A Bodipy as a luminescent probe for detection of the G protein estrogen receptor (GPER) (original) (raw)
Related papers
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.