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.

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).

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.

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.

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.

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.

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.

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.

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.

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 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.

Estrogen receptor ligands. Part 13: Dihydrobenzoxathiin SERAMs with an optimized antagonist side chain.

An optimized side chain for dihydrobenzoxathiin SERAMs was discovered and attached to four dihydrobenzoxathiin platforms. The novel SERAMs show exceptional estrogen antagonist activity in uterine tissue and an MCF-7 breast cancer cell assay.

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.

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 ligands. Part 10: Chromanes: old scaffolds for new SERAMs.

The discovery, synthesis, and SAR of chromanes as ER alpha subtype selective ligands are described. X-ray studies revealed that the origin of the ER alpha-selectivity resulted from a C-4 trans methyl substitution to the cis-2,3-diphenyl-chromane platform. Selected compounds from this class demonstrated very potent in vivo antagonism of estradiol in an immature rat uterine weight assay, effectively inhibited ovariectomy-induced bone resorption in a 42 days treatment paradigm, and lowered serum cholesterol levels in ovx'd adult rat models. The best antagonists 8F and 12F also exhibited potent inhibition of MCF-7 cell growth and were shown to be estrogen receptor down-regulators (SERDs).

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.

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.

Estrogen receptor ligands. Part 9: Dihydrobenzoxathiin SERAMs with alkyl substituted pyrrolidine side chains and linkers.

A series of dihydrobenzoxathiin SERAMs with alkylated pyrrolidine side chains or alkylated linkers was prepared. Minor modifications in the side chain or linker resulted in significant effects on biological activity, especially in uterine tissue.

Identification of neutral 4-O-alkyl quinolone nonpeptide GnRH receptor antagonists.

A series of neutral, nonbasic quinolone GnRH antagonists were prepared via Mitsunobu alkylation of protected and unprotected 4-hydroxy quinolone intermediates. The synthetic route was improved by utilization of unique reactivity and convergency afforded by the use of mono and bis-trimethylsilylethyl protected quinolones. Potent neutral GnRH antagonists were identified, including ether and lactam derivatives, that show similar in vitro binding affinity and functional activity as compared to the earlier basic 4-aminoalkyl quinolone series of nonpeptide GnRH antagonists.