TR-FRET Research Articles

Development of a time-resolved fluorescence resonance energy transfer assay for cyclin-dependent kinase 4 and identification of its ATP-noncompetitive inhibitors

Protein kinases are recognized as important drug targets due to the pivotal roles they play in human disease. Many kinase inhibitors are ATP competitive, leading to potential problems with poor selectivity and significant loss of potency in vivo due to cellular ATP concentrations being much higher than Km. Consequently, there has been growing interest in the development of ATP-noncompetitive inhibitors to overcome these problems. There are challenges to identifying ATP-noncompetitive inhibitors from compound library screens because ATP-noncompetitive inhibitors are often weaker and commonly excluded by potency-based hit selection criteria in favor of abundant and highly potent ATP-competitive inhibitors in screening libraries. Here we report the development of a time-resolved fluorescence resonance energy transfer (TR–FRET) assay for protein kinase cyclin-dependent kinase 4 (CDK4) and the identification of ATP-noncompetitive inhibitors by high-throughput screening after employing a strategy to favor this type of inhibitors. We also present kinetic characterization that is consistent with the proposed mode of inhibition.

Ursolic Acid Suppresses Interleukin-17 (IL-17) Production by Selectively Antagonizing the Function of RORγt Protein

Th17 cells have recently emerged as a major player in inflammatory and autoimmune diseases via the production of pro-inflammatory cytokines IL-17, IL-17F, and IL-22. The differentiation of Th17 cells and the associated cytokine production is directly controlled by RORγt. Here we show that ursolic acid (UA), a small molecule present in herbal medicine, selectively and effectively inhibits the function of RORγt, resulting in greatly decreased IL-17 expression in both developing and differentiated Th17 cells. In addition, treatment with UA ameliorated experimental autoimmune encephalomyelitis. The results thus suggest UA as a valuable drug candidate or leading compound for developing treatments of Th17-mediated inflammatory diseases and cancer.

Abstract 64: Development of homogeneous non-radioactive assays for studying histone 3 methyltransferases and demethylases

Abstract Post-translational modifications of histones are among the epigenetic mechanisms that can affect chromatin structure and function. Disruption of epigenetic processes can lead to altered gene expression and malignant cellular transformation. Epigenetic changes including histone acetylation, histone methylation and DNA methylation are now considered to play important roles in the initiation and progression of cancer. Histone methylation and demethylation are enzymatically dynamic processes controlled respectively by histone methyltransferases (HMTs) and histone demethylases (HDMs). Several assay methods have been developed for quantifying the activity of HMTs and HDMs. These include radioactive assays, enzyme-linked immunoassays (ELISA), mass spectrometry, and enzyme-coupled detection of reaction co-products (e.g. S-adenosylhomocysteine, formaldehyde, hydrogen peroxide). These assays suffer from various drawbacks such as low throughput, lack of sensitivity, generation of hazardous waste, requirement for expensive equipment, or artifacts associated with the use of enzyme-coupled assays (generation of false positives/negatives). In this study, we describe homogeneous (mix-and-read) assays for measuring the catalytic activity of both HMTs and HDMs acting on histone H3 using two different non-radioactive technologies: amplified luminescent proximity homogeneous assay (AlphaLISA) and time-resolved fluorescence energy transfer (LANCE). The EZH2 complex (which methylates histone H3 on lysine 27) and LSD1 and JMJD2A (which demethylate di-methyl lysine 4 and tri-methyl lysine 36, respectively, on histone H3) were selected as model enzymes due to their association with various human diseases. Assays were developed in 384-well format and used as substrates two synthetic biotinylated peptides derived from the N-terminus of histone H3 (amino acids 1 to 21 for LSD1 and 21 to 44 for EZH2 and JMJD2A). All assays were designed as signal-increase homogeneous assays, where direct detection of product formation was conducted using methyl-state selective antibodies conjugated to either AlphaLISA acceptor beads or LANCE europium chelate. Results demonstrated that all assays were sensitive and robust. Furthermore, profiling of known inhibitors for each epigenetic enzyme showed the expected potency with either technology. These assays will therefore be ideal for the identification of selective small molecule inhibitors.Although these studies focused on EZH2, JMJD2A and LSD1 enzymes, the approach described here is broadly suitable for measuring the catalytic activity of other HMTs and HDMTs by combining the appropriate biotinylated histone-derived peptides and methyl-state selective antibodies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 64. doi:10.1158/1538-7445.AM2011-64

Single-label time-resolved luminescence assay for estrogen receptor–ligand binding

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.

Structural Dynamics of SERCA and Phospholamban by TR-FRET and TIRF

We used fluorescence to investigate the structure and dynamics of phospholamban (PLB) and its inhibited target, sarcoplasmic reticulum Ca-ATPase (SERCA). Time-resolved fluorescence energy transfer (TR-FRET) was used to study the oligomeric interaction between PLB and SERCA. Previous work on our lab has shown that PLB is primarily pentameric but SERCA binds preferentially to the monomeric form in lipid vesicles. Recent EM studies suggest that the PLB pentamer might also bind to SERCA. We tested this hypothesis by labeling SERCA at C674 with a fluorescent donor (TMRIA) and labeling PLB at K3 with a non-fluorescent acceptor (MGITC), then reconstituting the proteins into lipid vesicles and performing TR-FRET as a function of the fraction of acceptor-labeled PLB (xA), keeping the total PLB/SERCA molar ratio constant at 10. Simulations showed that if a PLB monomer binds to SERCA, the dependence of FRET on xA should be linear, but the binding of a PLB oligomer should produce distinct curvature in the plot. The observed plot was quite linear, and was indistinguishable from control experiments using a monomeric mutant of PLB. We conclude that PLB binds to SERCA only as a monomer. We used polarized TIRF to study the structure of PLB in the presence and absence of SERCA in a supported lipid bilayer. Polarized TIRF data shows that the cytoplasmic helix of PLB, labeled with bifunctional rhodamine, is close to be parallel to the membrane surface. SERCA binding lifts the cytoplasmic helix away from the membrane surface. These results have important implications for the design of PLB mutants to be used in gene therapy for heart failure.

Geometrical analysis of cytochrome c unfolding

A geometrical model has been developed to study the unfolding of iso-1 cytochrome c. The model draws on the crystallographic data reported for this protein. These data were used to calculate the distance between specific residues in the folded state, and in a sequence of extended states defined by n = 3, 5, 7, 9, 11, 13, and 15 residue units. Exact calculations carried out for each of the 103 residues in the polypeptide chain demonstrate that different regions of the chain have different unfolding histories. Regions where there is a persistence of compact structures can be identified, and this geometrical characterization is fully consistent with analyses of time-resolved fluorescence energy-transfer (TrFET) data using dansyl-derivatized cysteine side-chain probes at positions 39, 50, 66, 85, and 99. The calculations were carried out assuming that different regions of the polypeptide chain unfold synchronously. To test this assumption, lattice Monte Carlo simulations were performed to study systematically the possible importance of asynchronicity. Calculations show that small departures from synchronous dynamics can arise if displacements of residues in the main body of the chain are much more sluggish than near-terminal residues.

Ursodeoxycholic Acid Amides As Novel Glucocorticoid Receptor Modulators

Ursodeoxycholic acid (UDCA) is used for the treatment of hepatic inflammatory diseases. Recent studies have shown that UDCA's biological effects are partly glucocorticoid receptor (GR) mediated. UDCA derivatives were synthesized and screened for ability to induce GR translocation in a high content analysis assay using the esophageal cancer SKGT-4 cell line. UDCA derivatives induced GR translocation in a time dependent manner with equal efficacy to that of dexamethasone (Dex) and with greatly increased potency relative to UDCA. The cyclopropylamide 1a suppressed TNF-α induced NF-κB activity and it induced GRE transactivation. 1a was unable to displace Dex from the GR ligand binding domain (LBD) in a competition experiment but was capable of coactivator recruitment in a time-resolved fluorescence energy transfer assay (TR-FRET). This represents a novel mechanism of action for a GR modulator. These derivatives could result in a new class of GR modulators.

Terbium chelate as donor label in time-resolved fluorescence energy transfer study of DNA hybridization

A terbium chelate formed with diethylenetriaminepentaacetic acid and p-aminosalicylic acid (Tb–DTPA–pAS) was labeled on a 23-mer DNA strand and used as donor in the fluorescence resonance energy transfer (FRET) study of DNA hybridization. The change in donor fluorescence lifetime with and without DNA-bind acceptor carboxytetramethylrhodamine (TAMRA) showed high energy transfer efficiency (87%). The distance between the two ends of the 23-mer double-stranded DNA was estimated. This study provides a possibility of using the Tb-chelate as a probe in the studies of the geometry-dependent interaction between DNA strands or proteins.

Monitoring a coordinated exchange process in a four-component biological interaction system: development of a time-resolved terbium-based one-donor/three-acceptor multicolor FRET system.

Hormonal regulation of cellular function involves the binding of small molecules with receptors that then coordinate subsequent interactions with other signal transduction proteins. These dynamic, multicomponent processes are difficult to track in cells and even in reconstituted in vitro systems, and most methods can monitor only two-component interactions, often with limited capacity to follow dynamic changes. Through a judicious choice of three organic acceptor fluorophores paired with a terbium donor fluorophore, we have developed the first example of a one-donor/three-acceptor multicolor time-resolved fluorescence energy transfer (TR-FRET) system, and we have exemplified its use by monitoring a ligand-regulated protein-protein exchange process in a four-component biological system. By careful quantification of the emission from each of the three acceptors at the four channels for terbium donor emission, we demonstrate that any of these donor channels can be used to estimate the magnitude of the three FRET signals in this terbium-donor triple-acceptor system with minimal bleedthrough. Using this three-channel terbium-based, TR-FRET assay system, we show in one experiment that the addition of a fluorescein-labeled estrogen agonist displaces a SNAPFL-labeled antiestrogen from the ligand binding pocket of a terbium-labeled estrogen receptor, at the same time causing a Cy5-labeled coactivator to be recruited to the estrogen receptor. This experiment demonstrates the power of a four-color TR-FRET experiment, and it shows that the overall process of estrogen receptor ligand exchange and coactivator binding is a dynamic but precisely coordinated process.

A Dual Estrogen Receptor TR-FRET Assay for Simultaneous Measurement of Steroid Site Binding and Coactivator Recruitment

The human estrogen receptors (hER) are members of the nuclear hormone receptor (NHR) superfamily and represent important drug targets for the pharmaceutical industry. Initially, ligand binding assays were used to identify novel ligands using receptors purified from native tissues. With the advent of molecular cloning techniques, cell-based transactivation assays have been the gold standard for many years of drug discovery. With the elucidation of the structural mechanisms underlying the activation of NHRs, cell-free assays with purified receptors have become important tools to directly assess different binding sites (e.g., the hormone binding site or the cofactor binding site). The available cell-free assays have so far facilitated the study of one binding site at a time. With the introduction of Terbium (Tb(3+))-based time-resolved fluorescence energy transfer (TR-FRET), it has become possible to measure 2 different interactions within 1 test tube in parallel. The authors have applied this technology to develop a dual readout system for the simultaneous monitoring of steroid hormone site binding and cofactor peptide recruitment. They took advantage of a commercially available fluorescent tracer as an indicator for classical steroid site binding and designed a novel peptide derived from the peroxisome proliferator-activated receptor gamma coactivator-1a (PGC1a) as an indicator for functional agonistic behavior of a test compound. The established assay is able to differentiate between agonists, antagonists, partial agonists, and compounds binding to the cofactor recruitment site. The IC(50) values obtained for a number of reference compounds in the multiplexed assay are in concordance with published data. The simple 1-step mix-and-measure protocol gives excellent quality and robustness and can be miniaturized to 5-microL volume.

A fluorescence resonance energy transfer-based binding assay for characterizing kinase inhibitors: Important role for C-terminal biotin tagging of the kinase

Novel biochemical strategies are needed to identify the next generation of protein kinase inhibitors. One promising new assay format is a competition binding approach that employs time-resolved fluorescence resonance energy transfer (TR–FRET). In this assay, a FRET donor is bound to the kinase via a purification tag, whereas a FRET acceptor is bound via a tracer-labeled inhibitor. Displacement of the tracer by an unlabeled inhibitor eliminates FRET between the fluorophores and provides a readout on binding. Although promising, this technique has so far been limited in applicability in part by a lack of signal strength is some cases and also by an inability to predict whether a particular tagging strategy will show robust FRET. In this work, we sought to better understand the factors that give rise to a strong FRET signal in this assay. We determined the magnitude of FRET for several tyrosine kinases using different purification tags (biotin, glutathione S-transferase [GST], and His) placed at either the N terminus or C terminus of the kinase. It was observed that coupling the FRET acceptor to the kinase C terminus using a biotin/streptavidin interaction resulted in the greatest increase in FRET. Specifically, for multiple kinases, the signal/background ratio was at least 3-fold better using C-terminal biotinylation compared with tagging at the N terminus using a His/anti-His antibody or GST/anti-GST antibody interaction. In one case, the FRET signal using C-terminal biotin tagging was more than 150-fold over background. This strong FRET signal facilitated development of improved inhibitor binding assays that required only tens of picomolar enzyme or tracer-labeled inhibitor. Together, these results indicate that C-terminal biotinylation is a promising tagging strategy for developing an optimal FRET-based competition binding assay for tyrosine kinases.

Folding energy landscape of cytochrome cb 562

Cytochrome cb(562) is a variant of an Escherichia coli four-helix bundle b-type heme protein in which the porphyrin prosthetic group is covalently ligated to the polypeptide near the terminus of helix 4. Studies from other laboratories have shown that the apoprotein folds rapidly without the formation of intermediates, whereas the holoprotein loses heme before native structure can be attained. Time-resolved fluorescence energy transfer (TRFET) measurements of cytochrome cb(562) refolding triggered using an ultrafast continuous-flow mixer (150 micros dead time) reveal that heme attachment to the polypeptide does not interfere with rapid formation of the native structure. Analyses of the TRFET data produce distributions of Trp-59-heme distances in the protein before, during, and after refolding. Characterization of the moments and time evolution of these distributions provides compelling evidence for a refolding mechanism that does not involve significant populations of intermediates. These observations suggest that the cytochrome b(562) folding energy landscape is minimally frustrated and able to tolerate the introduction of substantial perturbations (i.e., the heme prosthetic group) without the formation of deep misfolded traps.

A time-resolved fluorescence resonance energy transfer-based assay for DEN1 peptidase activity

Neural precursor cell expressed, developmentally down-regulated gene 8 (NEDD8) is a recently discovered ubiquitin-like posttranslational modifier. NEDD8 acts predominantly as a regulator of ubiquitin–protein ligases and as a decoy for proteins targeted for proteasomal degradation. It thereby controls key events in cell cycle progression and embryogenesis. Deneddylase-1 (DEN1/NEDP1/SENP8) features a selective peptidase activity converting the proNEDD8 precursor to its mature form and an isopeptidase activity deconjugating NEDD8 from substrates such as cullins and p53. In this study, we describe a high-throughput screening (HTS)-compatible time-resolved fluorescent resonance energy transfer (TR–FRET) assay measuring the peptidase activity of DEN1.

Electrostatic effects on funneled landscapes and structural diversity in denatured protein ensembles

The denatured state of proteins is heterogeneous and susceptible to general hydrophobic and electrostatic forces, but to what extent does the funneled nature of protein energy landscapes play a role in the unfolded ensemble? We simulate the denatured ensemble of cytochrome c using a series of models. The models pinpoint the efficacy of incorporating energetic funnels toward the native state in contrast with models having no native structure-seeking tendency. These models also contain varying strengths of electrostatic effects and hydrophobic collapse. The simulations based on these models are compared with experimental distributions for the distances between a fluorescent donor and the heme acceptor that were extracted from time-resolved fluorescence energy transfer experiments on cytochrome c. Comparing simulations to detailed experimental data on several labeling sites allows us to quantify the dominant forces in denatured protein ensembles.

PTH Receptor Responds To Shear Stress Perturbation of Plasma Membrane

The precise molecular mechanisms by which bone cells transduce mechanical stimulus into intracellular biochemical response have not been established yet. Here we show that mechanical perturbation of the plasma membrane leads to ligand‐independent conformational transitions in G protein coupled receptor (GPCR) such as parathyroid hormone type 1 receptor (PTH1r). By using picosecond time‐resolved fluorescence microscopy and GPCR conformation‐sensitive fluorescence energy transfer (FRET) we found that stimulation of MC3T3 cells with fluid shear stress or membrane fluidizing agent leads to a significant changes in conformational equilibrium of PTH1r in MC3T3 cells. The PTH1r conformational dynamics was detected by monitoring redistribution of GPCRs between inactive and active conformations in a single cell under fluid shear stress in real time using genetically engineered PTH1r containing intramolecular FRET pair. Our data demonstrate that changes in cell membrane tension and membrane fluidity affect conformational dynamics of PTH1r. Therefore we suggest that PTH1r is involved in mediating primary mechanochemical signal transduction in MC3T3 cells. Furthermore we show that conformational transitions induced by mechanical stress are different from those induced by its ligand PTH(1‐34).This research was supported by the NIH grant HL086943.

Development and Implementation of a Miniaturized High-Throughput Time-Resolved Fluorescence Energy Transfer Assay to Identify Small Molecule Inhibitors of Polo-Like Kinase 1

Polo-like kinase (Plk) 1 is a key enzyme involved in regulating the mammalian cell cycle that is also a validated anticancer drug target. Nonetheless, there are relatively few readily available potent and selective small molecule inhibitors of Plk1. To increase the availability of pharmacologically valuable Plk1 inhibitors, we describe herein the development, variability assessment, validation, and implementation of a 384-well automated, miniaturized high-throughput time-resolved fluorescence energy transfer screening assay designed to identify Plk1 kinase inhibitors. Using a small molecule library of pharmaceutically active compounds to gauge high-throughput assay robustness and reproducibility, we found nine general kinase inhibitors, including H-89, which was selected as the minimum control. We then interrogated a 97,101 compound library from the National Institutes of Health repository for small molecule inhibitors of Plk1 kinase activity. The initial primary hit rate in a single 10 microM concentration format was 0.21%. Hit compounds were subjected to concentration-response confirmation and interference assays. Identified in the screen were seven compounds with 50% inhibitory concentration (IC50) values below 1 microM, 20 compounds with IC50 values between 1 microM and 5 microM, and eight compounds with IC50 values between 5 and 10 microM, which could be assigned to seven distinct chemotype classes. Hit compounds were also examined for their ability to inhibit other kinases such as protein kinase D, focal adhesion kinase, rho-associated coiled coil protein kinase 2, c-jun NH2-terminal kinase 3, and protein kinase A via experimentation or data-mining. These compounds should be useful as probes for the biological activity of Plk1 and as leads for the development of new selective inhibitors of Plk1.

High-throughput cellular assays for regulated posttranslational modifications

We have developed a set of high-throughput screening (HTS)-compatible assays capable of measuring regulated, target-specific posttranslational modifications in a mammalian cell-based format. We chose the NFκB signal transduction cascade as a model system to validate this approach because specific target proteins in this signaling pathway undergo a multitude of posttranslational modifications in response to pathway stimulation. In this pathway, TNFα induces the phosphorylation, ubiquitination, and proteasomal degradation of IκBα, which leads to the release and translocation of the NFκB transcriptional complex into the nucleus. To measure these cellular processes, we describe the use of a stable cell line expressing a fusion of green fluorescent protein (GFP) with IκBα that can be interrogated for either ubiquitination or phosphorylation using a unique set of terbium-labeled antibodies in a time-resolved Förster resonance energy transfer (TR–FRET)-based readout. Concurrently, we have engineered a β-lactamase–IκBα reporter cell line that can be used to quantify proteasomal degradation of IκBα in living cells. Both TR–FRET and β-lactamase reporter technologies provide a convenient, sensitive, and robust means to interrogate the chronological steps in NFκB signaling in a physiologically relevant cellular context without the need to overexpress any enzyme involved in this pathway. Cellular HTS assays that interrogate such processes will provide a unique integrated approach to dissecting intermediate steps in NFκB activation and could serve as examples of broadly applicable pathway analysis tools for target-based drug discovery.

Crystal Structure of Human Estrogen-related Receptor α in Complex with a Synthetic Inverse Agonist Reveals Its Novel Molecular Mechanism

Inverse agonists of the constitutively active human estrogen-related receptor alpha (ERRalpha, NR3B1) are of potential interest for several disease indications (e.g. breast cancer, metabolic diseases, or osteoporosis). ERRalpha is constitutively active, because its ligand binding pocket (LBP) is practically filled with side chains (in particular with Phe(328), which is replaced by Ala in ERRbeta and ERRgamma). We present here the crystal structure of the ligand binding domain of ERRalpha (containing the mutation C325S) in complex with the inverse agonist cyclohexylmethyl-(1-p-tolyl-1H-indol-3-ylmethyl)-amine (compound 1a), to a resolution of 2.3A(.) The structure reveals the dramatic multiple conformational changes in the LBP, which create the necessary space for the ligand. As a consequence of the new side chain conformation of Phe(328) (on helix H3), Phe(510)(H12) has to move away, and thus the activation helix H12 is displaced from its agonist position. This is a novel mechanism of H12 inactivation, different from ERRgamma, estrogen receptor (ER) alpha, and ERbeta. H12 binds (with a surprising binding mode) in the coactivator groove of its ligand binding domain, at a similar place as a coactivator peptide. This is in contrast to ERRgamma but resembles the situation for ERalpha (raloxifene or 4-hydroxytamoxifen complexes). Our results explain the novel molecular mechanism of an inverse agonist for ERRalpha and provide the basis for rational drug design to obtain isotype-specific inverse agonists of this potential new drug target. Despite a practically filled LBP, the finding that a suitable ligand can induce an opening of the cavity also has broad implications for other orphan nuclear hormone receptors (e.g. the NGFI-B subfamily).

Development of a coactivator displacement assay for the orphan receptor estrogen-related receptor-γ using time-resolved fluorescence resonance energy transfer

The estrogen-related receptor-γ (ERRγ) 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, ERRγ 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 ERRγ 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 ERRγ 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 PGC1α, D22, and SRC1-4, known as class III coregulators, interacted most strongly with ERRγ in the absence of ligand. Given its assay window and biological relevance in energy metabolism and obesity, further studies were conducted with PGC1α. Fluorescein-labeled PGC1α peptide was displaced from the ERRγ LBD in the presence of increasing concentrations of 4-OHT and tamoxifen, but DES was less effective in PGC1α displacement. The statistical parameter Z′ factor that measures the robustness of the assay was greater than 0.8 for displacement of PGC1α from ERRγ 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 ERRγ results in differential affinity of coregulators for ERRγ due to unique ligand-induced conformations.

Use of Homogeneous Time-Resolved Fluorescence Energy Transfer in the Measurement of Nuclear Receptor Activation

Nuclear receptors (NRs) are a superfamily of ligand-dependent transcription factors that mediate the effects of hormones and other endogenous ligands to regulate the expression of specific genes. NRs are clearly important targets for drug discovery. Ligand-dependent protein-protein interactions between NRs and NR coactivators (NRCoAs) are a critical step in regulation of transcription. Homogeneous time-resolved fluorescence (HTRF) energy transfer technology is sensitive, homogeneous, and nonradioactive. These characteristics make this approach attractive for developing high-throughput screening assays. The long-lived nature of the fluorescence of europium cryptate combined with a time delay in reading facilitates the homogeneous nature of the assay. Importantly, the introduction of lanthanides (with R0 values as great as 90 Å in HTRF) make HTRF amenable to be used for protein-protein interactions. In this article we review, using peroxisome proliferator-activated receptor (PPAR)γ as a model system, a novel approach for characterizing the ligand-dependent interaction between NR and NRCoA using HTRF technology and its potential uses in small-molecule screening, profiling selectivity of NR-NRCoA paired interactions, and profiling NR ligands as agonists versus partial agonists or antagonists.