Control of rat tail skin temperature regulation by estrogen receptor-beta selective ligand.

OBJECTIVE: To test the role of ERbeta in the control of estrogen-dependent thermoregulation in rats. METHODS: Test the ability of an ERbeta-selective ligand to suppress the elevation in basal rat tail skin temperature (TST) caused by ovariectomy (OVX). RESULTS: ERbeta-19 is a tetrahydrofluorenone ERbeta-selective ligand that displaces 0.1 nM estradiol from ERbeta with an IC50 of 1.8 nM compared to an IC50 of 141 nM for ERalpha. Like estradiol, it acts as an agonist on ERbeta-mediated transactivation and transrepression with 25- and 60-fold selectivity, respectively, over ERalpha-controlled transcription. Administration of estradiol to estrogen-depleted rats suppresses the ovariectomy-induced elevation of TST. Similar treatment of OVX rats with ERbeta-19 also results in suppression of elevated TST. However, in contrast to estradiol, ERbeta-19 does not suppress body weight, does not increase uterine weight, nor does it stimulate uterocalin biomarker expression which is under the control of ERalpha. Thus, the ERbeta-19 suppression of rat TST is mediated by ERbeta without eliciting the activity of ERalpha. CONCLUSION: Estrogen-sensitive thermoregulation in ovariectomized rats can be controlled by an ERbeta-selective ligand.

Agonist-like SERM effects on ERalpha-mediated repression of MMP1 promoter activity predict in vivo effects on bone and uterus.

Estradiol receptors (ER), ERalpha and ERbeta, are ligand-dependent transcription factors that regulate gene expression. Human and murine genetics suggest that ERalpha is the key target for estradiol action on bone, uterus and breast. To date, the molecular mode of action of estradiol and selective estradiol receptor modulators (SERMs) on bone is not fully understood. This is exemplified by a lack of in vitro assays that reliably predict SERM agonist activities in vivo. We hypothesized that ligand-dependent ERalpha transrepression, via protein-protein interactions at AP1, may predict estrogenic effects on bone. We modeled this using the MMP1 promoter, which encodes an AP1 binding site. We show that ICI-182780, raloxifene, 4-hydroxytamoxifen and estradiol all exhibit differential agonistic activities on the MMP1 promoter by suppressing activity by 20-80%. Transrepression efficacy and potency correlated with both uterotrophic (R(2)=0.98) and osteoprotective (R(2)=0.80) potential in the ovariectomized rat. This identifies MMP1 promoter transrepression as an agonist activity commonly shared by AF2 agonists and "antagonists" alike. Mutation analysis showed that the repression by estradiol and SERMs required correct amino acid sequences in the AF-2 domain. For instance, L540Q AF2 mutation did not alter responses to raloxifene, although it greatly increased responses to ICI-182780 (threefold) and reduced estradiol's effect by 20%. Furthermore, all tested ligands repressed the MMP1 promoter through the L540Q mutant with identical efficacy. Together, these data suggest that estradiol and SERMs share common agonist transcriptional activity via protein-protein interactions at AP1.

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.

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.

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 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.

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.

Estrogen receptor ligands. Part 7: Dihydrobenzoxathiin SERAMs with bicyclic amine side chains.

A series of benzoxathiin SERAMs with bicyclic amine side chains was prepared. Minor modifications in the side chain resulted in significant effects on biological activity, especially in uterine tissue.

Estrogen receptor ligands. Part 8: Dihydrobenzoxathiin SERAMs with heteroatom-substituted side chains.

A series of benzoxathiin SERAMs with heteroatom-substituted amine side chains was prepared. Minor modifications in the side chain resulted in significant effects on biological activity, especially in uterine tissue.

Estrogen receptor ligands. Part 6: Synthesis and binding affinity of dihydrobenzodithiins.

Dihydrobenzodithiin compounds (1-6) were prepared to explore the expansion of the dihydrobenzoxathiin lead compounds I-III as SERAMs (Selective Estrogen Receptor Alpha Modulators). The dihydrobenzodithiin compounds generally maintained a high degree of selectivity for ERalpha over ERbeta, however, they lacked the in vivo antagonism/agonism activity exhibited by the lead class in an immature rat uterine growth model.

Estrogen receptor ligands. Part 5: The SAR of dihydrobenzoxathiins containing modified basic side chains.

Dihydrobenzoxathiin analogs (1-11) with modifications on the basic side chain region were prepared and evaluated for estrogen/anti-estrogen activity in both in vitro and in vivo models. The compounds generally maintained a high degree of selectivity for ERalpha over ERbeta, similar to the original lead compound I. Many of the compounds also maintained high potency in the inhibition of human carcinoma MCF-7 cell growth. However, all were less potent in the inhibition of estradiol-triggered uterine growth. This work demonstrates the sensitive nature of modification to the antagonist basic side chain region.

Estrogen receptor ligands. Part 4: The SAR of the syn-dihydrobenzoxathiin SERAMs.

A series of estrogen receptor ligands based on a dihydrobenzoxathiin scaffold is described and evaluated for estrogen/anti-estrogen activity in both in vitro and in vivo models. The most active analogue, 22, was found to be 40-fold ERalpha selective in a competitive binding assay, and 22 demonstrated very potent in vivo antagonism of estradiol driven proliferation in an immature rat uterine weight gain assay.