Capacity of type I and II ligands to confer to estrogen receptor alpha an appropriate conformation for the recruitment of coactivators containing a LxxLL motif-Relationship with the regulation of receptor level and ERE-dependent transcription in MCF-7 cells.

Estrogen receptor alpha (ERalpha) belongs to the superfamily of nuclear receptors and as such acts as a ligand-modulated transcription factor. Ligands elicit in ERalpha conformational changes leading to the recruitment of coactivators required for the transactivation of target genes via cognate response elements. In many cells, activated ERalpha also undergoes downregulation by proteolysis mediated by the ubiquitin/proteasome system. Although these various molecular processes have been well characterized, little is known as to which extent they are interrelated. In the present study, we used a panel of type I (estradiol derivatives and "linear", non-steroidal ligands) and type II ("angular" ligands) estrogens, in order to identify possible relationships between ligand binding affinity, recruitment of LxxLL-containing coactivators, ERalpha downregulation in MCF-7 cells and related transactivation activity of ligand-bound ERalpha. For type I estrogens, there was a clear-cut relationship between ligand binding affinity, hydrophobicity around C-11 of estradiol and ability of ERalpha to associate with LxxLL motifs, both in cell-free condition and in vivo (MCF-7 cells). Moreover, LxxLL motif recruitment by ERalpha seemed to be a prerequisite for the downregulation of the receptor. By contrast, type II ligands, as well as estradiol derivatives bearing a bulky side chain at 11beta, had much less tendency to promote ERalpha-LxxLL interaction or even behaved as antagonists in this respect, in agreement with the well known partial estrogenicity/antiestrogenicity of some of these compounds. Interestingly, some type II ligands which antagonized LxxLL motif recruitment were nonetheless able to enhance ERalpha-mediated gene transactivation.

Effect of fluorination on the pharmacological profile of 11beta isomers of fulvestrant in breast carcinoma cells.

We describe the synthesis of an 11beta isomer 3 of the steroidal antiestrogen fulvestrant 2. Partial fluorination of the 11beta side chain in 3 leads to 4, which still shows strong antiproliferative activity on MCF-7 cells. However, unlike 2 and 3, compound 4 fails to down-regulate estrogen receptor alpha (ERalpha). This result suggests that ERalpha down-regulation is not a sine qua non condition for the antitumor activity of steroidal antiestrogens.

Fluorous tolerance of the estrogen receptor alpha as probed by 11-polyfluoroalkylestradiol derivatives.

The concern of this work was to try to delineate factors, inherent to fluorination, susceptible to influence estradiol binding to the estrogen receptor alpha (ERalpha). For this purpose, fluorinated chains were linked at 11beta position of the steroid (i.e., C(6)F(13), CH(2)CH(2)C(4)F(9), CH(2)CH(2)C(8)F(17)). Relative binding affinity (RBA) for ERalpha of these compounds and of other related fluorinated derivatives was compared to those of non-fluorinated analogs. Despite being relatively well accepted by the receptor, investigated compounds exhibited lower RBA values at 0 degrees C than their non-fluorinated counterparts. Nevertheless, heavily fluorinated chains were tolerated in so far as they are not too long (C-4) and insulated from the steroidal core by a two methylene spacer unit. Increase of the temperature of our binding assay (25 degrees C) failed to change the RBA values of two selected polyfluorohexyl derivatives while it drastically enhanced the value of the corresponding non-fluorinated analogs. Rigidity of the chain induced by fluorination as well as the oleophilic (fluorophobic) nature of the estradiol binding cavity of ERalpha is proposed to explain these properties.

Effects of (R,S)/(S,R)-4,5-bis(2-chloro-4-hydroxyphenyl)-2-imidazolines and (R,S)/(S,R)-2,3-bis(2-chloro-4-hydroxyphenyl)piperazines on estrogen receptor alpha level and transcriptional activity in MCF-7 cells.

4,5-Diaryl-2-imidazolines (Im(s)) and 2,3-diarylpiperazines (Pip(s)) belong to the type II class of estrogens. These compounds enhance ERalpha-mediated transcription of ERE-driven reporter genes in MCF-7 cells but do not compete with [(3)H]estradiol (E(2)) for receptor binding, because of distinct anchoring modes. The present study examined whether the estrogenic action of Im(s) and Pip(s) is associated with a down regulation of ERalpha, as reported for conventional agonists. Im and Pip derivatives displaying a large spectrum of activities in three distinct ERE-dependent transactivation systems were selected for that purpose. ERalpha immunostaining as well as Western blotting analysis revealed that both classes of compounds down regulated ERalpha with an efficiency closely related to their transactivation potency. MG-132 abrogated this down regulation, pointing to a proteasomal degradation process. Im(s) and Pip(s) with strong transactivation potency also altered [(3)H]E(2) binding parameters, leading to a progressive decrease of cellular estrogen binding capacity. This property occurred largely before ERalpha down regulation and persisted even in presence of MG-132, indicating that it did not result from ERalpha breakdown but rather from a conformational change of the receptor. The additional finding that the most active agonist tested in this study enhanced the capacity of a purified ERalpha recombinant to recruit LxxLL co-activators, while its inactive counterpart failed to do so confirmed this hypothesis. Altogether, our data indicate that the association of Im(s) and Pip(s) with ERalpha elicits similar responses to conventional agonists, even if they interact with distinct residues of the binding pocket.

Synthesis, structure, and estrogenic activity of 4-amino-3-(2-methylbenzyl)coumarins on human breast carcinoma cells.

A number of coumarins exhibit interesting pharmacological activities and are therefore of therapeutic use. We report here the synthesis and the structural analysis of new N-substituted 4-amino-3-(2-methylbenzyl)coumarins (compounds 8a-8e) that present structural analogies with estrothiazine and 11- or 7-substituted 17beta-estradiol. These derivatives were tested with respect to estrogenic activity on the estrogen receptor positive (ER+) human MCF-7 breast cancer cell line. Two of the reported compounds (8a and 8b) stimulated specifically the proliferation of MCF-7 cells, but not that of estrogen receptor negative (ER-) human MDA-MB-231 breast cancer cells, suggesting that their mitogenic activity is mediated by ER. Accordingly, the stimulating effect of 8a and 8b was suppressed by the pure antiestrogen fulvestrant. Besides, 8a and 8b induced ER down-regulation similar to that produced by classical ER agonists or pure antagonists. The effects of the compounds under study on ER-mediated transcription were assessed on (ER+) MVLN cells, that is, MCF-7 cells stably transfected with a pVit-tk-Luc reporter plasmid. Derivatives 8a and 8b, and surprisingly compound 8c, enhanced ER-mediated gene transactivation in that model. Finally, no coumarin was able to compete with tritiated 17beta-estradiol ([(3)H]E(2)) for ER binding, suggesting unconventional interactions with the receptor, such as interactions with the second binding pocket or with the coactivator-binding region. To conclude, observations performed in this study on compound 8c reveal that estrogenic activity can be dissociated from enhancement of cell proliferation. Furthermore, ERE-driven transactivation of transcription seems to be a condition necessary, but not sufficient, for estrogen-induced stimulation of cell growth.

Calmodulin-independent, agonistic properties of a peptide containing the calmodulin binding site of estrogen receptor alpha.

Calmodulin (CaM) contributes to estrogen receptor alpha (ER)-mediated transcription. In order to study the underlying mechanisms, we synthesized a peptide including the CaM binding site: ERalpha17p (P(295)-T(311)). This peptide inhibited ER-CaM association, unlike two analogs in which two amino acids required for CaM binding were substituted. Exposure of MCF-7 cells to ERalpha17p down regulated ER, stimulated ER-dependent transcription and enhanced the proliferation of ER-positive breast cancer cell lines. Interestingly, ERalpha17p analogs unable to bind to CaM induced similar responses, demonstrating that ERalpha17p-mediated effects are mainly relevant to mechanisms independent of ER-CaM dissociation. The P(295)-T(311) motif is indeed a platform for multiple post-translational modifications not necessarily CaM-dependent. The additional finding that deletion of the P(295)-T(311) sequence in ER produced a constitutive transcriptional activity revealed that this platform motif has autorepressive functions. With regard to cell function, association of CaM to ER would counteract this autorepression, leading thereby to enhanced ER-mediated transactivation.

Loss of antagonistic activity of tamoxifen by replacement of one N-methyl of its side chain by fluorinated residues.

Efforts to limit the metabolic alteration of the aminoalkyl side chain of tamoxifen by fluorination largely decrease its ER-mediated antagonistic properties in MCF-7 cells (i.e., ability to inhibit growth, to stabilize ER, and to modulate ERE and AP-1 transcriptional activity). This loss is associated with an enhancement of agonistic activity. Loss of interaction between Asp 351 and the nitrogen atom of tamoxifen provoked by the fluorination of its side chain may explain this property.

Synthesis, characterization and biological evaluation of 7 alpha-perfluoroalkylestradiol derivatives.

Linkage of a long CH(2 )side chain ('spacer') onto C-7 alpha of estradiol-17 beta (E(2)) does not abrogate the binding affinity of this hormone for its receptor. Our purpose was to assess whether the linkage of a CF(2 )side chain, which is more bulky and rigid, could also be accommodated by the estrogen receptor (ER). We describe here the synthesis of 7 alpha-perfluorohexylestradiol 7 by perfluoroalkylation of a key silylenolether 2 with FITS-6 (perfluorohexyl-phenyliodoniurn trifluoromethanesulfonate). 7 alpha-Trifluoromethylestradiol 10a was prepared as a fluorinated control compound by UV-light induced trifluoromethylation of 2 with Umemoto reagent (S-trifluoromethyldibenzo[b,d]thiophenium trifluoromethanesulfonate). Endocrine properties of these two E(2 )derivatives were tested on the MCF-7 breast cancer cell line. Our data reveal that rigidity of the side chain of 7 affected the association of its hormone moiety with the ER to the same extent as a long alkyl side chain. Rigidity also failed to abrogate estrogenicity, as demonstrated by the ability of 7 to enhance ERE-dependent transcription and cell growth. Compound retained the capacity of inducing down regulation of the receptor. Interestingly, no evidence of antiestrogenicity was recorded since this compound behaved like a weak estrogen, exerting a mitogenic effect at high concentration. Of note, control 10a displayed a higher binding affinity than 7 for ER and consequently acted like the latter, albeit with a higher efficiency. Selection of appropriate residues to be linked at the end of a long 7 alpha alkyl side chain is known to be essential for generating strong antiestrogenicity. One may hope that such a property may also hold for perfluorinated chains to produce antiestrogens with strong metabolic stability.

Pharmacological profile of 6,12-dihydro-3-methoxy-1-benzopyrano[3,4-b] [1,4]benzothiazin-6-one, a novel human estrogen receptor agonist.

Pharmacological studies were carried out to characterize further the endocrinological profile and the binding mode to the estrogen receptor (ER) of 6,12-dihydro-3-methoxy-1-benzopyrano[3,4-b][1,4]benzothiazin-6-one (1). Binding experiments were conducted with highly purified recombinant human estrogen receptors hERa and beta. Potent estrogenic activity of compound 1 was assessed by testing its ability to down-regulate ERs and to enhance estrogen receptor element (ERE)-dependent transcription. The latest step of our work dealt with the synthesis of the 9-fluorinated derivative 15 for ionic microscopy experiments to determine the intracellular localization of compound 1. Although 1 failed to compete with [3H]E2 for binding to both ER isoforms, evidence was reported that it interacted with hERalpha in MCF-7 cells (ER down-regulation/ERE-dependent luciferase induction). Hence, an appropriate conformation of the hormone binding domain, most probably conferred by co-regulators of ER, is required for the onset of an activity of the compound 1. Estrogenic activity was weak but on the order of magnitude of that of coumestrol (slightly weaker). The synthesis of the 9-methoxylated derivative 16 and its pharmacological evaluation led us to propose a binding mode of 1 on hERalpha. Compound 1 appears to interact with ERa mainly through interactions of its 3-methoxy substituent with the residue His-524 of the hormone binding domain.