Binding characteristics of estrogen receptor (ER) in Atlantic croaker (Micropogonias undulatus) testis: different affinity for estrogens and xenobiotics from that of hepatic ER.

An estrogen receptor (ER) was identified in cytosolic and nuclear fractions of the testis in a marine teleost, Atlantic croaker (Micropogonias undulatus). A single class of high affinity, low capacity, and displaceable binding sites was identified by saturation analysis, with a Kd of 0.40 nM in cytosolic extracts and a Kd of 0.33 nM in nuclear extracts. Competition studies demonstrated that the receptor was highly specific for estrogens (diethylstilbestrol > estradiol > estriol = estrone) and also bound several antiestrogens. Testosterone and 5alpha-dihydrotestosterone had much lower affinities for the receptor, whereas no displacement of specific binding occurred with 11-ketotestosterone or any of the C21 maturation-inducing steroids. A variety of xenoestrogens, including o,p'-dichlorodiphenyltrichloroethane (DDT), chlordecone (Kepone), nonylphenol, hydroxylated polychlorinated biphenyls (PCBs), and the mycotoxin zearalenone, bound to the receptor with relatively low binding affinities, 10(-3) to 10(-5) that of estradiol. A comparison of the binding affinities of various ligands for the testicular ER and the hepatic ER in this species revealed that the testicular ER was saturated at a lower [3H]estradiol concentration (1 nM vs. 4 nM). The binding affinities of several compounds, including testosterone and nafoxidine, exhibited marked differences for the two ERs; and most of the estrogens and xenoestrogens tested had higher binding affinities for the testicular receptor. Minor amounts of estradiol (0.12 ng/g tissue/h) were produced by testicular tissue fragments incubated in vitro, and estradiol was detected in male Atlantic croaker plasma. The identification of a testicular ER and evidence that estradiol is produced by the testes in croaker suggest that estrogens participate in the hormonal control of testicular function in teleosts.     

Synthesis and evaluation of 17α-triazolyl and 9α-cyano derivatives of estradiol

A variety of 17α-triazolyl and 9α-cyano derivatives of estradiol were prepared and evaluated for binding to human ERβ in both a TR-FRET assay, as well as ERβ and ERα agonism in cell-based functional assays. 9α-Cyanoestradiol (5) was nearly equipotent as estradiol as an agonist for both ERβ and ERα. The potency of the 17α-triazolylestradiol analogs is considerably more variable and depends on the nature of the 4-substituent of the triazole ring. While rigid protein docking simulations exhibited significant steric clashing, induced fit docking providing more protein flexibility revealed that the triazole linker of analogs 2d and 2e extends outside of the traditional ligand binding domain with the benzene ring located in the loop connecting helix 11 to helix 12.     

BC-spiro-estradiols. Synthesis and estrogen receptor binding affinity of four new estradiol isomers

The synthesis of four new isomers of estradiol in which the ring A to ring C planes are perpendicular to each other as a result of a spiro BC ring junction is described. Heterocyclic analogs and carbocyclic homologs of these compounds are also reported. Estrogen receptor binding studies show that the spiro compounds with the natural stereochemistry at C9 bind almost as strongly as estradiol but with greater β to α selectivity. These studies show that the estrogen receptors can readily accommodate isomers of estrogen with substantially different fixed shapes than the native ligand.     

Peroxidase-catalyzed displacement of tritium from regiospecifically labeled estradiol and 2-hydroxyestradiol

Estradiol and 2-hydroxyestradiol with 3H at different positions in rings A, B or D were incubated with lactoperoxidase without added H2O2 and their oxidative transformation was followed by transfer of 3H into 3H2O. With estradiol, 3H loss from different positions in the aromatic ring was almost equal and also occurred to a lesser extent from the alicyclic portion of the molecule. Glutathione had less effect on the formation of 3H2O for the aromatic ring of estradiol than from that of the catechol estrogen where it increased the yield 6-fold. The rate of 3H loss was also very much greater from tritiated 2-hydroxyestradiol than from estradiol and NADPH was inhibitory with both steroids. Conditions for the release of 3H from estradiol and 2-hydroxyestradiol by peroxidase as well as the effect of some biochemical inhibitors were also investigated. The possible contribution of peroxidative formation of 3H2O during the radiometric assay for catechol estrogen biosynthesis by tissue monooxygenases is discussed.     

New progesterone receptor antagonists: phosphorus-containing 11beta-aryl-substituted steroids

A new series of phosphorus-containing 11beta-aryl-substituted steroids have been synthesized in an eight-step sequence involving a palladium-catalyzed coupling reaction to introduce a phosphorus group onto the aromatic ring. The compounds were evaluated for progesterone receptor (PR) antagonist activity in a T47D cell-based assay and for glucocorticoid receptor (GR) antagonist activity in an A549 cell-based assay. The structure-activity relationships of these compounds are discussed. Selected compounds were tested in vivo in a rat complement C3 assay.     

Synthesis and receptor binding in trans-CD ring-fused A-CD estrogens: Comparison with the cis-fused isomers

Ligands which selectively activate only one of the estrogen receptors, ERα or ERβ, are current pharmaceutical targets. Previously, we have reported on substituted cis A-CD ligands in which the B-ring of the steroidal structure has been removed and cis refers the stereochemistry of the CD ring junction as compared to trans in estradiol. These compounds often showed good potency and selectivity for ERβ. Here we report the synthesis and binding affinities for a similar series of trans A-CD ligands, and compare them to the cis-series. Counterintuitively, trans A-CD ligands, which are structurally more closely related to the natural ligand estradiol, show weaker binding and less β-selectivity than their cis-counterparts.     

Biological activity and ligand binding mode to the progesterone receptor of A-homo analogues of progesterone

The biological activity of two seven-membered A-ring (A-homo) analogues of progesterone was evaluated by transactivation assays in Cos-1 cells and by determination of Bcl-x(L) expression levels in T47D cells. The results show that both compounds act as selective progesterone receptor (PR) agonists but lack mineralocorticoid receptor (MR) activity. Molecular modelling using semiempirical AM1 and ab initio HF/6-31G** calculations, showed that the A-ring of the A-homo steroids may adopt five different conformations, although only three correspond to low energy conformers. The low energy conformers of each analogue were introduced into the ligand binding pocket of the PR ligand binding domain (LBD) obtained from the PR LBD-progesterone crystal structure. The steroid binding mode was then analyzed using 10 ns of molecular dynamics (MD) simulation. The PR LBD-progesterone complex was also simulated as a control system. The MD results showed that both A-homo steroids have one conformer that may be properly recognized by the PR, in agreement with the observed progestagen activity. Moreover, the simulation revealed the importance of a water molecule in the formation of a hydrogen bonding network among specific receptor residues and the steroid A-ring carbonyl.     

Identification of novel estrogen receptor alpha antagonists

We have identified novel estrogen receptor alpha (ERalpha) antagonists using both cell-based and computer-based virtual screening strategies. A mammalian two-hybrid screen was used to select compounds that disrupt the interaction between the ERalpha ligand binding domain (LBD) and the coactivator SRC-3. A virtual screen was designed to select compounds that fit onto the LxxLL peptide-binding surface of the receptor, based on the X-ray crystal structure of the ERalpha LBD complexed with a LxxLL peptide. All selected compounds effectively inhibited 17-beta-estradiol induced coactivator recruitment with potency ranging from nano-molar to micromolar. However, in contrast to classical ER antagonists, these novel inhibitors poorly displace estradiol in the ER-ligand competition assay. Nuclear magnetic resonance (NMR) suggested direct binding of these compounds to the receptors pre-complexed with estradiol and further demonstrated that no estradiol displacement occurred. Partial proteolytic enzyme digestion revealed that, when compared with 17-beta-estradiol- and 4 hydroxy-tamoxifen (4-OHT) bound receptors, at least one of these compounds might induce a unique receptor conformation. These small molecules may represent new classes of ER antagonists, and may have the potential to provide an alternative for the current anti-estrogen therapy.     

Ligand-specific structural changes in the vitamin D receptor in solution

Vitamin D receptor (VDR) is a member of the nuclear hormone receptor superfamily. When bound to a variety of vitamin D analogues, VDR manifests a wide diversity of physiological actions. The molecular mechanism by which different vitamin D analogues cause specific responses is not understood. The published crystallographic structures of the ligand binding domain of VDR (VDR-LBD) complexed with ligands that have differential biological activities have exhibited identical protein conformations. Here we report that rat VDR-LBD (rVDR-LBD) in solution exhibits differential chemical shifts when bound to three ligands that cause diverse responses: the natural hormone, 1,25-dihydroxyvitamin D(3) [1,25(OH)?D?], a potent agonist analogue, 2-methylene-19-nor-(20S)-1,25-dihydroxyvitamin D? [2MD], and an antagonist, 2-methylene-(22E)-(24R)-25-carbobutoxy-26,27-cyclo-22-dehydro-1α,24-dihydroxy-19-norvitamin D? [OU-72]. Ligand-specific chemical shifts mapped not only to residues at or near the binding pocket but also to residues remote from the ligand binding site. The complexes of rVDR-LBD with native hormone and the potent agonist 2MD exhibited chemical shift differences in signals from helix-12, which is part of the AF2 transactivation domain that appears to play a role in the selective recruitment of coactivators. By contrast, formation of the complex of rVDR-LBD with the antagonist OU-72 led to disappearance of signals from residues in helices-11 and -12. We present evidence that disorder in this region of the receptor in the antagonist complex prevents the attachment of coactivators.     

Molecular structure of the rat vitamin D receptor ligand binding domain complexed with 2-carbon-substituted vitamin D3 hormone analogues and a LXXLL-containing coactivator peptide

We have determined the crystal structures of the ligand binding domain (LBD) of the rat vitamin D receptor in ternary complexes with a synthetic LXXLL-containing peptide and the following four ligands: 1alpha,25-dihydroxyvitamin D(3); 2-methylene-19-nor-(20S)-1alpha,25-dihydroxyvitamin D(3) (2MD); 1alpha-hydroxy-2-methylene-19-nor-(20S)-bishomopregnacalciferol (2MbisP), and 2alpha-methyl-19-nor-1alpha,25-dihydroxyvitamin D(3) (2AM20R). The conformation of the LBD is identical in each complex. Binding of the 2-carbon-modified analogues does not change the positions of the amino acids in the ligand binding site and has no effect on the interactions in the coactivator binding pocket. The CD ring of the superpotent analogue, 2MD, is tilted within the binding site relative to the other ligands in this study and to (20S)-1alpha,25-dihydroxyvitamin D(3) [Tocchini-Valentini et al. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 5491-5496]. The aliphatic side chain of 2MD follows a different path within the binding site; nevertheless, the 25-hydroxyl group at the end of the chain occupies the same position as that of the natural ligand, and the hydrogen bonds with histidines 301 and 393 are maintained. 2MbisP binds to the receptor despite the absence of the 25-hydroxyl group. A water molecule is observed between His 301 and His 393 in this structure, and it preserves the orientation of the histidines in the binding site. Although the alpha-chair conformer is highly favored in solution for the A ring of 2AM20R, the crystal structures demonstrate that this ring assumes the beta-chair conformation in all cases, and the 1alpha-hydroxyl group is equatorial. The peptide folds as a helix and is anchored through hydrogen bonds to a surface groove formed by helices 3, 4, and 12. Electrostatic and hydrophobic interactions between the peptide and the LBD stabilize the active receptor conformation. This stablization appears necessary for crystal growth.     

Estrogen and antiestrogen interaction with estrogen receptor of MCF-7 cells--relationship between processing and estrogenicity

Overnight preincubation of MCF-7 cells with 2 x 10(-10) M estradiol (E2) produces a dramatic reduction of their specific [3H]E2 binding capacity. Scatchard plot analysis revealed that this loss of estrogen receptor (ER) concentration, usually termed "processing", occurs without any significant modification of binding properties of the unprocessed receptors. Direct measurement of ER (ER-EIA from Abbott) gave residual receptor concentrations close to those established by binding assay indicating that processing involves the loss of at least one epitope other than the steroid binding site. Incubation with increasing amounts of E2 (0.1 to 5 x 10(-10) M) resulted in an increasing reduction of binding capacity indicating that the extent of processing is associated with the hormone concentration. Steroidal estrogens other than E2 as well as antiestrogens of the triphenylethylene category behaved similarly in this regard although the latter compounds usually acted only when at higher concentrations. The processing capacity of a large series of ligands was compared with the corresponding binding affinity for ER as assessed by classical competitive inhibition of [3H]E2 binding in both cytosol and whole cells. For steroidal estrogens, a large spectrum of concordant values was found which correlated with the known uterotrophic activity of the compounds. On the contrary, weak estrogen and antiestrogens of the triphenylethylene category displayed low processing capacities which were in the order of magnitude of the binding affinities established in whole cells; these values were considerably lower than the corresponding values measured in the cytosol. These observations are consistent with the concept that the capacity of a ligand to process ER is related to its agonistic activity. They also support our hypothesis (J. steroid Biochem. 25 (1986) 677-682) that assessment of the ability of a ligand to inhibit the binding of [3H]E2 in whole cells provides an estimate of its agonistic activity, an estimate which can not be established in the corresponding cytosol assay.     

Structural and functional characterization of solute binding proteins for aromatic compounds derived from lignin: p‐Coumaric acid and related aromatic acids

Lignin comprises 15–25% of plant biomass and represents a major environmental carbon source for utilization by soil microorganisms. Access to this energy resource requires the action of fungal and bacterial enzymes to break down the lignin polymer into a complex assortment of aromatic compounds that can be transported into the cells. To improve our understanding of the utilization of lignin by microorganisms, we characterized the molecular properties of solute binding proteins of ATP‐binding cassette transporter proteins that interact with these compounds. A combination of functional screens and structural studies characterized the binding specificity of the solute binding proteins for aromatic compounds derived from lignin such as p‐coumarate, 3‐phenylpropionic acid and compounds with more complex ring substitutions. A ligand screen based on thermal stabilization identified several binding protein clusters that exhibit preferences based on the size or number of aromatic ring substituents. Multiple X‐ray crystal structures of protein–ligand complexes for these clusters identified the molecular basis of the binding specificity for the lignin‐derived aromatic compounds. The screens and structural data provide new functional assignments for these solute‐binding proteins which can be used to infer their transport specificity. This knowledge of the functional roles and molecular binding specificity of these proteins will support the identification of the specific enzymes and regulatory proteins of peripheral pathways that funnel these compounds to central metabolic pathways and will improve the predictive power of sequence‐based functional annotation methods for this family of proteins.Proteins 2013; 81:1709–1726. © 2013 Wiley Periodicals, Inc.     

Differential estrogen receptor binding of estrogenic substances: a species comparison

The study investigated the ability of 34 natural and synthetic chemicals to compete with [3H]17beta-estradiol (E2) for binding to bacterially expressed glutathione-S-transferase (GST)-estrogen receptors (ER) fusion proteins from five different species. Fusion proteins consisted of the ER D, E and F domains of human alpha (GST-hERalphadef), mouse alpha (GST-mERalphadef), chicken (GST-cERdef), green anole (GST-aERdef) and rainbow trout ERs (GST-rtERdef). All five fusion proteins displayed high affinity for E2 with dissociation constants (K(d)) ranging from 0.3 to 0.9 nM. Although, the fusion proteins exhibited similar binding preferences and binding affinities for many of the chemicals, several differences were observed. For example, alpha-zearalenol bound with greater affinity to GST-rtERdef than E2, which was in contrast to other GST-ERdef fusion proteins examined. Coumestrol, genistein and naringenin bound with higher affinity to the GST-aERdef, than to the other GST-ERdef fusion proteins. Many of the industrial chemicals examined preferentially bound to GST-rtERdef. Bisphenol A, 4-t-octylphenol and o,p' DDT bound with approximately a ten-fold greater affinity to GST-rtERdef than to other GST-ERdefs. Methoxychlor, p,p'-DDT, o,p'-DDE, p,p'-DDE, alpha-endosulfan and dieldrin weakly bound to the ERs from the human, mouse, chicken and green anole. In contrast, these compounds completely displaced [3H]E2 from GST-rtERdef. These results demonstrate that ERs from different species exhibit differential ligand preferences and relative binding affinities for estrogenic compounds and that these differences may be due to the variability in the amino acid sequence within their respective ER ligand binding domains.     

Identification of immunoassayable estrogen receptor lacking hormone binding ability in tamoxifen-treated rat uterus

Using two different monoclonal antibodies to human estrogen receptor (ER), the enzymeimmunoassay was performed. The values of ER contents in human breast cancer and untreated rat uteri obtained by this procedure were correlated well with those by [3H] estradiol binding assay. When estradiol was injected to immature rats, the enzymeimmunoassay showed the uterine receptor dynamic pattern similar to those analyzed by exchange assays. In contrast, tamoxifen administration induced the immunoassayable but nonsteroid binding form of ER. This ER-like antigen was the heat-labile molecule with the sedimentation constant of 7 S while ER in untreated rat uterine cytosol sedimented at 9 S. These results suggest the presence of unique molecular state of ER induced by tamoxifen.     

Synthesis and biologic activities of 11 beta-substituted estradiol as potential antiestrogens

The effect of attachment of a dimethylaminoethoxy or a dimethylaminopropoxy group at the 11 beta-position of estradiol (E2) on its relative binding affinity (RBA) to estrogen receptor (ER) and intrinsic biologic activity is described. The binding of 11 beta-[2-(N,N-dimethylamino) ethoxy]estra-1,3,5(10)-triene-3,17 beta-diol (4) and 11 beta-[3-(N,N- dimethylamino)propoxy]estra-1,3,5(10)-triene-3,17 beta-diol (5) to the ER from immature rat uterine tissue was measured relative to that of [3H]E2 by a competitive binding assay. It was found that the 11 beta-substituted E2 analogs have considerably lower RBA to ER than the corresponding parent compound. The intrinsic activity of compounds 4 and 5 were studied in terms of uterotrophic and antiuterotrophic activity. It was found that the uterotrophic activity of these compounds was drastically reduced compared with E2. However, no antiuterotrophic activity was observed in these compounds at dosages ranging from 1 to 100 micrograms/rat/d.