2,5-Diphenylfuran-based pure antiestrogens with selectivity for the estrogen receptor alpha

The estrogen receptor alpha (ERalpha) is understood to play an important role in the progression of breast cancer. Therefore, pure antiestrogens with a preference for this receptor form are of interest as new agents for the treatment of this malignancy. Several chemical structures with selective binding affinity for ERalpha have been identified and might be useful for the synthesis of ERalpha-selective pure antiestrogens. In this study we applied the 2,5-diphenylfuran system which is closely related to the triphenylfurans described by others. Various side chains with amino and/or sulfur functions were linked to C3 to convert the furans to estrogen antagonists without residual estrogenic activity. The degree of alpha-selectivity which ranges from 2.5- to 236-fold is strongly influenced by the alkyl group at C4. Antiestrogenic potency was determined in MCF-7/2a breast cancer cells stably transfected with a luciferase gene under the control of an ERE. The 2,5-bis(4-hydroxyphenyl)furan with an ethyl substituent and a 6-[N-methyl-N-(3-pentylthiopropyl)amino]hexyl side chain exerted the strongest antiestrogenic effect in this series with an IC(50) value of 50 nM in cells stimulated with 1 nM estradiol. The RBA values of this derivative were 18% (ERalpha) and 3.4% (ERbeta) of estradiol, respectively. It inhibited the growth of wild-type MCF-7 cells with an IC(50) value of 22 nM. The data show that the 2,5-diphenylfuran system is appropriate for the development of pure antiestrogens with preference for ERalpha.     

Benzopyrans as selective estrogen receptor beta agonists (SERBAs). Part 4: functionalization of the benzopyran A-ring

Benzopyrans are selective estrogen receptor (ER) beta agonists (SERBAs), which bind the ER receptor subtypes alpha and beta in opposite orientations. We have used structure based drug design to show that this unique phenomena can be exploited via substitution at the 8-position of the benzopyran A-ring to disrupt binding to ERalpha, thus improving ERbeta subtype selectivity. X-ray cocrystal structures with ERalpha and ERbeta are supportive of this approach to improve selectivity in this structural class.     

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.     

Aza analogues of equol: novel ligands for estrogen receptor beta

3-Aryl-tetrahydroquinolines, aza analogues of equol, are synthesized and evaluated for their binding properties to the estrogen receptors ERalpha and ERbeta. Several of these compounds exhibited binding selectivity for ER similar to that of genistein. Compounds 8c and 8d were found to have dual actions: antagonists for ERalpha and agonists for ERbeta in a yeast two-hybrid assay. These compounds have no estrogenic effects on the uterus and bone in vivo.     

Ligands differentially modulate the protein interactions of the human estrogen receptors alpha and beta

The interactions of human estrogen receptor subtypes ERalpha and ERbeta with DNA and a 210 amino acid residue fragment of the coactivator protein SRC-1 bearing three nuclear receptor interaction motifs were investigated quantitatively using fluorescence anisotropy in the presence of agonist and antagonist ligands. ERalpha and ERbeta were found to bind in a similar manner to DNA, and both salt and temperature affected the affinity and/or stoichiometry of these interactions. The agonist ligands estradiol, estrone and estriol did not modify the binding of ERalpha to the fluorescein-labeled target estrogen response element. However, in the case of ERbeta, these ligands led to the formation of some higher-order protein-DNA complexes and a small decrease in affinity. The partial agonist 4-hydroxytamoxifen had little effect on either ER subtype, whereas the pure antagonist ICI 182,780 led to the cooperative formation of protein-DNA complexes of higher order than dimer, as further demonstrated by competition experiments and gel mobility-shift assays. In addition to DNA binding, the interaction of both ER subtypes with the Alexa488-labeled SRC-1 coactivator fragment was investigated by fluorescence anisotropy. The agonist ligands estrone, estradiol, estriol, genistein and ethynyl estradiol exhibited distinct capacities for inducing the recruitment of SRC-1 that were not correlated with their affinity for the receptor. Moreover, estrone and genistein exhibited subtype specificity in that they induced SRC-1 recruitment to ERbeta with much higher efficiency than in the case of ERalpha. The differential coactivator recruitment capacities of the ER agonists and their receptor subtype coactivator recruitment specificity may be linked to the molecular structure of the agonists with respect to their interactions with a specific histidine residue located at the back of the ligand-binding pocket. Altogether, these quantitative in vitro studies of ER interactions reveal the complex energetic and stoichiometric consequences of changes in the chemical structures of these proteins and their ligands.     

Dissecting physiological roles of estrogen receptor alpha and beta with potent selective ligands from structure-based design

The distinct roles of the two estrogen receptor (ER) isotypes, ERalpha and ERbeta, in mediating the physiological responses to estrogens are not completely understood. Although knockout animal experiments have been aiding to gain insight into estrogen signaling, additional information on the function of ERalpha and ERbeta will be provided by the application of isotype-selective ER agonists. Based on the crystal structure of the ERalpha ligand binding domain and a homology model of the ERbeta-ligand binding domain, we have designed steroidal ligands that exploit the differences in size and flexibility of the two ligand binding cavities. Compounds predicted to bind preferentially to either ERalpha or ERbeta were synthesized and tested in vitro using radio-ligand competition and transactivation assays. This approach directly led to highly ER isotype-selective (approximately 200-fold) and potent ligands. To unravel physiological roles of the two receptors, in vivo experiments with rats were conducted using the ERalpha- and ERbeta-selective agonists in comparison to 17beta-estradiol. The ERalpha agonist induced uterine growth, caused bone-protective effects, reduced LH and FSH plasma levels, and increased angiotensin I, whereas the ERbeta agonist did not at all or only at high doses lead to such effects, despite high plasma levels. It can thus be concluded that estrogen effects on the uterus, pituitary, bone, and liver are primarily mediated via ERalpha. Simultaneous administration of the ERalpha and ERbeta ligand did not lead to an attenuation of ERalpha-mediated effects on the uterus, pituitary, and liver parameters.     

ERβ Binds N-CoR in the Presence of Estrogens via an LXXLL-like Motif in the N-CoR C-terminus

Nuclear receptors (NRs) usually bind the corepressors N-CoR and SMRT in the absence of ligand or in the presence of antagonists. Agonist binding leads to corepressor release and recruitment of coactivators. Here, we report that estrogen receptor beta (ERbeta) binds N-CoR and SMRT in the presence of agonists, but not antagonists, in vitro and in vivo. This ligand preference differs from that of ERalpha interactions with corepressors, which are inhibited by estradiol, and resembles that of ERbeta interactions with coactivators. ERbeta /N-CoR interactions involve ERbeta AF-2, which also mediates coactivator recognition. Moreover, ERbeta recognizes a sequence (PLTIRML) in the N-CoR C-terminus that resembles coactivator LXXLL motifs. Inhibition of histone deacetylase activity specifically potentiates ERbeta LBD activity, suggesting that corepressors restrict the activity of AF-2. We conclude that the ER isoforms show completely distinct modes of interaction with a physiologically important corepressor and discuss our results in terms of ER isoform specificity in vivo.     

Benzopyrans as selective estrogen receptor beta agonists (SERBAs). Part 3: synthesis of cyclopentanone and cyclohexanone intermediates for C-ring modification

Benzopyrans are selective estrogen receptor (ER) beta agonists (SERBAs), which bind the ER subtypes alpha and beta in opposite orientations. Here we describe the syntheses of cyclopentanone and cyclohexanone intermediates for SAR studies of the C-ring on the benzopyran scaffold. Modification of the C-ring disrupts binding to ERalpha, thus improving ERbeta selectivity up to 100-fold. X-ray cocrystal structures confirm previously observed binding modes.     

2-Amino-4,6-diarylpyridines as novel ligands for the estrogen receptor.

We have prepared a novel series of 2-amino-4,6-diarylpyridines that function as ligands of estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta). These compounds bind to both ERalpha and ERbeta with a modest selectivity for the alpha subtype. The most potent of these analogues, compound 19, has a K(i)=20nM at ERalpha. These molecules represent a novel template for designing potentially useful ligands for the estrogen receptor.     

Identification of ligands with bicyclic scaffolds provides insights into mechanisms of estrogen receptor subtype selectivity

Estrogen receptors alpha (ERalpha) and beta (ERbeta) have distinct functions and differential expression in certain tissues. These differences have stimulated the search for subtype-selective ligands. Therapeutically, such ligands offer the potential to target specific tissues or pathways regulated by one receptor subtype without affecting the other. As reagents, they can be utilized to probe the physiological functions of the ER subtypes to provide information complementary to that obtained from knock-out animals. A fluorescence resonance energy transfer-based assay was used to screen a 10,000-compound chemical library for ER agonists. From the screen, we identified a family of ERbeta-selective agonists whose members contain bulky oxabicyclic scaffolds in place of the planar scaffolds common to most ER ligands. These agonists are 10-50-fold selective for ERbeta in competitive binding assays and up to 60-fold selective in transactivation assays. The weak uterotrophic activity of these ligands in immature rats and their ability to stimulate expression of an ERbeta regulated gene in human U2OS osteosarcoma cells provides more physiological evidence of their ERbeta-selective nature. To provide insight into the molecular mechanisms of their activity and selectivity, we determined the crystal structures of the ERalpha ligand-binding domain (LBD) and a peptide from the glucocorticoid receptor-interacting protein 1 (GRIP1) coactivator complexed with the ligands OBCP-3M, OBCP-2M, and OBCP-1M. These structures illustrate how the bicyclic scaffolds of these ligands are accommodated in the flexible ligand-binding pocket of ER. A comparison of these structures with existing ER structures suggests that the ERbeta selectivity of OBCP ligands can be attributed to a combination of their interactions with Met-336 in ERbeta and Met-421 in ERalpha. These bicyclic ligands show promise as lead compounds that can target ERbeta. In addition, our understanding of the molecular determinants of their subtype selectivity provides a useful starting point for developing other ER modulators belonging to this relatively new structural class.