Homogeneous Time-resolved Fluorescence Resonance Energy Research Articles

Affinities between the Binding Partners of the HIV-1 Integrase Dimer-Lens Epithelium-derived Growth Factor (IN Dimer-LEDGF) Complex

The interaction between lens epithelium-derived growth factor/transcriptional co-activator p75 (LEDGF) and human immunodeficiency virus type 1 (HIV-1) integrase (IN) is essential for HIV-1 replication. Homogeneous time-resolved fluorescence resonance energy transfer assays were developed to characterize HIV-1 integrase dimerization and the interaction between LEDGF and IN dimers. Using these assays in an equilibrium end point dose-response format with mathematical modeling, we determined the dissociation constants of IN dimers (K(dimer) = 67.8 pm) and of LEDGF from IN dimers (K(d) = 10.9 nm). When used in a kinetic format, the assays allowed the determination of the on- and off-rate constants for these same interactions. Integrase dimerization had a k(on) of 0.1247 nm(-1) x min(-1) and a k(off) of 0.0080 min(-1) resulting in a K(dimer) of 64.5 pm. LEDGF binding to IN dimers had a k(on) of 0.0285 nm(-1).min(-1) and a k(off) of 0.2340 min(-1) resulting in a K(d) of 8.2 nm. These binding assays can also be used in an equilibrium end point competition format. In this format, the IN catalytic core domain produced a K(i) of 15.2 nm while competing for integrase dimerization, confirming the very tight interaction of IN with itself. In the same format, LEDGF produced a K(i) value of 35 nm when competing for LEDGF binding to IN dimers. In summary, this study describes a methodology combining homogeneous time-resolved fluorescence resonance energy transfer and mathematical modeling to derive the affinities between IN monomers and between LEDGF and IN dimers. This study revealed the significantly tighter nature of the IN-IN dimer compared with the IN-LEDGF interaction.

Identification of Small Molecule Inhibitors of the Mitotic Kinase Haspin by High-Throughput Screening Using a Homogeneous Time-Resolved Fluorescence Resonance Energy Transfer Assay

Haspin/Gsg2 is a kinase that phosphorylates histone H3 at Thr-3 (H3T3ph) during mitosis. Its depletion by RNA interference results in failure of chromosome alignment and a block in mitosis. Haspin, therefore, is a novel target for development of antimitotic agents. We report the development of a high-throughput time-resolved fluorescence resonance energy transfer (TR-FRET) kinase assay for haspin. Histone H3 peptide was used as a substrate, and a europium-labeled H3T3ph phosphospecific monoclonal antibody was used to detect phosphorylation. A library of 137632 small molecules was screened at K(m) concentrations of ATP and peptide to allow identification of diverse inhibitor types. Reconfirmation of hits and IC( 50) determinations were carried out with the TR-FRET assay and by a radiometric assay using recombinant histone H3 as the substrate. A preliminary assessment of specificity was made by testing inhibition of 2 unrelated kinases. EC( 50) values in cells were determined using a cell-based ELISA of H3T3ph. Five compounds were selected as leads based on potency and chemical structure considerations. These leads form the basis for the development of specific inhibitors of haspin that will have clear utility in basic research and possible use as starting points for development of antimitotic anticancer therapeutics.

A homogeneous time-resolved fluorescence resonance energy transfer assay for phosphatidylserine exposure on apoptotic cells

A simple, “mix-and-measure” microplate assay for phosphatidylserine (PtdSer) exposure on the surface of apoptotic cells is described. The assay exploits the fact that annexin V, a protein with high affinity and specificity for PtdSer, forms trimers and higher order oligomers on binding to membranes containing PtdSer. The transition from soluble monomer to cell-bound oligomer is detected using time-resolved fluorescence resonance energy transfer from europium chelate-labeled annexin V to Cy5-labeled annexin V. PtdSer detection is achieved by a single addition of a reagent mix containing labeled annexins and calcium ions directly to cell cultures in a 96-well plate, followed by a brief incubation before fluorescence measurement. The assay can be used to quantify PtdSer exposure on both suspension cells and adherent cells in situ. This method is simpler and faster than existing annexin V binding assays based on flow cytometry or microscopy, and it yields precise data with Z’ values of 0.6–0.7.

High-Throughput Screen for Small Molecule Inhibitors of Mint1-PDZ Domains

Several hundred PDZ (postsynaptic density-95, Drosophila disks-large, ZO-1) domain-containing proteins have been identified in the human genome. PDZ domains play a critical role in organization and function of cellular signaling pathways. Thus, small molecule inhibitors of PDZ domain association with their targets have wide potential applications as research and therapeutic agents. PDZ domains typically bind to a carboxyl-terminal tail of the target protein. Here we describe a high-throughput screening (HTS) assay for small molecule inhibitors of association between Mint1-PDZ domains and N-type Ca2+ channel carboxyl-terminal peptide (NC peptide). The performance of a homogeneous time-resolved fluorescence resonance energy transfer (HTRF) and an amplified luminescent proximity homogeneous assay (ALPHA) were systematically compared in parallel pilot HTS experiments with glutathione S-transferase-Mint1-PDZ1/2 protein and biotinylated NC peptide. Both of the two assays showed similar sensitivities in our target protein assay. Using HTRF-based assay we screened a library of 100,000 small molecule compounds and identified a number of potential "hits." The activity of isolated "hits" was confirmed by ALPHA assay. However, further evaluation revealed that isolated "hits" most likely act as "promiscuous binders," not as specific Mint-PDZ inhibitors, and that additional screening will be required to identify the true Mint-PDZ inhibitors. The assays described provided an example of HTS for a small molecule inhibitor of Mint-PDZ domain that can be easily adapted to other PDZ domain-mediated interactions.

High Capacity Homogeneous Non-Radioactive Cortisol Detection Assays for Human 11β-Hydroxysteroid Dehydrogenase Type 1

11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) catalyzes the interconversion of inert glucocorticoid (cortisone) to the active glucocorticoid (cortisol) and is enriched in liver and fat tissues. Increasing evidence suggests that selective inhibition of 11beta-HSD1 may reduce the excess glucocorticoid levels that underlie the etiology of many common disorders that constitute the metabolic syndrome. Measurement of 11beta-HSD1 activity has historically involved the detection of cortisol by methods unfavorable for large-scale screening, such as high performance liquid chromatography or thin layer chromatography. Here we describe the development and validation of novel homogeneous time-resolved fluorescence resonance energy transfer (TR-FRET) and electrochemiluminescence assays for the measurement of cortisol. These non-radioactive assays were easy to perform and produced robust results with reference compound values comparable to those obtained by conventional methods. The TR-FRET assay was easily automated and was successfully employed for the high-throughput screening of a large compound library for inhibitors of purified human recombinant 11beta-HSD1.

Assay Concordance between SPA and TR-FRET in High-Throughput Screening

High-throughput screening (HTS) of large chemical libraries has become the main source of new lead compounds for drug development. Several specialized detection technologies have been developed to facilitate the cost- and time-efficient screening of millions of compounds. However, concerns have been raised, claiming that different HTS technologies may produce different hits, thus limiting trust in the reliability of HTS data. This study was aimed to investigate the reliability of the authors most frequently used assay techniques: scintillation proximity assay (SPA) and homogeneous time-resolved fluorescence resonance energy transfer (TR-FRET). To investigate the data concordance between these 2 detection technologies, the authors screened a large subset of the Schering compound library consisting of 300,000 compounds for inhibitors of a nonreceptor tyrosine kinase. They chose to set up this study in realistic HTS scale to ensure statistical significance of the results. The findings clearly demonstrate that the choice of detection technology has no significant impact on hit finding, provided that assays are biochemically equivalent. Data concordance is up to 90%. The little differences in hit findings are caused by threshold setting but not by systematic differences between the technologies. The most significant difference between the compared techniques is that in the SPA format, more false-positive primary hits were obtained.

A homogeneous time-resolved fluorescence resonance energy transfer assay for IL-13/IL-13Rα1 interaction

A homogeneous time-resolved fluorescence resonance energy transfer assay for IL-13/IL-13Rα1 interaction

Identification and characterization of a constitutively T-loop phosphorylated and active recombinant S6K1: Expression, purification, and enzymatic studies in a high capacity non-radioactive TR-FRET Lance assay

The p70 S6 ribosomal protein kinase 1 (S6K) is a substrate and effector of the mammalian target of rapamycin (mTOR). The mTOR/S6K pathway is implicated in cancer and metabolic disorders. To study the molecular regulation of S6K and identify specific inhibitors, availability of active recombinant S6K and robust enzyme assays are critically needed. To date, however, expression of active recombinant S6K has not been feasible as S6K activation requires a cascade of phosphorylation events. We have compared several engineered S6K enzymes. Expression of the Flag-S6KΔCT(T389E) in HEK293 cells resulted in a highly active S6K that was constitutively phosphorylated on T229 in the activation-loop (T-loop). The active enzyme was readily purified in large scale by anti-Flag affinity chromatography achieving a high purity. We developed a high capacity homogeneous time-resolved fluorescence resonance energy transfer. Lance assay for measurement of substrate phosphorylation and analysis of kinetic parameters. The Michaelis constant ( K m) values of S6K for ATP and the Biotin-S6 substrate peptide were determined to be 21.4 ± 0.29 and 0.9 ± 0.48 μM, respectively. The Lance assay was further validated with a diverse panel of literature inhibitors, in which the PKC inhibitors staurosporine, Ro-318220, and the PKA inhibitor Balanol potently inhibited S6K. Dose–response and inhibition mechanism by these inhibitors were also studied. Our data provide a new simplified strategy to achieve rapid production of active S6K and demonstrate utility of the Lance assay for S6K enzyme screen in searching for specific inhibitors.

Cell surface detection of membrane protein interaction with homogeneous time-resolved fluorescence resonance energy transfer technology

Direct or indirect interactions between membrane proteins at the cell surface play a central role in numerous cell processes, including possible synergistic effects between different types of receptors. Here we describe a method and tools to analyze membrane protein–protein interaction at the surface of living cells. This technology is based on the use of specific antibodies directed against each partner and labeled either with europium cryptate or with Alexa Fluor 647. This allows the measurement of a fluorescence resonance energy transfer (FRET) signal in a time-resolved manner if both antibodies are in close proximity. This approach is here validated using the heterodimeric γ-aminobutyrate B receptor as a model. We show that after washing out the unbound antibodies, the time-resolved FRET signal can be measured together with the expression level of both partners via the quantification of the donor and the acceptor fluorophores bound to the cells. Thanks to the high sensitivity of this method and to the low concentration of antibodies required, we show that the signal can also be measured directly after the incubation period without washing out the unbound antibody (homogeneous time-resolved FRET). As such, this method is highly sensitive, reproducible, and compatible with the development of high-throughput screening protocols.

Cell surface detection of membrane protein interaction with homogeneous time-resolved fluorescence resonance energy transfer technology

Direct or indirect interactions between membrane proteins at the cell surface play a central role in numerous cell processes, including possible synergistic effects between different types of receptors. Here we describe a method and tools to analyze membrane protein–protein interaction at the surface of living cells. This technology is based on the use of specific antibodies directed against each partner and labeled either with europium cryptate or with Alexa Fluor 647. This allows the measurement of a fluorescence resonance energy transfer (FRET) signal in a time-resolved manner if both antibodies are in close proximity. This approach is here validated using the heterodimeric γ-aminobutyrate B receptor as a model. We show that after washing out the unbound antibodies, the time-resolved FRET signal can be measured together with the expression level of both partners via the quantification of the donor and the acceptor fluorophores bound to the cells. Thanks to the high sensitivity of this method and to the low concentration of antibodies required, we show that the signal can also be measured directly after the incubation period without washing out the unbound antibody (homogeneous time-resolved FRET). As such, this method is highly sensitive, reproducible, and compatible with the development of high-throughput screening protocols.

Development and comparison of two nonradioactive kinase assays for I kappa B kinase

In response to diverse stimuli, the transcription factor NF-κB is activated by the IKK kinase complex containing two kinases (IKKα and IKKβ) that phosphorylate IκB, an inhibitory protein of NF-κB. The phosphorylation of IκB results in ubiquitination and degradation of IκB, allowing NF-κB to translocate to the nucleus where it regulates its target genes. To elucidate the role of IKK in the NF-κB signaling pathway, we have developed and characterized two quantitative, sensitive, and nonradioactive assays for evaluating IKKβ activity: a dissociation-enhanced lanthanide fluorescence immunoassay called DELFIA and a homogeneous time-resolved fluorescence resonance energy transfer assay called LANCE. We show that the two assays have similar sensitivity and Michaelis constants ( K m ) for adenosine 5 ′-triphosphate and substrate; however, the LANCE format was far more efficient and easier to perform. Additionally, the assays were validated with the known kinase inhibitor K252a and several other kinase inhibitors, which showed that the IC 50 values of the two assays were comparable. In summary, both assays are quantitative, sensitive, reproducible, and amenable to high-throughput screening with improved waste management over radioactive assays.

Testing and validating a homogeneous immunometric assay for interference.

We analyzed 95 sera, demonstrating interference in a previous study, with the Kryptor homogeneous time-resolved fluorescence resonance energy transfer carcinoembryonic antigen (CEA) immunoassay (Brahms AG, Berlin, Germany). Only one serum differed, i.e., 6.0 microg/l for Kryptor vs. 13.3 microg/l for a microtiter plate in-house immunofluorometric assay (IFMA), using both aggregated mouse immunoglobulins as blocker and capture monoclonal antibody (Mab) F(ab')2-fragments to reduce interference. Sera were further analyzed with experimental IFMAs with intact capture Mabs and without blocker. The Kryptor-CEA assay Mab GFR44 (capture) had elevated results in 71% of sera with the Kryptor Mab G15 (tracer) or in 81% with our tracer Mab. G15 (capture) had 65% elevated results with GFR44 (tracer) or 99% with our tracer Mab. The latter was reduced from 99% to 62% by adding Kryptor blocker to the buffer or to 30% by adding our blocker. Finally, combining both assay Mabs and assay buffer from Kryptor still gave interference in 16% of sera. Kryptor-CEA assay results thus agreed with our in-house CEA assay results, showing no interference. As the Kryptor-CEA assay antibodies were sensitive to interference and the Kryptor-CEA assay buffer did not reduce interference as efficiently as our in-house assay buffer, the Kryptor-CEA assay format was crucial for the absence of interference.

Development of a fluorescence resonance energy transfer assay for measuring the activity of Streptococcus pneumoniae DNA ligase, an enzyme essential for DNA replication, repair, and recombination

DNA ligase is an enzyme essential for DNA replication, repair, and recombination in all organisms. Bacterial DNA ligases catalyze a NAD +-dependent DNA ligation reaction, i.e., the formation of a phosphodiester bond between adjacent 3 ′-OH and 5 ′-phosphate termini of dsDNA. Due to their essential nature, unique cofactor requirement, and widespread existence in nature, bacterial DNA ligases appear to be valuable targets for identifying novel antibacterial agents. To explore bacterial DNA ligases as antibacterial targets and further characterize them, we developed a simple, robust, homogeneous time-resolved fluorescence resonance energy transfer assay (TR-FRET) for measuring Streptococcus pneumoniae DNA ligase activity. This assay involves the use of one dsDNA molecule labeled with biotin and another dsDNA molecule labeled with Cy5, an acceptor fluorophore. During ligation reactions, the donor fluorophore europium (Eu 3+) labeled with streptavidin was added to the assay mixtures, which bound to the biotin label on the ligated products. This in turn resulted in the FRET from Eu 3+ to Cy5 due to their close proximity. The formation of ligation products was measured by monitoring the emission at 665 nm. This assay was validated by the experiments showing that the DNA ligase activity required NAD + and MgCl 2, and was inhibited by NMN and AMP, products of the ligase reaction. Using this assay, we determined the K m values of the enzyme for dsDNA substrates and NAD +, and the IC 50 values of NMN and AMP, examined the effects of MgCl 2 and PEG 8000 on the enzyme activity, optimized the concentrations of Eu 3+ in the assay, and validated its utilities for high-throughput screening and biochemical characterizations of this class of enzymes.

Comparison of Assay Technologies for a Tyrosine Kinase Assay Generates Different Results in High Throughput Screening

In today's high-throughput screening (HTS) environment, an increasing number of assay detection technologies are routinely utilized in lead finding programs. Because of the relatively broad applicability of several of these technologies, one is often faced with a choice of which technology to utilize for a specific assay. The aim of this study was to address the question of whether the same compounds would be identified from screening a set of samples in three different versions of an HTS assay. Here, three different versions of a tyrosine kinase assay were established using scintillation proximity assay (SPA), homogeneous time-resolved fluorescence resonance energy transfer (HTR-FRET), and fluorescence polarization (FP) technologies. In this study, 30,000 compounds were evaluated in each version of the kinase assay in primary screening, deconvolution, and dose-response experiments. From this effort, there was only a small degree of overlap of active compounds identified subsequent to the deconvolution experiment. When all active compounds were then profiled in all three assays, 100 and 101 active compounds were identified in the HTR-FRET and FP assays, respectively. In contrast, 40 compounds were identified in the SPA version of the kinase assay, whereas all of these compounds were detected in the HTR-FRET assay only 35 were active in the FP assay. Although there was good correlation between the IC(50) values obtained in the HTR-FRET and FP assays, poor correlations were obtained with the IC(50) values obtained in the SPA assay. These findings suggest that significant differences can be observed from HTS depending on the assay technology that is utilized, particularly in assays with high hit rates.