A Bodipy as a luminescent probe for detection of the G protein estrogen receptor (GPER) (original) (raw)
Novel biosensors for the detection of estrogen receptor ligands
The Journal of Steroid Biochemistry and Molecular Biology, 2005
There exists a significant need for the detection of novel estrogen receptor (ER) ligands for pharmaceutical uses, especially for treating complications associated with menopause. We have developed fluorescence resonance energy transfer (FRET)-based biosensors that permit the direct in vitro detection of ER ligands. These biosensors contain an ER ligand-binding domain (LBD) flanked by the FRET donor fluorophore, cyan fluorescent protein (CFP), and the acceptor fluorophore, yellow fluorescent protein (YFP). The ER-LBD has been modified so that Ala 430 has been changed to Asp, which increases the magnitude of the FRET signal in response to ligand-binding by more than four-fold compared to the wild-type LBD. The binding of agonists can be distinguished from that of antagonists on the basis of the distinct ligand-induced conformations in the ER-LBD. The approach to binding equilibrium occurs within 30 min, and the FRET signal is stable over 24 h. The biosensor demonstrates a high signal-to-noise, with a Z value (a statistical determinant of assay quality) of 0.72. The affinity of the ER for different ligands can be determined using a modified version of the biosensor in which a truncated YFP and an enhanced CFP are used. Thus, we have developed platforms for high-throughput screens for the identification of novel estrogen receptor ligands. Moreover, we have demonstrated that this FRET technology can be applied to other nuclear receptors, such as the androgen receptor.
Review of fluorescent steroidal ligands for the estrogen receptor 1995–2018
Steroids, 2019
The development of fluorescent ligands for the estrogen receptor (ER) continues to be of interest. Over the past 20 years, most efforts have focused on appending an expanding variety of fluorophores to the B-, C-and D-rings of the steroidal scaffold. This review highlights the synthesis and evaluation of derivatives substituted primarily at the 6-, 7α-and 17α-positions, culminating with our recent work on 11β-substituted estradiols, and proposes an approach to new fluorescent imaging agents that retain high ER affinity.
Analytical Chemistry, 2004
A genetically encoded fluorescent indicator was developed for the detection and characterization of estrogen agonists and antagonists. Two different color mutants of green fluorescent protein were joined by a tandem fusion domain composed of LXXLL (L) leucine, X) any amino acid) motif from the nuclear receptor-box II of steroid receptor coactivator 1, a flexible linker sequence, and the estrogen receptor r ligand binding domain (ERr LBD). Monitoring real-time ligand-induced conformational change in the ERr LBD to recruit the LXXLL motif allowed screening of natural and synthetic estrogens in single living cells using fluorescence resonance energy-transfer technique. The indicator was named SCCoR (single cellcoactivator recruitment). The high sensitivity of the present indicator made it possible to distinguish between estrogen strong and weak agonists in a dose-dependent fashion, immediately after adding ligand to live cells. Discrimination of agonists from antagonists was efficiently achieved using the present study. The approach described here can be applied to develop biosensors for other hormone receptors as well.
Single-label time-resolved luminescence assay for estrogen receptor–ligand binding
Analytical Biochemistry, 2011
Homogeneous luminescence-based microplate assays are desirable in high-throughput screening of new nuclear receptor regulators. Time-resolved fluorescence resonance energy transfer (TR-FRET) assays provide high sensitivity due to low background signal. The TR-FRET concept requires labeling of both ligand and receptor, making the assay format and its development relatively expensive and complex compared with single-label methods. To overcome the limitations of the multilabel methods, we have developed a single-label method for estrogen receptor (ER)-ligand binding based on quenching resonance energy transfer (QRET), where estradiol labeled with luminescent europium(III) chelate (Eu-E 2 ) is quenched using soluble quencher molecules. The luminescence signal of Eu-E 2 on binding to full-length ER is protected from quenching while increasing competitor concentrations displace Eu-E 2 from the receptor, reducing the signal. The QRET method was paralleled with a commercial fluorescence polarization (FP) assay. The measured signal-to-background (S/B) values for estradiol, estrone, fulvestrant, and tamoxifen obtained for the QRET assay (5.8-9.2) were clearly higher than the S/B values for the FP assay (1.3-1.5). A K d value of 30 nM was calculated for binding of Eu-E 2 to ER from a saturation binding isotherm. The QRET method provides an attractive new single-label assay format for nuclear receptor ligand screening.
Bioorganic & medicinal chemistry letters, 2017
In vivo imaging of estrogen receptor (ER) densities in human breast cancer is a potential tool to stage disease, guide treatment protocols and follow-up on treatment outcome. Both positron emission tomography (PET) and fluorescence imaging have received ample attention to detect ligand-ER interaction. In this study we prepared BODIPY-estradiol conjugates using 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) as fluorescent probe and estradiol derivatives as ligand and established their relative binding affinity (RBA) for the ERα. The synthesis of the conjugates involves attachment of a BODIPY moiety to the C17α-position of estradiol using Sonogashira or click reactions of iodo-BODIPY or aza-BODIPY with various 17α-ethynylestradiol (EE2) derivatives. The highest RBA for the ERα was observed with the EE2-BODIPY conjugate (7) featuring a linear eight carbon spacer chain. Cell uptake studies and in vivo imaging experiments in an ER-positive mouse tumor model are in progress.
Toxicological Sciences, 2000
A reporter gene sequence was constructed within a eukaryotic expression vector. The altered plasmid contained 2 sequential estrogen response elements (ERE) coupled to a human phosphoglycerate kinase (PGK) promoter inserted upstream from a cDNA sequence encoding enhanced green fluorescent protein (GFP) with a 3-polyadenylation signal. The plasmid was linearized and transfected into MCF-7 cells, a human breast cancer-derived line that expresses the estrogen receptor (ER). No selectable marker was present in the plasmid, requiring stably transfected cells to be selected by fluorescence-activated cell sorting based on GFP expression after the cells were treated with 10-9 M 17-estradiol (E2). Stably transfected MCF-7 cells (MCF7-ERE) exhibited 2000-3000 times more fluorescence at 488 nm excitation and 512 nm emission than non-transfected cells. MCF7-ERE cells exhibited a linear increase in GFP expression induced over a range of 10-12 M E2, a concentration giving 2 times the background expression, to maximal expression at 3 ؋ 0-10 M E2. From the maximal level, GFP expression plateaued, and then declined when E2 was increased to the highest concentration tested, 10-7 M. 4-Hydroxytamoxifen (TFN-OH) treatment of cells produced a dosedependent inhibition of E2-induced GFP expression, indicating the interaction of ER in the regulation of GFP gene expression. A series of estrogenic chemicals were evaluated for their capacity to induce GFP expression in MCF7-ERE cells, showing induced expression of GFP at concentrations 2-4 log units higher than the E2 concentration giving maximal GFP expression. The ERE-PGK-GFP reporter gene system is capable of rapid GFP expression in the presence of low concentrations of E2, and of quantifying estrogenicity of chemicals compared with a standard curve of the natural ligand, 17-estradiol.
Preparation and preliminary characterization of new monoclonal antibodies versus estradiol receptor
European Journal of Cancer and Clinical Oncology, 1991
Fluorescence is one of the most versatile and powerful tools for the study of macromolecules. While most proteins are intrinsically fluorescent, working at crystallization concentrations require the use of covalently prepared derivatives added as tracers. This approach requires derivatives that do not markedly affect the crystal packing. We have prepared fluorescent derivatives of chicken egg white lysozyme with probes bound to one of two different sites on the protein molecule. Lucifer yellow and 5-(2-aminoethyl)aminonapthalene-1-sulfonic acid (EDANS) have been attached to the side chain carboxyl of Asp 101 using a carbodiimide coupling procedure. Asp 101 lies within the active site cleft, and it is believed that the probes are ''buried'' within that cleft. Lucifer yellow and EDANS probes with iodoacetamide reactive groups have been bound to His 15 , located on the ''back side'' of the molecule relative to the active site. All the derivatives fluoresce in the solution and the crystalline states. Fluorescence characterization has focused on determination of binding effects on the probe quantum yield, lifetime, absorption and emission spectra, and quenching by added solutes. Quenching studies show that, as postulated, the Asp 101 -bound probes are partially sheltered from the bulk solution by their location within the active site cleft. Probes bound to His 15 have quenching constants about equal to those for the free probes, indicating that this site is highly exposed to the bulk solution. #
Molecular Endocrinology, 1995
We have analyzed four fluorescent nonsteroidal estrogens for their potential to serve as vital cytological stains to visualize the estrogen receptor (ER) in a model receptor expression system. The novel estrogen fluorophores are based on the rigidified stilbene-like structure of 5,6,11,12-tetrahydrochrysene (THC), and they embody electrondonor (hydroxyl) and electron-acceptor groups (nitrile, amide, ester, or ketone) that afford efficient, long wavelength, and environment-sensitive fluorescence. These probes bind with high affinity to human ER (relative binding affinity, 22-65 vs. estradiol, loo), and they stimulate the transcriptional activity of this receptor. The strong fluorescence of the estrogenic THCs permits visuali-zation, using conventional epifluorescence microscopy, of ER in transfected Cos-7 cells that express elevated levels of receptor. Cell staining by the donor-acceptor THCs characteristically displays a nonuniform pattern of nuclear fluorescence that can be fully inhibited by nonfluorescent estrogens such as estradiol or diethylstilbestrol. Additionally, this staining appears to be specific for ER, since it coincides with the distribution of receptor as determined by indirect immunofluorescence analysis using an ERspecific monoclonal antibody. Using these probes, we have analyzed the intracellular distribution of ER mutants containing a variety of deletions. Evidence is presented to show that removal of aminoterminal sequences within the ER polypeptide results in an altered pattern of intranuclear distribution with preferential accumulation of receptor protein within the nucleolus. These THC fluorophores therefore represent excellent probes for cytolological studies involving ER expressed in cultured cells and represent an important advance oma-8809/95/53.00/0 Molecular Endocrmology Copyright 0 1995 by The Endocrme Soclety toward the goal of exploiting fluorescence technology to analyze the expression and distribution of ER within tissue samples.
Imaging local estrogen production in single living cells with recombinant fluorescent indicators
Biosensors and Bioelectronics, 2011
Estrogens are steroid hormones with many systemic effects in addition to development and maintenance of the female reproductive system, and ligands of estrogen receptors are of clinical importance because of their use as oral contraceptive, hormone replacement and antitumoral therapy. In addition, tumoral tissues have been found to express aromatase and other steroidogenic enzymes synthesizing estradiol. To aid in the understanding of these processes, we have developed assays to image the local production of estrogens in isolated living mammalian cells. We constructed biosensors based on estrogen receptor ␣ ligand binding domain and fluorescent proteins by following two approaches. First, the ligand binding domain and a short fragment of steroid receptor coactivator-1 were appended to a circularly permuted yellow fluorescent protein to construct an excitation ratio estrogen indicator. In the second strategy, we constructed emission ratio sensors based on fluorescence resonance energy transfer, containing the ligand binding domain flanked by donor and acceptor fluorescent proteins. Estrogens altered the fluorescence signal of cells transfected with the indicators in a dose-dependent manner. We imaged local estrogen production in adrenocortical H295 cells expressing aromatase and transfected with the fluorescent sensors. In addition, paracrine detection was observed in HeLa cells harboring the indicators and co-cultured with H295 cells. This imaging approach may allow detection of physiological levels of these hormones in suitable animal models.
PLoS ONE, 2012
The discovery of the G protein-coupled estrogen receptor GPER (also GPR30) and the resulting development of selective chemical probes have revealed new aspects of estrogen receptor biology. The potential clinical relevance of this receptor has been suggested from numerous studies that have identified GPER expression in breast, endometrial, ovarian and other cancers. Thus GPER can be considered a candidate biomarker and target for non-invasive imaging and therapy. We have designed and synthesized a series of organometallic tricarbonyl-rhenium complexes conjugated to a GPER-selective small molecule derived from tetrahydro-3H-cyclopenta[c]quinoline. The activity and selectivity of these chelates in GPERmediated signaling pathways were evaluated. These results demonstrate that GPER targeting characteristics depend strongly on the structure of the chelate and linkage. Ethanone conjugates functioned as agonists, a 1,2,3-triazole spacer yielded an antagonist, and derivatives with increased steric volume exhibited decreased activities. Promising GPER selectivity was observed, as none of the complexes interacted with the nuclear estrogen receptors. Radiolabeling with technetium-99m in aqueous media was efficient and gave radioligands with high radiochemical yields and purity. These chelates have favorable physicochemical properties, show excellent stability in biologically relevant media, exhibit receptor specificity and are promising candidates for continuing development as diagnostic imaging agents targeting GPER expression in cancer.
Design and synthesis of fluorescently labeled steroidal antiestrogens
Steroids, 2019
A set of derivatives of 11β-(4-oxyphenyl)estradiol were prepared as potential fluorescent imaging agents for the evaluation of the estrogen receptor. The compounds were designed based on the established affinity and selectivity of 11β-[4-(dimethylethoxy)phenyl]estradiol (RU39411) as an estrogen receptor (ER) antagonist. The 5-(dimethylamino) naphathalene-1-sulfonyl (dansyl) and 7-nitrobenzo[c][1,2,5] oxadiaol-4-yl (NBD) moieties were selected based on their fluorescent and physicochemical properties. A convergent synthesis was developed that culminated in the [3 + 2] copper (I) assisted alkyne-azide cycloaddition coupling of the steroidal and fluorescent components. Good yields were obtained for the intermediates and final products, and the structural variations in the steroid component will permit evaluation of ER affinity and selectivity.
Biochemistry, 1992
We have examined the binding behavior and fluorescence characteristics of a series of novel ligands for the estrogen receptor (ER). These ligands are derivatives of 5,6,11,12-tetrahydrochrysene (THC), a structure that embodies a stilbene chromophore, found in many nonsteroidal estrogens, within a rigid tetracyclic system where it cannot easily be distorted from planarity, thus providing the conjugation and rigidity required for efficient fluorescence. Additional steric bulk, as trans-disposed ethyl substituents at the internal C-5 and C-1 1 positions, is required for the highest relative binding affinity (RBA), and the trans-5,ll-diethyl-2,8-dihydroxy-THC derivative binds to ER with an affinity greater than that of estradiol. The replacement of one of the phenolic hydroxyl groups of this THC derivative with an electron-withdrawing group (COMe, COOMe, CONH2, CN, or NOz) yields unsymmetrical THCs with binding affinities 15-40% that of estradiol (E2). The fluorescence emission shifts from about 380 nm for the dihydroxy THC to 475-688 nm for the donor-acceptor THCs. The emission of these donor-acceptor THCs is highly solvatochromic and shifts to longer wavelengths as the solvent polarity increases. In ethanol, the fluorescence quantum yield of the first four of these compounds is high (@f = 0.43-0.69), but the fifth compound, the nitro-THC, is almost nonemissive in protic solvents. When they are incubated with protein solutions containing ER (N M), the emission from the donor-acceptor THCs bound specifically to ER is in the 500-570-nm range, whereas fluorescence from non-receptor-bound fluorophores is in the 425-460-nm range. Thus, fluorescence from these probes bound specifically to ER could be measured under equilibrium conditions as well as after the removal of free and non-receptor-bound material by treatment with charcoal4extran. This is one of the first demonstrations of ligands whose fluorescence is distinctly different when free, when We are grateful for support of this research through a grant from the National Institutes of Health (PHS 5R37 DK15556).
Journal of Fluorescence, 2014
The goal of the present study was to design small, functionalized green-emitting BODIPY dyes, which can readily be coupled to target molecules such as receptor ligands, or even be integrated into their pharmacophores. A simple twostep one-pot procedure starting from 2,4-dimethylpyrrole and ω-bromoalkylcarboxylic acid chlorides was used to obtain new ω-bromoalkyl-substituted BODIPY fluorophores (1a-1f) connected via alkyl spacers of different length to the 8position of the fluorescent dye. The addition of radical inhibitors reduced the amount of side products. The ω-bromoalkylsubstituted BODIPYs were further converted to introduce various functional groups: iodo-substituted dyes were obtained by Finkelstein reaction in excellent yields; microwave-assisted reaction with methanolic ammonia led to fast and clean conversion to the amino-substituted dyes; a hydroxyl-substituted derivative was prepared by reaction with sodium ethylate, and thiol-substituted BODIPYs were obtained by reaction of 1a -1f with potassium thioacetate followed by alkaline cleavage of the thioesters. Watersoluble derivatives were prepared by introducing sulfonate groups into the 2-and 6-position of the BODIPY core. The synthesized BODIPY derivatives showed high fluorescent yields and appeared to be stable under basic, reducing and oxidative conditions. As a proof of concept, 2-thioadenosine was alkylated with bromoethyl-BODIPY 1b. The resulting fluorescent 2-substituted adenosine derivative 15 displayed selectivity for the A 3 adenosine receptor (ARs) over the other AR subtypes, showed agonistic activity, and may thus become a useful tool for studying A 3 ARs, or a lead structure for further optimization. The new functionalized dyes may be widely used for fluorescent labeling allowing the investigation of biological targets and processes.
Plant Molecular Biology Reporter, 2015
Cell-based screening methods for nuclear receptor ligands that use transgenic plant cells expressing a single human NR may have advantages over other eukaryotic systems which express multiple NRs. For example, signal-to-noise ratio might be improved because ligands would be less likely to bind to other NRs and/or less likely to cause confounding functional changes in plant cells. As a first step toward this aim we have expressed in plants truncated human estrogen receptor (ER) constructs linked to reporters, or selective markers such as luciferase, green fluorescent protein (GFP) and hygromycin. A variety of ligands for the ER (including estradiol and known phytoestrogens) have then been tested for their ability to over-express the linked marker gene(s) which could be measured (luciferase activity), visualized under fluorescent microscopy (GFP activity), or selected on antibiotic-containing media (Hygromycin B). Our results show a close association between the effects of ER ligands in the transgenic plant roots and their effects on native ERs in mammalian cells. With the stable expression of an ERalpha-GFP ligand detection system in A. thaliana, the estradiol-mediated response in transgenic roots is inhibited by an ER partial agonist (tamoxifen) and an antagonist (fulvestrant) at concentrations relevant to their use in breast cancer. We conclude that it is possible to express human NRs in plants in a form that can report on exogenous or endogenous ER ligands and that these constructs have a pharmacology which is relevant to ligands for the native NRs in human cells.
Journal of Biomolecular Screening, 2014
We have completed a robust high-content imaging screen for novel estrogen receptor α (ERα) agonists and antagonists by quantitation of cytoplasmic to nuclear translocation of an estrogen receptor chimera in 384-well plates. The screen was very robust, with Z′ values >0.7 and coefficients of variation (CV) <5%. The screen utilized a stably transfected green fluorescent protein-tagged glucocorticoid/estrogen receptor (GFP-GRER) chimera, which consisted of the N-terminus of the glucocorticoid receptor fused to the human ERα ligand binding domain. The GFP-GRER exhibited cytoplasmic localization in the absence of ERα ligands and translocated to the nucleus in response to stimulation with ERα agonists and antagonists. The BD Pathway 435 imaging system was used for image acquisition, analysis of translocation dynamics, and cytotoxicity measurements. We screened 224,891 samples from our synthetic, pure natural product libraries, prefractionated natural product extracts library, and crude natural product extracts library, which produced a 0.003% hit rate. In addition to identifying several known ER ligands, five compounds were discovered that elicited significant activity in the screen. Transactivation potential studies demonstrated that two hit compounds behave as agonists, while three compounds elicited antagonist activity in MCF-7 cells.
Analytical Chemistry, 2004
A new bioluminescence resonance energy transfer (BRET) homogeneous assay to evaluate the presence of estrogenlike compounds has been developed and optimized. The assay is based on the direct evaluation of estrogen r receptor (ERr) homodimerization as a result of estrogenlike compound binding. ERr monomer was genetically fused either to Renilla luciferase (Rluc) or to enhanced yellow fluorescent protein (EYFP). In the presence of estrogens, ERr dimerization brings Rluc and EYFP molecules close enough for an energy transfer. An in vitro BRET assay was first developed using purified fusion proteins (ERr-Rluc and ERr-EYFP) expressed in Escherichia coli to evaluate and optimize the analytical performances of the assay in the presence of 17-estradiol. The "in vivo" BRET quantitative assay was then developed by coexpressing the two fusion proteins in live HepG2 cells. The assay can be performed in 96-well microplate format with a 30-min incubation and allows detection with adequate accuracy and precision of as low as 1 nM of 17-estradiol. This new "in vivo" BRET assay allows evaluating the estrogen-like activity and synthetic xenoestrogens from biological and environmental samples.
Endocrine Abstracts, 2019
FSH receptor has been shown to be specifically expressed only in the Sertoli cells in males. In one of our studies that consisted of deprival of endogenous FSH in immature rats and adult bonnet monkeys, atrophy of the epididymis was observed, cauda region being the most affected. Although epididymis is an androgen-dependent tissue, the changes in histology of the cauda region were observed without any associated change in the levels of testosterone in FSH-deprived animals. Considering this, it was of interest to evaluate the possibility of epididymis being a direct target for FSH action. In the present study, we have examined the expression of FSH receptor in the epididymis of rat and monkey. In the cauda region of rat epididymis, FSH receptor expression was demonstrated by RT-PCR and Northern and Western blot analyses. FSH receptor was found to be functional as observed by its ability to bind 125 IoFSH, by an increase in cAMP production, and by BrdU incorporation following addition of FSH under in vitro conditions. These results suggest the possibility of a role for FSH in regulating the growth of the epididymis.
Analytical Biochemistry, 2006
The estrogen-related receptor-c (ERRc) is a constitutively active orphan receptor that belongs to the nuclear receptor superfamily and is most closely related to the estrogen receptors. Although its physiological ligand is unknown, ERRc has been shown to interact with synthetic estrogenic compounds such as 4-hydroxytamoxifen (4-OHT), tamoxifen, and diethylstilbestrol (DES). To assess how coregulator proteins interact with ERRc in response to ligand, an in vitro interaction methodology using time-resolved fluorescence resonance energy transfer (TR-FRET) was developed using glutathione S-transferase (GST)-tagged ERRc ligand-binding domain (LBD), a terbium-labeled anti-GST antibody, a fluorescein-labeled peptide containing sequences derived from coregulator proteins, and various ligands. An initial screen of these coregulator peptides bearing the coactivator LXXLL motif, the corepressor LXXI/HIXXXI/L motif, or other interaction motifs from natural coactivator sequences or random phage display peptides indicated that the peptides PGC1a, D22, and SRC1-4, known as class III coregulators, interacted most strongly with ERRc in the absence of ligand. Given its assay window and biological relevance in energy metabolism and obesity, further studies were conducted with PGC1a. Fluorescein-labeled PGC1a peptide was displaced from the ERRc LBD in the presence of increasing concentrations of 4-OHT and tamoxifen, but DES was less effective in PGC1a displacement. The statistical parameter Z 0 factor that measures the robustness of the assay was greater than 0.8 for displacement of PGC1a from ERRc LBD in the presence of saturating 4-OHT over an assay incubation time of 1-6 h, indicating an excellent assay. These findings also suggest that binding of 4-OHT, tamoxifen, or DES to ERRc results in differential affinity of coregulators for ERRc due to unique ligand-induced conformations.
2004
Estrogen receptor (ER) is a ligand-activated transcriptional factor, able to dimerize after activation and to bind specific DNA sequences (estrogen response elements), thus activating gene target transcription. Since ER homo- and hetero-dimerization (giving a-a and a-b isoforms) is a fundamental step for receptor activation, we developed an assay for detecting compounds that induce human ERa homo-dimerization based on bioluminescence resonance energy transfer (BRET). BRET is a non-radiative energy transfer, occurring between a luminescent donor and a fluorescent acceptor, that strictly depends on the closeness between the two proteins and can therefore be used for studying protein-protein interactions. We cloned ERa coding sequence in frame with either a variant of the green fluorescent protein (enhanced yellow fluorescent protein, EYFP) or Renilla luciferase (RLuc). Upon ERa homo-dimerization, BRET process takes place in the presence of the RLuc substrate coelenterazine resulting i...