Triazolo-tetrahydrofluorenones as selective estrogen receptor beta agonists.

Several tetrahydrofluorenones with a triazole fused across C7-C8 showed high levels of ERbeta-selectivity and were found to be potent ERbeta-agonists. As a class they demonstrate improved oral bioavailability in the rat over a parent class of 7-hydroxy-tetrahydrofluorenones. The most selective agonist displayed 5.7 nM affinity and 333-fold selectivity for ERbeta.

Androstenediol analogs as ER-beta-selective SERMs.

A series of 19-substituted androstenediol derivatives was prepared. Some of the novel analogs were surprisingly potent and selective ligands for ER-beta.

Biomarker discovery in rat plasma for estrogen receptor-alpha action.

To support in vivo screening efforts for estrogen receptor (ER) subtype selective therapeutic agents, we initiated work to discover surrogate markers (biomarkers) in blood plasma that would change in response to ER subtype-specific action. We used a proteomic approach employing strong anion exchange chromatography (SAX), PAGE, and MS to identify potential plasma markers for selective ER-alpha action. The methodology was used to compare blood from vehicle-treated rats to blood from rats treated with either 17beta-estradiol (an ER-alpha/ER-beta agonist) or compound 1 (17alpha-ethynyl-[3,2-c]pyrazolo-19-nor-4-androstene-17beta-ol, an ER-alpha-selective agonist). Blood samples were first fractionated by SAX to separate fractions containing dominant common plasma proteins from fractions enriched for less-abundant plasma proteins. 1-D PAGE analysis of fractions depleted of dominant plasma proteins revealed treatment-specific changes in protein profiles. Protein bands that changed reproducibly in response to ER-alpha action were excised from the gel, separated by capillary LC, and identified by microspray ESI-MS. Using this method, the plasma levels of two proteins, transthyretin and apolipoprotein E, were shown to decrease in response to ER-alpha agonism. The method lacked the sensitivity to identify the known, 1000-fold less-abundant, estrogenic marker prolactin (PRL). However, using a commercial RIA and immunoblots, we showed that PRL levels increase significantly in response to treatment with the ER-alpha selective agonist, compound 1.

Estrogen receptor ligands. Part 11: Synthesis and activity of isochromans and isothiochromans.

The ring oxygen and sulfur analogs of lasofoxifene, 1a and 1b, were synthesized in an attempt to impart ERalpha selectivity, as found in the closely related dihydrobenzoxathiin compound I, recently discovered in these laboratories. The resulting isochroman and isothiochroman compounds were found to exhibit equipotent binding affinities to the ER isoforms and were less active in the inhibition of estradiol-triggered uterine growth when compared to I and lasofoxifene.

Estrogen receptor ligands. Part 14: application of novel antagonist side chains to existing platforms.

Two novel side chains which had previously been found to enhance antagonist activity in the dihydrobenzoxathiin SERM series were applied to three existing platforms. The novel side chains did not improve the antagonist activity of the existing platforms.