CoMSIA and docking study of rhenium based estrogen receptor ligand analogs

OPLS all atom force field parameters were developed in order to model a diverse set of novel rhenium based estrogen receptor ligands whose relative binding affinities (RBA) to the estrogen receptor alpha isoform (ERalpha) with respect to 17beta-estradiol were available. The binding properties of these novel rhenium based organometallic complexes were studied with a combination of Comparative Molecular Similarity Indices Analysis (CoMSIA) and docking. A total of 29 estrogen receptor ligands consisting of 11 rhenium complexes and 18 organic ligands were docked inside the ligand-binding domain (LBD) of ERalpha utilizing the program Gold. The top ranked pose was used to construct CoMSIA models from a training set of 22 of the estrogen receptor ligands which were selected at random. In addition scoring functions from the docking runs and the polar volume (PV) were also studied to investigate their ability to predict RBA ERalpha. A partial least-squares analysis consisting of the CoMSIA steric, electrostatic and hydrophobic indices together with the polar volume proved sufficiently predictive having a correlation coefficient, r(2), of 0.94 and a cross-validated correlation coefficient, q(2), utilizing the leave-one-out method of 0.68. Analysis of the scoring functions from Gold showed particularly poor correlation to RBA ERalpha which did not improve when the rhenium complexes were extracted to leave the organic ligands. The combined CoMSIA and polar volume model ranked correctly the ligands in order of increasing RBA ERalpha, illustrating the utility of this method as a prescreening tool in the development of novel rhenium based estrogen receptor ligands.     

Control of estrogen receptor ligand binding by Hsp90

The molecular chaperone Hsp90 interacts with unliganded steroid hormone receptors and regulates their activity. We have analyzed the function of yeast and mammalian Hsp90 in regulating the ability of the human estrogen receptor (ER) to bind ligands in vivo and in vitro. Using the yeast system, we show that the ER expressed in several different hsp82 mutant strains binds reduced amounts of the synthetic estrogen diethylstilbestrol compared to the wild type. This defect in hormone binding occurs without any significant change in the steady state levels of ER protein. To analyze the role of mammalian Hsp90, we synthesized the human ER in rabbit reticulocyte lysates containing geldanamycin, an Hsp90 inhibitor. At low concentrations of geldanamycin we observed reduced levels of hormone binding by the ER. At higher concentrations, we found reduced synthesis of the receptor. These data indicate that Hsp90 functions to maintain the ER in a high affinity hormone-binding conformation.     

Design,synthesis and biological evaluation of novel indole-xanthendione hybrids as selective estrogen receptor modulators

Ground breaking clinical therapeutic advances in the treatment of breast cancer (BC) is the introduction of selective estrogen receptor modulators (SERMs). We have expeditiously designed and synthesized indole-xanthendione hybrids by coalescing the indole nucleus with xanthendione. All the compounds were first screened for anti-proliferative activity, cytotoxicity and ER-α binding affinity by utilizing ER-α dominant T47D BC cell lines, PBMCs and ER-α competitor assay kit. From this study, two representative compounds 6e and 6f showing most promising activity were advanced for gene expression studies for targeting ER-α. Cell imaging experiment undoubtedly indicate that both the compounds were able to cross cellular bio membrane and accumulate thus instigating cytotoxicity. RT-PCR and Western blotting experiments further strengthened that both compounds altered the expression of mRNA and receptor protein of ER-α, thereby forestalling downstream transactivation and signalling pathway in T47D cells line. Structural investigation from induced fit simulation study suggest that indole moiety of the compounds 6e and 6f helps in the anchoring of the xanthendione moiety in the hydrophobic region of the cavity thus enabling the compound to bind in antagonistic conformation similar to bazedoxifene by extensive hydrogen bonding and Van der Waals forces. All these finding collectively imply that compound 6e and 6f represents a novel potent ER-α antagonist and in the development of SERMs for the management of BC.     

Acquired resistance to selective estrogen receptor modulators (SERMs) in clinical practice (tamoxifen & raloxifene) by selection pressure in breast cancer cell populations

Tamoxifen, a pioneering selective estrogen receptor modulator (SERM), has long been a therapeutic choice for all stages of estrogen receptor (ER)-positive breast cancer. The clinical application of long-term adjuvant antihormone therapy for the breast cancer has significantly improved breast cancer survival. However, acquired resistance to SERM remains a significant challenge in breast cancer treatment. The evolution of acquired resistance to SERMs treatment was primarily discovered using MCF-7 tumors transplanted in athymic mice to mimic years of adjuvant treatment in patients. Acquired resistance to tamoxifen is unique because the growth of resistant tumors is dependent on SERMs. It appears that acquired resistance to SERM is initially able to utilize either E₂ or a SERM as the growth stimulus in the SERM-resistant breast tumors. Mechanistic studies reveal that SERMs continuously suppress nuclear ER-target genes even during resistance, whereas they function as agonists to activate multiple membrane-associated molecules to promote cell growth. Laboratory observations in vivo further show that three phases of acquired SERM-resistance exists, depending on the length of SERMs exposure. Tumors with Phase I resistance are stimulated by both SERMs and estrogen. Tumors with Phase II resistance are stimulated by SERMs, but are inhibited by estrogen due to apoptosis. The laboratory models suggest a new treatment strategy, in which limited-duration, low-dose estrogen can be used to purge Phase II-resistant breast cancer cells. This discovery provides an invaluable insight into the evolution of drug resistance to SERMs, and this knowledge is now being used to justify clinical trials of estrogen therapy following long-term antihormone therapy. All of these results suggest that cell populations that have acquired resistance are in constant evolution depending upon selection pressure. The limited availability of growth stimuli in any new environment enhances population plasticity in the trial and error search for survival.     

Sensitive detection of estradiol based on ligand binding domain of estrogen receptor and gold nanoparticles

With increasing concerns of estrogenic effects of endocrine disrupting compounds, the development of simple detection assay for these compounds is an ongoing need. Herein, a simple, rapid, and highly sensitive assay for estradiol (E2) detection was developed using the ligand binding domain of estrogen receptor α (LBD-ERα), the receptor interacting domain of steroid receptor co-activator 1 (RID-SRC1), and gold nanoparticles (AuNPs). The colloidal AuNPs could be stabilized against a salt-induced aggregation by adding LBD-ERα protein. However, with the presence of E2, the specific binding of LBD-ERα protein and E2 led to a salt-induced aggregation of AuNPs as seeing from a color change from red to blue. This developed assay exhibited a high sensitivity for E2 detection with the limit of detection (LOD) of 2.62 × 10⁻¹⁴ M. When the RID-SRC1 protein was included, the detection sensitivity was increased, which the LOD for E2 was at 1.20 × 10⁻¹⁵ M. This assay was specific for a detection of E2 but not progesterone, the negative control ligand. Results of this work clearly showed the efficiency of developed assay for E2 detection, which possibly further developed for an onsite monitoring of E2.     

Toward the active conformations of rhodopsin and the beta2-adrenergic receptor

Using sets of experimental distance restraints, which characterize active or inactive receptor conformations, and the X-ray crystal structure of the inactive form of bovine rhodopsin as a starting point, we have constructed models of both the active and inactive forms of rhodopsin and the beta2-adrenergic G-protein coupled receptors (GPCRs). The distance restraints were obtained from published data for site-directed crosslinking, engineered zinc binding, site-directed spin-labeling, IR spectroscopy, and cysteine accessibility studies conducted on class A GPCRs. Molecular dynamics simulations in the presence of either "active" or "inactive" restraints were used to generate two distinguishable receptor models. The process for generating the inactive and active models was validated by the hit rates, yields, and enrichment factors determined for the selection of antagonists in the inactive model and for the selection of agonists in the active model from a set of nonadrenergic GPCR drug-like ligands in a virtual screen using ligand docking software. The simulation results provide new insights into the relationships observed between selected biochemical data, the crystal structure of rhodopsin, and the structural rearrangements that occur during activation.     

雌激素受体亚型及其配体调节基因转录机制的研究

本文综述雌激素受体亚型（ERα和ERβ）的结构,功能,组织分布,生理作用及雌激素受体配体调节基因转录的机制,目的是深入系统地了解植物雌激素和选择性雌激素受体调节剂的作用路径及其组织特异性的发生机制,最终为提高雌激素类药物的选择性,优化以临床为基础的药物设计提供一条较为系统的思路。结果表明,ERα和ERβ对不同雌激素类化合物产生不同应答,配体的结构不同,调节基因转录的路径不同和募集的辅调节蛋白的不同是雌激素受体两种亚型组织特异性激活或抑制的主要原因。     

Comparative analysis of estrogen receptor gene polymorphisms in apes

Polymorphic microsatellite repeats in the promoter region of estrogen receptor α gene (ESRα and the intron 6 region of estrogen receptor β gene (ESRβ) have been reported in human populations. To examine the evolutional state of both repeats, we surveyed the corresponding regions in DNA sequences from the following great apes and gibbons: 56 chimpanzees, 3 bonobos, 16 gorillas, 20 orangutans and 60 gibbons (four species: 17 of Hylobates agilis, 11 of H. lar, 15 of H. muelleri, and 17 of H. syndactylus). In the corresponding region of the TA repeat of human ESRα, chimpanzees and bonobos had two motifs in the repeat tract, (TA)_7–9 and (CA)_4–6. Gorillas had the (TA)_9–10 repeat tracts and orangutans had monomorphic (TA)₇ repeats. Although all great apes maintained the TA expansion, all gibbon sequences contained (TA)₂, implying that the CA dinucleotide expansion arose in the ancestor of chimpanzees and bonobos. The nucleotide sequences of ESRβ showed a very complex repeat pattern in apes. The human sequences had a non-variable preceding sequence at (CA) _n , (GA)₂(TA)₈(CA)₄(TA). In apes that region included {(TA) _n (CA) _n } _n . Gibbon sequences included (TATG) _n and (TATC) _n and no regular construction was observed. A deletion event in the reverse primer site seems to have occurred in the orangutan lineage. In addition, a great diversity of allele length was detected in each gibbon species.     

Replacement of lysine-181 by aspartic acid in the third transmembrane region of endothelin type B receptor reduces its affinity to endothelin peptides and sarafotoxin 6c without affecting G protein coupling.

A conserved aspartic acid residue in the third transmembrane region of many of the G protein-coupled receptors has been shown to play a role in ligand binding. In the case of endothelin receptors, however, a lysine residue replaces this conserved aspartic acid residue. To access the importance of this residue in ligand binding, we have replaced it with an aspartic acid in the rat endothelin type B (ETb) receptor by PCR mediated mutagenesis. The binding characteristics and functional properties of both the wild type and mutant receptors were determined in COS-7 cells transiently expressing the cloned receptor cDNAs. Using 125I-ET-1 as the radioactive peptide ligand in displacement binding studies, the wild type receptor displayed a typical non-isopeptide-selective binding profile with similar IC50 values (0.2-0.6 nM) for all three endothelin peptides (ET-1, ET-2, and ET-3) and sarafotoxin 6c (SRTX 6c). Interestingly, the mutant receptor showed an increase in IC50 values for ET-1 (5 nM), ET-2 (27 nM), and ET-3 (127 nM) but displayed a much larger increase in IC50 value for SRTX 6c (> 10 uM). The lysine mutant receptor still elicited full inositol phosphate (IP) turnover responses in the presence of saturating concentrations of endothelins (10 nM of ET-1, 100 nM of ET-2, or 1 uM of ET-3), indicating that the mutation (K181D) did not affect the coupling of mutant receptor to the appropriate G protein. These results demonstrate that lysine-181 on the receptor is important for binding ET peptides; however, it is required for binding the ETb selective agonist-SRTX 6c.     

Ablation of estrogen receptor alpha (ERalpha) prevents upregulation of POMC by leptin and insulin

Diabetic Akita male mice are more hyperphagic because of downregulation of proopiomelanocortin (POMC) caused by hypoleptinemia. We investigated the role of estrogen receptor α (ERα) in the regulation of the hypothalamic POMC in females. ERaKOAkt mice consumed 30% greater food (g/3 weeks) than the Akita diabetic controls. Ovariectomized diabetic (AFO) and nondiabetic (B6FO) mice had significantly lower food intake and elevated serum leptin levels. ERaKOAkt and ERaKO mice also increased serum leptin concentrations, while hypoinsulinemia was observed in ERaKOAkt and hyperinsulinemia in ERaKO mice. RT-PCR showed a significant attenuation of POMC expression in both ERaKOAkt and ERaKO mice, irrespective of the elevated leptin serum levels or hyperinsulinemia, while elevated serum leptin levels in AFO and B6FO mice upregulated POMC gene expression. These results indicate that ERα plays an essential role in leptin- and insulin-stimulated upregulation of the POMC gene. This action of ERα is likely mediated in a ligand-independent manner.     

Conformational free energy of alkylsilanes by nonequilibrium-pulling Monte Carlo simulation

The stationary phase in supercritical fluid chromatography includes alkylsilanes, bearing typically 18-carbon alkane chains, bonded to silica. The silanes are in contact with supercritical carbon dioxide. Interaction of the stationary phase with analytes from the mobile phase depends on conformation of the silanes, whether they form a collapsed layer between the silica and the carbon dioxide or are extended into the carbon dioxide. Although equilibrium conformation of alkylsilanes can be determined by equilibrium Monte Carlo (MC) simulation, that is hampered by slow relaxation of the chains. An alternative is to pull alkylsilanes from collapsed to extended conformations, then calculate free energy change from the Jarzynski equality. This work compares conformational results from equilibrium MC simulation to free energies from nonequilibrium pulling simulations. Because both equilibrium and nonequilibrium simulations are faster for shorter silanes, this work also compares results from 8-carbon and 18-carbon silanes. Free energies from nonequilibrium pulling predict that alkylsilanes tend to bend over and form a layer between silica and carbon dioxide. Results from equilibrium simulations are qualitatively consistent with results from nonequilibrium pulling. Longer-chain silanes have greater tendency to extend slightly into the carbon dioxide.     

Partial purification of the nuclear estrogen receptor from chicken liver by affinity chromatography

The crude nuclear extract from the liver of estrogenized chickens contains 0.3–1 pmol/g tissue of the estrogen receptor. The receptor has been partially purified by ammonium sulphate precipitation and affinity chromatography on 17β-estradiol-17-hemisuccinyl-ovalbumin-Sepharose 4B. A 12% pure receptor preparation (2700-fold purification) with a yield of 17% could be obtained. The partially purified receptor has retained most properties which it displayed in cruder preparations, e.g. the dissociation constant of 10^?9?10^?10 M, the hormone specificity and the sedimentation coefficient of 3.9 S. The size (Stokes radius, 2.9 nm; molecular weight, 49 000) and the asymetry (f/f₀ = 1.10) of the receptor molecule, however, appear slightly reduced after the purification.     

Design and synthesis of estrogen receptor ligands with a 4-heterocycle-4-phenylheptane skeleton

The estrogen receptor (ER), a member of the nuclear receptor (NR) family, is involved in the regulation of physiological effects such as reproduction and bone homeostasis. Approximately 70% of human breast cancers are hormone-dependent and ERα-positive, and, thus, ER antagonists are broadly used in breast cancer therapy. We herein designed and synthesized a set of ER antagonists with a 4-heterocycle-4-phenylheptane skeleton.     

The influence of type I interferons on immune cells can be mediated through regulation of estrogen receptor alpha level

Autoimmune disorders are connected with the actions of sex hormones. Clinical observations have shown that especially estrogens are involved in these phenomena. In some cases the administration of estrogens can increase the pathological symptoms of a disorder, while in others they can cause disease remission. In multiple autoimmune diseases, type I interferons, a family of cytokines acting through the common receptor IFNAR1/IFNAR2, seem to have action convergent with that of estrogens. We hypothesize that this coincidence is not accidental and type I interferons can regulate the level of estrogen receptor alpha (ERα) and consequently change the sensitivity of immune cells to estrogen's action. There is evidence that ERα is responsible for the effects exerted by estrogens and that this phenomenon mainly involves antigen-presenting cells. On the other hand, research on IFN-tau, a type I interferon family members, showed that this cytokine can modulate ERα levels in ovine endometrium. Because of the common receptor for these interferons, we suspect that other type I interferons can act in this way not only in endometrial cells, but also in immune cells. If there is such a mechanism, it can be exploited in the therapy of immune disorders, especially autoimmune disease, for example through simultaneous administration of less toxic interferons and estrogens.