Synthesis and functional analysis of novel bivalent estrogens

Abstract

The steroid hormone estrogen plays a critical role in female development and homeostasis. Estrogen mediates its effects through binding and activation of specific estrogen receptors alpha (ERalpha) and beta (ERbeta), members of the steroid/nuclear receptor family of ligand-induced transcription factors. Due to their intimate roles in genomic and nongenomic signaling pathways, these hormones and their receptors have been also implicated in the pathologies of a variety of cancers and metabolic disorders, and have been the target of large therapeutic development efforts. The binding of estrogen to its respective receptors initiates a cascade of events that include receptor dimerization, nuclear localization, DNA binding and recruitment of co-regulatory protein complexes. In this manuscript, we investigate the potential for manipulating steroid receptor gene expression activity through the development of bivalent steroid hormones that are predicted to facilitate hormone receptor dimerization events. Data are presented for the development and testing of novel estrogen dimers, linked through their C-17 moiety, that can activate estrogen receptor alpha (ERalpha)-mediated transcription events with efficacy and potency equal to or greater than that of ERalpha's cognate ligand, 17beta-estradiol. These bivalent estrogen structures open the door to the development of a variety of steroid therapeutics that could dramatically impact future drug development in this area.

Fig.1

Monovalent and bivalent 17-oxime esters. Presented are the 6 monovalent (1, 2, 3, 4a, 4b and 4c) and 4 bivalent (5a-d) 17-oxime esters evaluated for their stability, specificity and ability to modulate ERα-mediated gene expression parameters.

Fig.2

Monovalent and bivalent Girard P-based oxime derivatives. Presented are the 2 monovalent (6 and 7) and 3 bivalent (8a, 8b, 8c) estrogens evaluated for their ability to modulate ERα-mediated gene expression events.

Fig.3

Monovalent and bivalent estrogens modified at their C-3 and C17 moieties analyzed for their ERα agonist activities. Compound number identifiers are as designated in Fig.1. ERα-mediated transcription was reconstituted in an HEK cell line through the co-transfection of an ERα mammalian expression plasmid and an ERE-driven luciferase reporter plasmid. As controls, parallel analyses were run to evaluate solvent impact as well as background attributable to the ERE reporter vector (ERE). ERα’s cognate ligand 17β-estradiol (E2) along with a series of monovalent and bivalent estrogens modified at their C-3 and C-17 positions (A) were analyzed (B) in a 96-well plate format. For normalization of data, included in all transfections was a mammalian expression construct for β-galactosidase (β-Gal). All compounds were analyzed at 100 nM, and data from 6 wells for each ligand was averaged and divided by the average β-Gal rate for these wells. Error bars represent the SEM for the 6 wells.

Fig.4

Dose Response analysis of the bivalent estrogens 5a and 5b. Utilizing the ERα-specific HEK co-transfection analysis described in Fig. 3, the bivalent estrogens 5a and 5b, along with the monovalent estrogen 4c and 17β-estradiol (E2), were analyzed at concentrations varying from 0.1 nM to 100 nM. Data are expressed as averaged reporter gene luciferase values for sextuplet wells divided by the average β-Gal rate for these wells.

Fig. 5

Efficacy comparison of the bivalent estrogen 5b to its monovalent oxime derivative 4c. Utilizing the ERα-specific HEK co-transfection analysis described in Fig. 3, the bivalent estrogen 5b and its monovalent oxime derivative 4c were analyzed in molar equivalents of estrogen for their ability to stimulate ERα-specific reporter gene expression. Data are expressed as averaged reporter gene luciferase values for sextuplet wells divided by the average β-Gal rate for these wells.

Fig. 6

Stability evaluation of the bivalent estrogen 5b. HEK cells, aliquoted at 5 ×10⁵ cells per well of a 96-well plate, were treated for 36 hours in the presence and absence of 100 nM bivalent estrogen 5b and analyzed by thin layer chromatography using 1:2 ethyl acetate-hexanes with 10% phosphomolybdic acid spray for detection. Lane 1: bivalent estrogen 5b + oxime 4a; lane 2: bivalent estrogen 5b; lane 3: oxime 4a; lane 4: extract from 25 wells; lane 5: extract from 10 wells; lane 6: extract from 5 wells; lane 7: extract from 1 well; lane 8: bivalent estrogen 5b; lane 9: bivalent estrogen 5b + oxime 4a.

Fig. 7

Analysis of the Girard-based bivalent estragens 8a, 8b, and 8c for their ERα agonist activity. Utilizing the ERα-specific HEK co-transfection analysis described in Fig. 3, the Girard-based bivalent estrogens 8a, 8b, and 8c along with 17β-estradiol (E₂), were analyzed at concentrations varying from 0.1 nM to 100 nM. Data are expressed as averaged reporter gene luciferase values for sextuplet wells divided by the average β-Gal rate for these wells.

Fig. 8

Specificity analysis of the Girard-based bivalent estrogen 8b. HEK cells were reconstituted with receptor/reporter systems for ERα, human progesterone receptor beta (PR), human androgen receptor (AR), and human glucocorticoid receptor (GR) through co-transfection of specific receptor and reporter plasmids as described in Fig. 3. Assays were performed in the presence of solvent, 8b and receptor-specific ligands [ER: 17β-estradiol (E₂); AR: dihydrotestosterone (Test.); GR: dexamethasone (Dex); PR: progesterone (Prog.)]. For normalization of data, included in all transfections was a mammalian expression construct for β-Gal. All compounds were analyzed at 100 nM, and data from 6 wells for each ligand was averaged and divided by the average β-Gal rate for these wells. Error bars represent the SEM for the 6 wells.