Time-resolved Fluorescence Resonance Energy Transfer Assay Research Articles

TR-FRET assays of Huntingtin protein fragments reveal temperature and polyQ length-dependent conformational changes.

Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) technology is a widely used immunoassay that enables high-throughput quantitative measurements of proteins of interest. One of the well established examples is the TR-FRET assay for mutant huntingtin protein (HTT), which is the major cause of the neurodegenerative Huntington's disease (HD). To measure the mutant HTT protein, the published assays utilize a polyQ antibody, MW1, paired with HTT N-terminal antibodies. MW1 has much higher apparent affinity to mutant HTT with expanded polyQ stretch than to wild-type HTT with shorter polyQ, and thus the assays detect mutant HTT preferentially. Here we report a reversible temperature dependent change of TR-FRET signals for HTT N-terminal fragments: the signals become higher when the temperature is lowered from room temperature to 4°C. Interestingly, the temperature sensitivity of the TR-FRET signals is much higher for the Q25 (wild-type) than for the Q72 (mutant) protein. We further revealed that it is likely due to a temperature and polyQ length-dependent structural or spatial change of HTT, which is potentially useful for understanding polyQ structure and toxicity.

Frontiers commentary on tallet et Al. Investigation of prolactin receptor activation and blockade using time-resolved fluorescence resonance energy transfer.

Frontiers commentary on tallet et Al. Investigation of prolactin receptor activation and blockade using time-resolved fluorescence resonance energy transfer.

TR-FRET Assays for Endogenous Huntingtin Protein Level in Mouse Cells.

High-throughput measurement of huntingtin (Htt) levels is useful for Huntington's disease research. For example, identification of genetic or chemical modifiers that reduce Htt levels by high-throughput screening provides promising strategy for HD drug discovery. In the human cells, high-throughput measurement of Htt levels has been established based on the Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) technology, using the 2B7/MW1 antibody pair. Unfortunately, application of this assay in the mouse cells has been problematic due to discrepancies between TR-FRET signals and Western-blots, possibly caused by non-specific antibody binding. Here we report TR-FRET assays that are able to detect endogenous Htt levels of the mouse striatal cell line (STHdh).

A Time-Resolved Fluorescence Resonance Energy Transfer Assay for High-Throughput Screening of 14-3-3 Protein–Protein Interaction Inhibitors

Protein-protein interaction networks mediate diverse biological processes by regulating various signaling hubs and clusters. 14-3-3 proteins, a family of phosphoserine/threonine-binding molecules, serve as major interaction hubs in eukaryotic cells and have emerged as promising therapeutic targets for various human diseases. In order to identify chemical probes for mechanistic studies and for potential therapeutic development, we have developed highly sensitive bioassays to monitor the interaction of 14-3-3 with a client protein. In this study, we describe a homogenous time-resolved fluorescence resonance energy transfer (TR-FRET) assay to detect the interaction of 14-3-3 with Bad, a proapoptotic member of the Bcl-2 family. Through a series of titration studies in which europium-labeled 14-3-3 serves as an FRET donor and a Dy647-labeled phosphorylated Bad, the peptide acts as an FRET acceptor, we have achieved a robust TR-FRET assay that is suitable for high-throughput screening (HTS) with an excellent signal-to-background ratio of >20 and Z' values >0.7. This assay was further miniaturized to a 1,536-well format for ultra-HTS (uHTS), and exhibited a similar robust performance. The utility and performance of the assay for uHTS were validated by (i) known inhibitors, including peptide R18 and small molecule FOBISIN101, and (ii) screening of a 51,200 compound library. This simple and robust assay is generally applicable to detect the interaction of 14-3-3 with other client proteins. It provides a sensitive and easy-to-use tool to facilitate the discovery of 14-3-3 protein inhibitors as well as to study 14-3-3-mediated protein-protein interactions.

Development of time resolved fluorescence resonance energy transfer-based assay for FXR antagonist discovery

FXR (farnesoid X receptor, NRIH4), a nuclear receptor, plays a major role in the control of cholesterol metabolism. FXR ligands have been investigated in preclinical studies for targeted therapy against metabolic diseases, but have shown limitations. Therefore, there is a need for new agonist or antagonist ligands of FXR, both for potential clinical applications, as well as to further elucidate its biological functions. Here we describe the use of the X-ray crystal structure of FXR complexed with the potent small molecule agonist GW4064 to design and synthesize a novel fluorescent, high-affinity probe (DY246) for time resolved fluorescence resonance energy transfer (TR-FRET) assays. We then used the TR-FRET assay for high throughput screening of a library of over 5000 bioactive compounds. From this library, we identified 13 compounds that act as putative FXR transcriptional antagonists.

Screening for SERCA Activators using a High-throughput Time-Resolved FRET Assay

We used a prototype time-resolved fluorescence lifetime microplate reader to carry out a high-throughput screen designed to identify compounds that interact with the sarcoplasmic reticulum calcium ATPase (SERCA). SERCA is essential for the Ca homeostasis in many cell types. Insufficient SERCA activity leads to cardiovascular disease, muscular dystrophy, skin disease, and diabetes. Our goal is to discover activators of SERCA that can be developed into drugs to treat diseases in which Ca transport is deficient. The fluorescence lifetime plate reader was made possible by our recent development of fast time-resolved fluorescence by direct waveform recording, which achieves 105 higher throughput than the conventional single-photon counting technology. using this plate reader, we detected fluorescence resonance energy transfer (FRET) between IAEDANS-labeled SERCA and nucleotide analog TNPADP in native sarcoplasmic reticulum membranes. This assay was designed to detect compounds that interact with SERCA and modify either the enzyme's structure or the binding affinity of TNP-ADP. Initial hit compounds were further analyzed in functional assays. Upon screening a small (1300 compound) library, we determined that the time-resolved microplate reader has at least 10x higher precision than a conventional intensity-based microplate reader, raising the quality index (z') of our assay from marginal, in the intensity reader, to excellent. A 384-well plate is read with high precision in 2 min, which allows screening of thousands of compounds/day. An important advantage of the time-resolved fluorescence measurement is that it provides detailed structural information, thus enabling discovery of multiple classes of compounds during the primary screen.

Lipid-Sensing High-Throughput ApoA-I Assays

Apolipoprotein A-I (ApoA-I), a primary protein component of high-density lipoprotein (HDL), plays an important role in cholesterol metabolism mediating the formation of HDL and the efflux of cellular cholesterol from macrophage foam cells in arterial walls. Lipidation of ApoA-I is mediated by adenosine triphosphate (ATP) binding cassette A1 (ABCA1). Insufficient ABCA1 activity may lead to increased risk of atherosclerosis due to reduced HDL formation and cholesterol efflux. The standard radioactive assay for measuring cholesterol transport to ApoA-I has low throughput and poor dynamic range, and it fails to measure phospholipid transfer. We describe the development of two sensitive, nonradioactive high-throughput assays that report on the lipidation of ApoA-I: a homogeneous assay based on time-resolved fluorescence resonance energy transfer (TR-FRET) and a discontinuous assay that uses the label-free Epic platform. The TR-FRET assay employs HiLyte Fluor 647–labeled ApoA-I with N-terminal biotin bound to streptavidin-terbium. When fluorescent ApoA-I was incorporated into HDL, TR-FRET decreased proportionally to the increase in the ratio of lipids to ApoA-I, demonstrating that the assay was sensitive to the amount of lipid bound to ApoA-I. In the Epic assay, biotinylated ApoA-I was captured on a streptavidin-coated biosensor. Measured resonant wavelength shift was proportional to the amount of lipids associated with ApoA-I, indicating that the assay senses ApoA-I lipidation.

Time-Resolved Fluorescence Resonance Energy Transfer as a Versatile Tool in the Development of Homogeneous Cellular Kinase Assays

Homogeneous cellular assays can streamline product detection in the drug discovery process. One commercially available assay employing time-resolved fluorescence resonance energy transfer (TR-FRET) that detects phosphorylated products was used to evaluate inhibitors of the receptor tyrosine kinase AXL in a cell line expressing an AXL-green fluorescent protein fusion protein. This TR-FRET assay was modified to evaluate the phosphorylation state of the AXL family member MER in a cell line expressing MER with a V5 tag by adding a fluorescein-labeled anti-V5 antibody. This homogeneous cellular assay was further modified to evaluate the nonreceptor tyrosine kinase focal adhesion kinase (FAK) in cell lines that expressed an untagged kinase by the inclusion of a commercially available anti-FAK antibody conjugated with an acceptor dye. The methods described here can be further adapted for TR-FRET detection of other cellular kinase activities.

High-Throughput Fluorescence Polarization Assay for Chemical Library Screening against Anti-Apoptotic Bcl-2 Family Member Bfl-1

Overexpression of the anti-apoptotic Bcl-2 family proteins occurs commonly in human cancers. Bfl-1 is highly expressed in some types of malignant cells, contributing significantly to tumor cell survival and chemoresistance. Therefore, it would be desirable to have chemical antagonists of Bfl-1. To this end, we devised a fluorescence polarization assay (FPA) using Bfl-1 protein and fluorescein-conjugated Bid BH3 peptide, which was employed for high-throughput screening of chemical libraries. Approximately 66 000 compounds were screened for the ability to inhibit BH3 peptide binding to Bfl-1, yielding 14 reproducible hits with ≥50% displacement. After dose-response analysis and confirmation using a secondary assay based on time-resolved fluorescence resonance energy transfer (TR-FRET), two groups of Bfl-1-specific inhibitors were identified, including chloromaleimide and sulfonylpyrimidine series compounds. FPAs generated for each of the six anti-apoptotic Bcl-2 proteins demonstrated selective binding of both classes of compounds to Bfl-1. Analogs of the sulfonylpyrimidine series were synthesized and compared with the original hit for Bfl-1 binding by both FPAs and TR-FRET assays. The resulting structure-activity relation analysis led to the chemical probe compound CID-2980973 (ML042). Collectively, these findings demonstrate the feasibility of using the HTS assay for discovery of selective chemical inhibitors of Bfl-1.

TR-FRET-Based High-Throughput Screening Assay for Identification of UBC13 Inhibitors

UBC13 is a noncanonical ubiquitin conjugating enzyme (E2) that has been implicated in a variety of cellular signaling processes due to its ability to catalyze formation of lysine 63–linked polyubiquitin chains on various substrates. In particular, UBC13 is required for signaling by a variety of receptors important in immune regulation, making it a candidate target for inflammatory diseases. UBC13 is also critical for double-strand DNA repair and thus a potential radiosensitizer and chemosensitizer target for oncology. The authors developed a high-throughput screening (HTS) assay for UBC13 based on the method of time-resolved fluorescence resonance energy transfer (TR-FRET). The TR-FRET assay combines fluorochrome (Fl)–conjugated ubiquitin (fluorescence acceptor) with terbium (Tb)–conjugated ubiquitin (fluorescence donor), such that the assembly of mixed chains of Fl- and Tb-ubiquitin creates a robust TR-FRET signal. The authors defined conditions for optimized performance of the TR-FRET assay in both 384- and 1536-well formats. Chemical library screens (total 456 865 compounds) were conducted in high-throughput mode using various compound collections, affording superb Z′ scores (typically >0.7) and thus validating the performance of the assays. Altogether, the HTS assays described here are suitable for large-scale, automated screening of chemical libraries in search of compounds with inhibitory activity against UBC13.

High-Throughput TR-FRET Assays for Identifying Inhibitors of LSD1 and JMJD2C Histone Lysine Demethylases

Lysine demethylase 1 (LSD1) and Jumonji C domain–containing oxygenase D2C (JMJD2C) participate in regulating the methylation status of histone H3 lysine residues. In some contexts, LSD1 and JMJD2C activity causes enhanced cellular proliferation, which may lead to tumorigenesis. The authors explored the utility of time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassays, which employed peptides consisting of the first 21 amino acids of histone H3 in which lysine 4 (H3K4) or lysine 9 (H3K9) was methylated (me) to quantify LSD1 and JMJD2C activity. The LSD1 assay monitored demethylation of the H3K4me1 peptide using an antibody that recognizes H3K4me1 but not the unmethylated peptide product. The JMJD2C assay measured demethylation of H3K9me3 with an antibody that selectively recognizes H3K9me2. The optimized conditions resulted in robust assays (Z′ > 0.7) that required only 3 to 6 nM of enzyme in a reaction volume of 6 to 10 µL. These assays were used to compare the activity of different LSD1 constructs and to determine the apparent Km of each JMJD2C substrate. Finally, both assays were used in a high-throughput setting for identifying demethylase inhibitors. Compounds discovered by these TR-FRET methods may lead to powerful tools for ascertaining the roles of demethylases in a cellular context and ultimately for potential cancer treatments.

Abstract C94: A novel binding assay to identify inhibitors that bind to inactive forms of Bruton's tyrosine kinase based on fluorescence resonance energy transfer.

Abstract Bruton's tyrosine kinase (Btk) is a member of the Tec family of non-receptor tyrosine kinases. Btk is one of the crucial kinases for the B cell maturation and also involved in mast cell activation through the high-affinity IgE receptor. Therefore Btk is an attractive target for the potential treatment of multiple therapeutic areas that involve B-cell and/or mast cell activation, such as B cell malignances, asthma, and rheumatoid arthritis. In order to develop a selective Btk inhibitor, it is important to identify a highly selective compound as the drug discovery starting point. Generally, activity-based high-throughput screening (HTS) using active kinase has been used to identify hit compounds by measuring the inhibition of substrate phosphorylation. However, the activity-based HTS campaign frequently results in the enrichment of classical ATP competitive inhibitors, which would often require considerable medicinal chemistry efforts to increase kinase selectivity, because the human protein kinase family consists of over 500 enzymes with very similar active sites. Here we describe a novel approach to identify inhibitors of inactive form of Btk. Method: Several compounds that inhibit Btk kinase activity have been described in the literature. One compound, CGI1746 is reported to bind to the inactive form of Btk, so we synthesized FITC-labeled CGI1746 derivatives with various linker groups as fluorescent probes that preferentially bind to an inactive form of Btk. To develop a screening system targeting the inactive form of Btk, we designed a competitive binding assay based on time-resolved fluorescence resonance energy transfer (TR-FRET) platform that measures the binding of the FITC-labeled CGI1746 probe to biotinylated Btk. Result: We produced a single endogenous biotinylated Btk protein (BTN-Btk), and the BTN-Btk protein was treated with ATP (autophosphorylation) or with Lambda Protein Phosophatase (dephosphorylation) to obtain the activated and unactivated BTN-Btk respectively. Interestingly, dephosphorylated BTN-Btk still showed weak catalytic activity indicating phosphatase-treatment might not able to remove phosphates of Btk completely. We synthesized various FITC-labeled CGI1746 derivatives with different linkers, and determined their IC50 value against both the activated and unactivated BTN-Btk by measuring substrate phosphorylation. Several compounds were found to maintain the original inhibitory potency even after introducing a FITC fluorophore. To test if the FITC-labeled CGI1746 derivatives can be applied to a competitive binding assay, we determined the Kd value of each probes with the TR-FRET assay platform. Most of the probes synthesized were found to be excellent FRET acceptors with high affinity and low background signals. Detailed data of this novel binding assay targeting the inactive form of Btk will be presented in this poster. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C94.

A Novel High-Throughput Screening Assay for Discovery of Molecules That Increase Cellular Tetrahydrobiopterin

Tetrahydrobiopterin (BH(4)) is an essential cofactor for the nitric oxide (NO) synthases and the aromatic amino acid hydroxylases. Insufficient BH(4) has been implicated in various cardiovascular and neurological disorders. GTP cyclohydrolase 1 (GTPCH-1) is the rate-limiting enzyme for de novo biosynthesis of BH(4). The authors have recently shown that the interaction of GTPCH-1 with GTP cyclohydrolase feedback regulatory protein (GFRP) inhibits endothelial GTPCH-1 enzyme activity, BH(4) levels, and NO production. They propose that agents that disrupt the GTPCH-1/GFRP interaction can increase cellular GTPCH-1 activity, BH(4) levels, and NO production. They developed and optimized a novel time-resolved fluorescence resonance energy transfer (TR-FRET) assay to monitor the interaction of GTPCH-1 and GFRP. This assay is highly sensitive and stable and has a signal-to-background ratio (S/B) greater than 12 and a Z' factor greater than 0.8. This assay was used in an ultra-high-throughput screening (uHTS) format to screen the Library of Pharmacologically Active Compounds. Using independent protein-protein interaction and cellular activity assays, the authors identified compounds that disrupt GTPCH-1/GFRP binding and increase endothelial cell biopterin levels. Thus, this TR-FRET assay could be applied in future uHTS of additional libraries to search for molecules that increase GTPCH-1 activity and BH(4) levels.

Abstract 2878: A sensitive cell signaling assay for low level activation of endogenous receptors – a comparison of AlphaScreen® SureFire® and TR-FRET assays

Abstract Two closely related proteins, ERK1 and ERK2, belong to a widely conserved family of serine/threonine protein kinases whose activities are regulated by phosphorylation. These kinases are components of signal transduction pathways which control critical cellular processes such as growth, differentiation, metabolism and apoptosis. In addition to normal cellular processes, these pathways have been associated with signaling in many cancers. Phosphorylation of ERK 1/2 has been shown to be mediated by signal transduction pathways which are activated by ligand binding to both receptor tyrosine kinase receptors (RTKs) and G protein-coupled receptors (GPCRs). The focus of cell signaling studies is increasingly shifting to endogenous cell surface receptors rather than recombinant, over-expressed receptors. The hope is that these model systems will have greater biological relevance, more closely reflecting the physiology of the organism studied. In such studies the sensitivity of the assay platform becomes critical for success, especially when the endogenous receptors are activated at low levels. Several technologies have been used to quantitate relative levels of protein phosphorylation, including western blot, time-resolved fluorescence resonance energy transfer (TR-FRET), and AlphaScreen® SureFire® assays. AlphaScreen® SureFire® assays are homogenous, bead-based assays designed to measure endogenous levels of intracellular phosphorylated proteins. Here we describe the performance of the AlphaScreen® SureFire® p-ERK 1/2 assay compared to that of a TR-FRET assay. For this study we used two different cell lines. The first was CHO-K1 cells stably transfected with the human somatostatin sst5 GPCR, which is stimulated by its natural ligand somatostatin-28. The second was wild-type HEK 293 cells, which express endogenous receptors for epidermal growth factor (an RTK), and endogenous GPCRs activated by acetylcholine and ATP. In general, the AlphaScreen® SureFire® assay gave higher S/B ratios than the TR-FRET assay in all conditions tested, which in turn can have a significant impact on the assay robustness. In situations that gave wide response windows, such as stimulation of the CHO over-expressed somatostatin receptor or the HEK 293 endogenous EGF receptor, both assay kits yielded excellent results. However, the HEK 293 endogenous GPCRs for acetylcholine and ATP are coupled to a lower production of p-ERK, and in those cases the AlphaScreen® SureFire® assay proved to be much more robust, with significantly higher overall signal levels and S/B ratios. 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 2878. doi:10.1158/1538-7445.AM2011-2878

Time-resolved Fluorescence Resonance Energy Transfer (TR-FRET) to Analyze the Disruption of EGFR/HER2 Dimers: A NEW METHOD TO EVALUATE THE EFFICIENCY OF TARGETED THERAPY USING MONOCLONAL ANTIBODIES

In oncology, simultaneous inhibition of epidermal growth factor receptor (EGFR) and HER2 by monoclonal antibodies (mAbs) is an efficient therapeutic strategy but the underlying mechanisms are not fully understood. Here, we describe a time-resolved fluorescence resonance energy transfer (TR-FRET) method to quantify EGFR/HER2 heterodimers on cell surface to shed some light on the mechanism of such therapies. First, we tested this antibody-based TR-FRET assay in NIH/3T3 cell lines that express EGFR and/or HER2 and in various tumor cell lines. Then, we used the antibody-based TR-FRET assay to evaluate in vitro the effect of different targeted therapies on EGFR/HER2 heterodimers in the ovarian carcinoma cell line SKOV-3. A simultaneous incubation with Cetuximab (anti-EGFR) and Trastuzumab (anti-HER2) disturbed EGFR/HER2 heterodimers resulting in a 72% reduction. Cetuximab, Trastuzumab or Pertuzumab (anti-HER2) alone induced a 48, 44, or 24% reduction, respectively. In contrast, the tyrosine kinase inhibitors Erlotinib and Lapatinib had very little effect on EGFR/HER2 dimers concentration. In vivo, the combination of Cetuximab and Trastuzumab showed a better therapeutic effect (median survival and percentage of tumor-free mice) than the single mAbs. These results suggest a correlation between the extent of the mAb-induced EGFR/HER2 heterodimer reduction and the efficacy of such mAbs in targeted therapies. In conclusion, quantifying EGFR/HER2 heterodimers using our antibody-based TR-FRET assay may represent a useful method to predict the efficacy and explain the mechanisms of action of therapeutic mAbs, in addition to other commonly used techniques that focus on antibody-dependent cellular cytotoxicity, phosphorylation, and cell proliferation.

A Homogeneous Single-Label Time-Resolved Fluorescence cAMP Assay

G-protein-coupled receptors (GPCRs) are an important class of pharmaceutical drug targets. Functional high-throughput GPCR assays are needed to test an increasing number of synthesized novel drug compounds and their function in signal transduction processes. Measurement of changes in the cyclic adenosine monophosphate (cAMP) concentration is a widely used method to verify GPCR activation in the adenylyl cyclase pathway. Here, a single-label time-resolved fluorescence and high-throughput screening (HTS)-feasible method was developed to measure changes in cAMP levels in HEK293(i) cells overexpressing either β(2)-adrenergic or δ-opioid receptors. In the quenching resonance energy transfer (QRET) technique, soluble quenchers reduce the signal of unbound europium(III)-labeled cAMP in solution, whereas the antibody-bound fraction is fluorescent. The feasibility of this homogeneous competitive assay was proven by agonist-mediated stimulation of receptors coupled to either the stimulatory G(s) or inhibitory G(i) proteins. The reproducibility of the assays was excellent, and Z' values exceeded 0.7. The dynamic range, signal-to-background ratio, and detection limit were compared with a commercial time-resolved fluorescence resonance energy transfer (TR-FRET) assay. In both homogeneous assays, similar assay parameters were obtained when adenylyl cyclase was stimulated directly by forskolin or via agonist-mediated activation of the G(s)-coupled β(2)AR. The advantage of using the single-label approach relates to the cost-effectiveness of the QRET system compared with the two-label TR-FRET assay as there is no need for labeling of two binding partners leading to reduced requirements for assay optimization.

A PCSK9-binding antibody that structurally mimics the EGF(A) domain of LDL-receptor reduces LDL cholesterol in vivo1[S

Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) regulates LDL cholesterol levels by inhibiting LDL receptor (LDLr)-mediated cellular LDL uptake. We have identified a fragment antigen-binding (Fab) 1D05 which binds PCSK9 with nanomolar affinity. The fully human antibody 1D05-IgG2 completely blocks the inhibitory effects of wild-type PCSK9 and two gain-of-function human PCSK9 mutants, S127R and D374Y. The crystal structure of 1D05-Fab bound to PCSK9 reveals that 1D05-Fab binds to an epitope on the PCSK9 catalytic domain which includes the entire LDLr EGF(A) binding site. Notably, the 1D05-Fab CDR-H3 and CDR-H2 loops structurally mimic the EGF(A) domain of LDLr. In a transgenic mouse model (CETP/LDLr-hemi), in which plasma lipid and PCSK9 profiles are comparable to those of humans, 1D05-IgG2 reduces plasma LDL cholesterol to 40% and raises hepatic LDLr protein levels approximately fivefold. Similarly, in healthy rhesus monkeys, 1D05-IgG2 effectively reduced LDL cholesterol 20%-50% for over 2 weeks, despite its relatively short terminal half-life (t(1/2) = 3.2 days). Importantly, the decrease in circulating LDL cholesterol corresponds closely to the reduction in free PCSK9 levels. Together these results clearly demonstrate that the LDL-lowering effect of the neutralizing anti-PCSK9 1D05-IgG2 antibody is mediated by reducing the amount of PCSK9 that can bind to the LDLr.

Identification of Novel Scaffolds for IκB Kinase Beta Inhibitor via a High Throughput Screening TR-FRET Assay

Control of NF-κB release through the inhibition of IκB kinase β (IKKβ) has been identified as a potential target for the treatment of inflammatory and autoimmune diseases. To screen the small molecule compound library against IKKβ, a high-throughput screening (HTS) campaign was carried out using immobilized metal affinity for phosphochemicals (IMAP)-based time-resolved fluorescence resonance energy transfer (TR-FRET) assay. Through serial optimization of assay conditions, the Z' value was achieved at 0.88 from the pilot library screening of the most diverse 7,243 compounds with reconfirmation rate of 63%. The results from this HTS campaign identified three novel scaffolds for the prospective IKKβ inhibitor, such as 7-benzoyl-4-phenylcyclopenta[1,2] oxazine, 1-(thiophen or furan)-2,3-dihydroimidazo[1,5] pyridine and 2-phenyloxazolo[5,4] pyridine. Particularly, 7-benzoyl-4-phenylcyclopenta[1,2] oxazine derivatives presented potent inhibitory activity and selectivity for IKKβ. These findings suggest that the current TR-FRET assay system for IKKβ was successful to identify hits for novel IKKβ inhibitors as a robust, reproducible and sensitive HTS system.

Multiplexed Random Peptide Library and Phospho-Specific Antibodies Facilitate Human Polo-Like Kinase 1 Inhibitor Screen

One of the challenges to develop time-resolved fluorescence resonance energy transfer (TR-FRET) assay for serine/threonine (Ser/Thr) protein kinase is to select an optimal peptide substrate and a specific phosphor Ser/Thr antibody. This report describes a multiplexed random screen-based development of TR-FRET assay for ultra-high-throughput screening (uHTS) of small molecule inhibitors for a potent cancer drug target polo-like kinase 1 (Plk1). A screen of a diverse peptide library in a 384-well plate format identified several highly potent substrates that share the consensus motif for phosphorylation by Plk1. Their potencies were comparable to FKD peptide, a designed peptide substrate derived from well-described Plk1 substrate Cdc25C. A specific anti-phosphor Ser/Thr antibody p(S/T)F antibody that detects the phosphorylation of FKD peptide was screened out of 87 antibodies with time-resolved fluorometry technology in a 96-well plate format. Using FKD peptide and p(S/T)F antibody, we successfully developed a robust TR-FRET assay in 384-well plate format, and further miniaturized this assay to 1,536-well plate format to perform uHTS. We screened about 1.2 million compounds for Plk1 inhibitors using a Plk1 deletion mutant that only has the kinase domain and subsequently screened the same compound library using a full-length active-mutant Plk1. These uHTSs identified a number of hit compounds, and some of them had selectivity to either the deletion mutant or the full-length protein. Our results prove that a combination of random screen for substrate peptide and phospho-specific antibodies is very powerful strategy to develop TR-FRET assays for protein kinases.

A Time-Resolved Fluorescence–Resonance Energy Transfer Assay for Identifying Inhibitors of Hepatitis C Virus Core Dimerization

Binding of hepatitis C virus (HCV) RNA to core, the capsid protein, results in the formation of the nucleocapsid, the first step in the assembly of the viral particle. A novel assay was developed to discover small molecule inhibitors of core dimerization. This assay is based on time-resolved fluorescence resonance energy transfer (TR-FRET) between anti-tag antibodies labeled with either europium cryptate (Eu) or allophycocyanin (XL-665). The N-terminal 106-residue portion of core protein (core106) was tagged with either glutathione-S-transferase (GST) or a Flag peptide. Tag-free core106 was selected as the reference inhibitor. The assay was used to screen the library of pharmacologically active compounds (LOPAC) consisting of 1,280 compounds and a 2,240-compound library from the Center for Chemical Methodology and Library Development at Boston University (CMLD-BU). Ten of the 28 hits from the primary TR-FRET run were confirmed in a secondary amplified luminescent proximity homogeneous assay (ALPHA screen). One hit was further characterized by dose-response analysis yielding an IC(50) of 9.3 microM. This 513 Da compound was shown to inhibit HCV production in cultured hepatoma cells.