Estrogen Receptor Alpha Ligand-binding Domain Research Articles

Phytochemical Characterization and Anticancer Screening of Cladophora goensis and Cladophora glomerata Extracts

Background Cladophora goensis and Cladophora glomerata has been widely identified in the Indian Oceans. Marine algae have been identified as potential pharmacological agents useful in the treatment of diabetes, hypertension, infections, and cancers. Objectives The present study aimed to evaluate the phytochemicals in C. goensis and C. glomerata using gas chromatography-mass spectroscopy (GC-MS), and to study the molecular interactions with cancer-related proteins using molecular docking techniques. Materials and methods GC-MS analysis was done using electron impact ionization at 70eV and the data was evaluated using total ion count for compound identification and quantification. AutoDock 4.0 version was used for the molecular docking analysis. Results The methanolic extracts of algae when subjected to GC-MS analysis, 19 compounds from C. goensis and 11 phytocompounds from C. glomerata were identified. The significant molecular interactions of phytochemicals of C. goensis (6-nitro-3 H-quinazolin-4-one, Isoquinoline, 1,2,3,4-tetrahydro-7-methoxy-2-methyl-8-(phenyl methoxy) and 9-Decen-1-ol, pentafluropropionate) and C. glomerata (phytol, palmitic acid, and octadec-9-enoic acid) against human epidermal growth factor receptor (4WRG), poly (ADP-ribose) polymerase-1 (4UND), human estrogen receptor alpha ligand-binding domain (3ERT), human peroxisome proliferator-activated receptor alpha ligand-binding domain (3VI8), and human topoisomerase (1EJ9) have been demonstrated. Conclusion The phytochemicals of methanolic extracts of C. goensis and C. glomerata showed potential interactions with cancer-related proteins.

Creation of a point-of-care therapeutics sensor using protein engineering, electrochemical sensing and electronic integration

Point-of-care sensors, which are low-cost and user-friendly, play a crucial role in precision medicine by providing quick results for individuals. Here, we transform the conventional glucometer into a 4-hydroxytamoxifen therapeutic biosensor in which 4-hydroxytamoxifen modulates the electrical signal generated by glucose oxidation. To encode the 4-hydroxytamoxifen signal within glucose oxidation, we introduce the ligand-binding domain of estrogen receptor-alpha into pyrroloquinoline quinone-dependent glucose dehydrogenase by constructing and screening a comprehensive protein insertion library. In addition to obtaining 4-hydroxytamoxifen regulatable engineered proteins, these results unveil the significance of both secondary and quaternary protein structures in propagation of conformational signals. By constructing an effective bioelectrochemical interface, we detect 4-hydroxytamoxifen in human blood samples as changes in the electrical signal and use this to develop an electrochemical algorithm to decode the 4-hydroxytamoxifen signal from glucose. To meet the miniaturization and signal amplification requirements for point-of-care use, we harness power from glucose oxidation to create a self-powered sensor. We also amplify the 4-hydroxytamoxifen signal using an organic electrochemical transistor, resulting in milliampere-level signals. Our work demonstrates a broad interdisciplinary approach to create a biosensor that capitalizes on recent innovations in protein engineering, electrochemical sensing, and electrical engineering.

RETooling the RET Inhibitor Pralsetinib for ESR1 Fusion-Positive Breast Cancer and Beyond.

Transcriptionally active fusions of ESR1 (ESR1-TAF) and somatic mutations in the estrogen receptor alpha (ERα) ligand-binding domain (LBD) cause endocrine therapy resistance in breast cancer. In searching for therapeutic target kinase(s) in these breast cancers, Gou and colleagues identified FLT4, RET, JAK1, and IGF1R as the top upregulated kinases induced by ESR1-TAFs and ERα LBD mutants in breast cancer cells. Among them, inhibition of RET by pralsetinib suppressed ESR1-TAF-driven and ERα LBD mutant-driven cell proliferation and patient-derived xenograft growth. Pralsetinib is an inhibitor of the RET protein tyrosine kinase that is approved for treating oncogenic RET mutation-positive and RET fusion-positive thyroid cancers and non-small cell lung cancer. The work by Gou and colleagues reinforces the knowledge of RET as an ESR1 target gene and highlights that RET interacts with ERα to promote breast cancer tumorigenesis and antiestrogen resistance. It also raises the prospect of repositioning pralsetinib to target wildtype RET in ER-positive breast cancer. See related article by Gou et al., p. 3237.

Design of Novel Selective Estrogen Receptor Inhibitors using Molecular Docking and Protein-Ligand Interaction Fingerprint Studies

Aims: The genomic and non-genomic actions of human estrogen receptor α (hERα) play a crucial role in breast epithelial cell proliferation and survival, as well as mammary tumorigenesis. hERα has been proved as a potential target for breast cancer therapy over the last 3 decades. Hence designing novel inhibitors targeting hERα can be a valuable approach in breast cancer therapy.
 Study Design: In the present study, the goal is to identify novel hERα inhibitors through molecular docking, AI based virtual screening and interaction fingerprint analysis.
 Place and Duration of Study: Department of Bioinformatics, Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh, India in between July 2021-sep 2021.
 Methodology: Molecular docking studies were performed using the human estrogen receptor alpha ligand-binding domain in complex with 4-hydroxytamoxifen (PDB: 3ERT) against existing compounds from literature. The best docked existing compound and co-crystal ligand were subjected to shape screening against 28 million compounds and resulted compounds constituted the hERα inhibitor dataset which was subjected to rigid receptor docking. Further, interaction fingerprint analysis was applied as complimentary method to docking for comparing the similarity of predicted binding poses of proposed leads with that of reference binding pose.
 Results: Co-crystal ligand (4-OHT) and E99 exhibited better binding affinity among existing ligand set. Rigid receptor docking studies resulted in four lead compounds possessing better docking scores than 4-OHT and E99. Moreover, leads showed favorable absorption, distribution, metabolism, excretion and toxicity properties within the range of 95% FDA approved drugs. Proposed leads showed interactions with binding site residues of hERα similar to that of 4-OHT with better binding affinity. The ability of obtained leads to retrieve actives was validated using receiver operative characteristic (ROC) curve.
 Conclusion: From above results, it has been observed that the proposed leads have potential to act as novel hERα inhibitors.

Synthesis, Docking, and Biological activities of novel Metacetamol embedded [1,2,3]-triazole derivatives

ERα controls the breast tissue development and progression of breast cancer. In our search for novel compounds to target Estrogen Receptor Alpha Ligand-Binding Domain, we identified “N-(3-((1H-1,2,3-triazol-4-yl)methoxy)phenyl)acetamide” derivatives as lead compounds. The Docking studies indicated good docking score for Metacetamol derivatives when docked into the 1XP6. A series of metacetamol derivatives have been synthesized, characterized and evaluated for cytotoxicity, anti bacterial and anti oxidant activities. Among the tested twelve hybrid compounds, “7a, 7g, 7h and 7i” derivatives showed promising cytotoxicity with IC50 value of <22 µg/mL against MCF-7 cell line. The Compounds “7a, 7d, 7e, 7f, 7h, 7i, 7j, 7k and 7l” showed outstanding anti oxidant activity with IC50 value of <30 µM, whereas Compounds “7a, 7b, 7c, 7d, 7g, 7j, 7k and 7l” showed moderate anti bacterial activity with the MIC value of <300 µM.

Synthesis, docking and biological activities of novel chromone linked [1,2,3]-triazole derivatives

Estrogen receptor-positive breast cancer is one of the commonly diagnosed cancers today. A series of chromone linked [1,2,3]-triazoles were designed based on literature followed by in silico studies. The in silico studies indicated good docking score for (2E)-(1R-1,2,3-triazol-4-yl)methyl 3-(6-methyl-4-oxo-4H-chromen-3-yl)acrylate derivatives when docked into the Human Estrogen Receptor Alpha Ligand-Binding Domain (PDB ID: 1XP6). So a new series of ten compounds have been synthesized, characterized and evaluated for cytotoxicity, antibacterial and antioxidant activities. The compounds 9f and 9a showed promising cytotoxicity with IC50 value of 18.61 and 20.39 μg/mL respectively. The Compounds 9b, 9c, 9f, 9 g and 9j showed encouraging antioxidant activity with IC50 value of < 30 μM i.e. DPPH scavenging activity levels more than that of positive control, whereas Compounds 9a, 9c, 9e, 9f, 9 h and 9j exhibited moderate antibacterial activities with the MIC value of <240 μM.

Abstract MIP-056: CONSTITUTIVELY ACTIVE ESTROGEN RECEPTOR–ALPHA LIGAND BINDING DOMAIN (ERA–LBD) MUTATIONS IN OVARIAN CARCINOMA

Abstract OBJECTIVES: Mutations in ESR1 that are acquired following treatment with aromatase inhibitors commonly underlie resistance to endocrine therapy in breast cancer. ERa-LBD mutations, especially at ESR1 Y537 and D538, confer constitutive activity and partial resistance to selective estrogen receptor modulators (SERMs), such as tamoxifen, and degraders (SERDs) such as fulvestrant in transcription assays. Some ovarian tumors also respond to anti-hormone therapy, and a subset which initially respond subsequently become resistant. The mechanisms of resistance to hormone therapy in ovarian cancer have not been previously described. Comprehensive genomic profiling (CGP) of ovarian tumors was used to analyze ESR1 and correlate responsiveness to aromatase inhibitors in a subset of these patients. METHODS: DNA was extracted from tumor specimens and sequenced using hybridization-capture (FoundationOne) targeting up to 315 genes, plus select introns frequently rearranged in cancer. Demographic and clinicopathologic data will be presented for a subset of patients whose tumors harbor ERa-LBD mutations associated with activation and resistance.. RESULTS: CGP of 3,641 ovarian and peritoneal carcinomas revealed 31 (0.9%) samples with amplification of ESR1 and 16 (0.4%) samples with acquired or de novo mutations affecting the ERa-LBD: Y537S (10), Y537N (1), D638G (4), and S341L (1). Within the 16 samples, ovarian serous carcinoma (10), ovarian endometrioid adenocarcinoma (3), ovarian epithelial carcinoma NOS (2), and peritoneal serous carcinoma (1) were represented. In cases of acquired mutations following prior hormone therapy, there was evidence of clinical benefit to SERM/SERDs. CaseAgeDiseaseESR1 MutationPrior HRTAcquiredSERM/SERD (*post mut if acquired)Clinical Benefit SERM/SERD159Ov serousY537SYYNN/A258Papillary serousY537NNNYY344Endometrioid ov adenoY537SNNYY445HG ov serousD538GNUYU564Endometrioid ov adenoY537SYUNU646LG ov serousY537SYUYU739Papillary serousY537SYUNN/A827LG ov serousY537SYYYY CONCLUSIONS: Acquired activating mutations in ESR1, previously described in breast cancer, can occur in ovarian serous carcinomas and endometrioid carcinomas initially responsive to hormone therapy and suggest a similar mechanism for acquired aromatase inhibitor resistance. However, ERa mutations can occur in gynecologic malignancies even in the absence of prior endocrine therapy. Tumors harboring these mutations may remain responsive to SERM/SERDs. Citation Format: J. A. Elvin, L. Gay, G. Colon-Otero, M. Jorgensen, L. Havrilesky, D. Zajchowski, L Shawver, F. A. Valea, S Aithal, J. S. Ross, M. Markman, S. Gaillard. CONSTITUTIVELY ACTIVE ESTROGEN RECEPTOR–ALPHA LIGAND BINDING DOMAIN (ERA–LBD) MUTATIONS IN OVARIAN CARCINOMA [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr MIP-056.

Molecular docking, QSAR and AMDET analysis of 6-methyl-1,3,8-trichlorodibenzofuran and its analogs against Estrogen receptor alpha

Estrogen receptor alpha (ERα) plays a key role in the malignancy of breast cancer. Therefore, inhibitors for ERα can act as useful agents for the treatment of breast cancer. In the present study, we describe docking, toxicity prediction as a rational strategy for identification of novel hits or leads. Pubmed search showed, 76 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) analogs. Molecular docking of these analogs and MCDF against human estrogen receptor alpha ligand-binding domain showed 48 compounds containing binding energy greater than MCDF. Further, in silico pharmacological and structural studies were successful in predicting two compounds 4,6-dichloro-2-methyl-1-benzofuran (CID 57722561) and 1-chlorodibenzofuran (CID 55293) as inhibitors against Estrogen receptor alpha.

A novel analytical tool for quantification of estrogenicity in river water based on fluorescence labelled estrogen receptor α

A novel combined procedure for estrogen-affinity purification and labelling of estrogen receptor alpha ligand-binding domain with Cy 5.5 cystein reactive dye was established. By using this procedure, mainly functional proteins are recovered. It can be easily adapted to a large variety of other proteins for which ligand-coated affinity materials are available. The labelled receptor was used in a total internal reflection fluorescence-based binding inhibition assay for determination of the impact of pollutants in river water on the receptor. The great advantage compared to conventional methods is that the total effect on the receptor is measured instead of concentrations of single compounds and that even currently unknown ligands are found as well. Therefore, the obtained signal is related to the response of the organism, which is exposed to the water. The limit of detection was found to be 0.139 nM of estradiol equivalents. The assay also provides a highly sensitive tool for pharmaceutical research and can be adapted to diagnostic applications.

GST-tagged mouse estrogen receptor α-transactivation domain fusion protein is specifically degraded during its over-expression in E. coli and purification

Escherichia coli BL21 (DE3) is commonly used for the overproduction of fusion proteins. Using this system, we recently reported the overproduction of histidine-tagged mouse estrogen receptor (ER) alpha-ligand binding domain as an intact 30 kD protein and its inhibitory effect on the growth of bacteria. However, when GST-tagged mouse ERalpha transactivation domain (TAD) was overproduced using this system, it showed no effect on the growth of bacteria but was specifically degraded during its expression and purification. Here we report the expression of 47 kD GST-tagged mouse ERalpha-TAD protein, which was degraded partially and specifically into 46 and 43 kD fragments. This fusion protein was further degraded into 37, 31, 29 and 26 kD fragments during its purification by affinity chromatography. Such specific degradation of GST-tagged mouse ERalpha-TAD during its overproduction in E. coli and purification indicates the induction of specific protease and suggests the modification of expression system.

Communication between the ERRα Homodimer Interface and the PGC-1α Binding Surface via the Helix 8–9 Loop

Although structural studies on the ligand-binding domain (LBD) have established the general mode of nuclear receptor (NR)/coactivator interaction, determinants of binding specificity are only partially understood. The LBD of estrogen receptor-alpha (ERalpha), for example, interacts only with a region of peroxisome proliferator-activated receptor coactivator (PGC)-1alpha, which contains the canonical LXXLL motif (NR box2), whereas the LBD of estrogen-related receptor-alpha (ERRalpha) also binds efficiently an untypical, LXXYL-containing region (NR box3) of PGC-1alpha. Surprisingly, in a previous structural study, the ERalpha LBD has been observed to bind NR box3 of transcriptional intermediary factor (TIF)-2 untypically via LXXYL, whereas the ERRalpha LBD binds this region of TIF-2 only poorly. Here we present a new crystal structure of the ERRalpha LBD in complex with a PGC-1alpha box3 peptide. In this structure, residues N-terminal of the PGC-1alpha LXXYL motif formed contacts with helix 4, the loop connecting helices 8 and 9, and with the C terminus of the ERRalpha LBD. Interaction studies using wild-type and mutant PGC-1alpha and ERRalpha showed that these contacts are functionally relevant and are required for efficient ERRalpha/PGC-1alpha interaction. Furthermore, a structure comparison between ERRalpha and ERalpha and mutation analyses provided evidence that the helix 8-9 loop, which differs significantly in both nuclear receptors, is a major determinant of coactivator binding specificity. Finally, our results revealed that in ERRalpha the helix 8-9 loop allosterically links the LBD homodimer interface with the coactivator cleft, thus providing a plausible explanation for distinct PGC-1alpha binding to ERRalpha monomers and homodimers.

Identification of Endocrine-Disrupting Compounds Using Nanoelectrospray Ionization Mass Spectrometry

A method using chip-based nanoelectrospray mass spectrometry (nanoESI-MS) is described to detect noncovalent ligand binding to the human estrogen receptor alpha ligand-binding domain (hER? LBD). This system represents an important environmental interest, because a wide variety of molecules, known as endocrine-disrupting compounds (EDCs), can bind to the estrogen receptor (ER) and induce adverse health effects in wildlife and humans. An efficient analytical method is therefore required to identify EDCs and characterize their solution-phase binding affinity and character (i.e. agonist or antagonist). Using proper experimental conditions, the nanoESI-MS approach allowed the detection of specific ligand interactions with hER? LBD. The best approach to evaluate solution-binding affinity by nanoESI-MS was to perform competitive binding experiments with 17?-estradiol (E2) as a reference ligand. Among the ligands tested, the relative binding affinity for hER? LBD measured by nanoESI-MS was 4-hydroxytamoxifen ? diethylstilbestrol &gt; E2 ? genistein ? bisphenol A, consistent with the order of the binding affinities in solution. To discern agonist from antagonist, we used the specificity of a coactivator peptide for agonist-bound receptor. A specific coactivator-hER? LBD complex was detected only in the presence of an agonist ligand. Therefore, the specificity of nanoESI-MS combined with its speed (1 min/ligand), low sample consumption (90 pmol protein/ligand), and its sensitivity for ligand (30 ng/ml) demonstrates that this method is promising for the identification and characterization of suspected ER ligands in a high-throughput manner.

Overproduction of mouse estrogen receptor alpha-ligand binding domain decreases bacterial growth

Escherichia coli (E. coli) is the most widely used prokaryotic host system for the synthesis of recombinant proteins. The overproduction of recombinant proteins is sometimes lethal to the host cells. In the present study, we expressed the ligand binding domain (LBD) of mouse estrogen receptor alpha (mouse ERalpha) using an expression vector (pIVEX) in E. coli BL21(DE3) and examined the effect of production of this protein on bacterial growth. The expressed protein was immunologically detected as a 30 kD histidine-tagged protein in the soluble part of the bacterial lysate. The overproduction of mouse ERalpha-LBD, as reflected by total protein content and expression pattern, resulted in the decrease of bacterial growth.

Estrogen receptor–ligand complexes measured by chip‐based nanoelectrospray mass spectrometry: An approach for the screening of endocrine disruptors

In the present report, a method based on chip-based nanoelectrospray mass spectrometry (nanoESI-MS) is described to detect noncovalent ligand binding to the human estrogen receptor alpha ligand-binding domain (hERalpha LBD). This system represents an important environmental interest, because a wide variety of molecules, known as endocrine disruptors, can bind to the estrogen receptor (ER) and induce adverse health effects in wildlife and humans. Using proper experimental conditions, the nanoESI-MS approach allowed for the detection of specific ligand interactions with hERalpha LBD. The relative gas-phase stability of selected hERalpha LBD-ligand complexes did not mirror the binding affinity in solution, a result that demonstrates the prominent role of hydrophobic contacts for stabilizing ER-ligand complexes in solution. The best approach to evaluate relative solution-binding affinity by nanoESI-MS was to perform competitive binding experiments with 17beta-estradiol (E2) used as a reference ligand. Among the ligands tested, the relative binding affinity for hERalpha LBD measured by nanoESI-MS was 4-hydroxtamoxifen approximately diethylstilbestrol > E2 >> genistein >> bisphenol A, consistent with the order of the binding affinities in solution. The limited reproducibility of the bound to free protein ratio measured by nanoESI-MS for this system only allowed the binding constants (K(d)) to be estimated (low nanomolar range for E2). The specificity of nanoESI-MS combined with its speed (1 min/ligand), low sample consumption (90 pmol protein/ligand), and its sensitivity for ligand (30 ng/mL) demonstrates that this technique is a promising method for screening suspected endocrine disrupting compounds and to qualitatively evaluate their binding affinity.

VISCANA: Visualized Cluster Analysis of Protein−Ligand Interaction Based on the ab Initio Fragment Molecular Orbital Method for Virtual Ligand Screening

We have developed a visualized cluster analysis of protein-ligand interaction (VISCANA) that analyzes the pattern of the interaction of the receptor and ligand on the basis of quantum theory for virtual ligand screening. Kitaura et al. (Chem. Phys. Lett. 1999, 312, 319-324.) have proposed an ab initio fragment molecular orbital (FMO) method by which large molecules such as proteins can be easily treated with chemical accuracy. In the FMO method, a total energy of the molecule is evaluated by summation of fragment energies and interfragment interaction energies (IFIEs). In this paper, we have proposed a cluster analysis using the dissimilarity that is defined as the squared Euclidean distance between IFIEs of two ligands. Although the result of an ordered table by clustering is still a massive collection of numbers, we combine a clustering method with a graphical representation of the IFIEs by representing each data point with colors that quantitatively and qualitatively reflect the IFIEs. We applied VISCANA to a docking study of pharmacophores of the human estrogen receptor alpha ligand-binding domain (57 amino acid residues). By using VISCANA, we could classify even structurally different ligands into functionally similar clusters according to the interaction pattern of a ligand and amino acid residues of the receptor protein. In addition, VISCANA could estimate the correct docking conformation by analyzing patterns of the receptor-ligand interactions of some conformations through the docking calculation.

Rapid Creation of a Novel Protein Function by in Vitro Coevolution

We have developed a simple and efficient method for creation of novel protein functions in an existing protein scaffold. The in vitro coevolution method involves design of a hypothetical pathway for the target function followed by stepwise directed evolution of the corresponding protein along the pathway. As a test case, this strategy was used to engineer variants of human estrogen receptor alpha ligand-binding domain (hERalphaLBD) with novel corticosterone activity. Two steroids, testosterone and progesterone, that provide a progressive structural bridge between 17beta-estradiol and corticosterone, were chosen to assist the directed evolution of hERalphaLBD. A total of approximately 10(6) variants were screened in four rounds of random mutagenesis, resulting in two hERalphaLBD variants that respond to corticosterone. Creation of this new ligand activity required the presence of four simultaneous mutations. In addition, several required mutations were located outside the ligand binding pocket and yet exerted important action on ligand binding. Our results demonstrate the ability of in vitro coevolution to create novel protein function that is difficult or impossible to achieve by existing protein engineering approaches and also shed light on the natural evolution of nuclear hormone receptors. This in vitro coevolution approach should provide a powerful, broadly applicable tool for engineering biological molecules and systems with novel functions.

Estrogen receptor dimerization: ligand binding regulates dimer affinity and dimer dissociation rate.

Nuclear receptors form strong dimers that are essential for their function as transcription factors, and it is thought that ligand binding can affect dimer stability. In this report, we describe convenient fluorescence resonance energy transfer (FRET)-based methods for measuring the thermodynamic and kinetic stability of dimers of the estrogen receptor-alpha ligand-binding domain (ERalpha-LBD). We have developed receptors that are chemically labeled with a single fluorophore in a site-specific manner. These fluorophore-labeled ERs are functional and can be used to measure directly the affinity and stability of ERalpha-LBD dimers. Our results indicate that unliganded ERalpha-LBDs exist as very stable dimers and that the dissociation rate of these dimers is slow (t(1/2)=39 +/- 3 min at 28 C) and is further slowed (< or =7-fold) by the addition of various ligands. Estrogen antagonists provide greater kinetic stabilization of the ER dimers than agonists. In addition, coactivator peptides containing the LXXLL motif selectively stabilize agonist-bound ERalpha-LBD dimers. These fluorescence-based assays for measuring the kinetic and thermodynamic stability of ER dimers provide a functional in vitro method for assessing the agonist or antagonist character of novel ligands.

Structural characterization of a subtype-selective ligand reveals a novel mode of estrogen receptor antagonism.

The R,R enantiomer of 5,11-cis-diethyl-5,6,11,12-tetrahydrochrysene-2,8-diol (THC) exerts opposite effects on the transcriptional activity of the two estrogen receptor (ER) subtypes, ER alpha and ER beta. THC acts as an ER alpha agonist and as an ER beta antagonist. We have determined the crystal structures of the ER alpha ligand binding domain (LBD) bound to both THC and a fragment of the transcriptional coactivator GRIP1, and the ER beta LBD bound to THC. THC stabilizes a conformation of the ER alpha LBD that permits coactivator association and a conformation of the ER beta LBD that prevents coactivator association. A comparison of the two structures, taken together with functional data, reveals that THC does not act on ER beta through the same mechanisms used by other known ER antagonists. Instead, THC antagonizes ER beta through a novel mechanism we term 'passive antagonism'.

Overexpression, purification, and crystal structure of native ER alpha LBD.

Several crystal structures of human estrogen receptor alpha ligand-binding domain (hERalpha LBD) complexed with agonist or antagonist molecules have previously been solved. The proteins had been modified in cysteine residues (carboxymethylation) or renatured in urea to circumvent aggregation and denaturation problems. In this work, high-level protein expression and purification together with crystallization screening procedure yielded high amounts of soluble protein without renaturation or modifications steps. The native protein crystallizes in the space group P3(2) 21 with three molecules in the asymmetric unit. The overall structure is very similar to that previously reported for the hERalpha LBD with cysteine carboxymethylated residues thus validating the modification approach. The present strategy can be adapted to other cases where the solubility and the proper folding is a difficulty.

Flexible Estrogen Receptor Modulators: Design, Synthesis, and Antagonistic Effects in Human MCF-7 Breast Cancer Cells

Although many series of estrogen receptor antagonists continue to be produced, the majority are direct structural analogues of existing modulators. To examine the tolerance of the estrogen receptor toward flexible ligands, a series of novel flexible estrogen receptor antagonists were prepared and their antiproliferative effects on human MCF-7 breast tumor cells investigated. Each of these compounds deviated from the traditional triphenylethylene backbone associated with common tamoxifen analogues through the introduction of a flexible methylene (benzylic) spacing group between one of the aryl rings and the ethylene group and through variations in the basic side chain moiety. The compounds prepared, when assayed in conjunction with a tamoxifen standard, demonstrated high potency in antiproliferative assays against an MCF-7 human breast cancer cell line with low cytotoxicity and high binding affinity. A computational study was undertaken to investigate the compounds' potential interactions with specific residues within the human estrogen receptor alpha ligand-binding domain (ER-LBD), predicting these compounds bind in an antiestrogenic fashion within the ER-LBD and interact with those important residues previously identified in the structures of ER-LBD agonist/antagonist cocrystals. These compounds further illustrate the eclectic nature of the estrogen receptor in terms of ligand flexibility tolerance.