scholarly journals Optimizing a Kinase Assay for IKKβ on an HTS Station

2009 ◽  
Vol 14 (10) ◽  
pp. 1263-1268 ◽  
Author(s):  
Nunzianna Doti ◽  
Daniela Marasco ◽  
Carlo Pedone ◽  
Marco Sabatella ◽  
Menotti Ruvo
Keyword(s):  
High Throughput Screening ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Kinase Assay ◽  
Time Resolved Fluorescence ◽  
Glutathione S Transferase ◽  
Time Resolved ◽  
Assay Procedure ◽  
Pure Solvent ◽  
Data Points

Using a commercially available time-resolved fluorescence resonance energy transfer (TR-FRET)—based assay for IKKβ, the authors have automated the assay procedure on a high-throughput screening station to carry out screening campaigns on multiwell plates. They have determined the Z′ factor and optimized volumes, times, and time-resolved fluorescence parameters. They have also compared 2 kinases with different fusion tags, the influence of different enzyme/substrate ratios and of DMSO presence at different concentration. The authors found that glutathione S-transferase (GST)—fused IKKβ shows better signal-to-noise (S/N) ratios over the poly-histidine-tagged variant. The substrate can be used at 50 nM with optimal performances when the enzyme is used at 2 nM. DMSO at 0.2% and 1% only slightly affects the S/N ratio, whereas when used at 2%, the final concentration deriving from a 50-fold dilution from a 5-mM stock solution in pure solvent, S/N undergoes a decrease of about 15%. Under the optimized conditions, the assay Z′ factor calculated over 192 data points has an optimized value of 0.881 and allows the testing of 94 molecules in quadruplicate in 140 min.

scholarly journals Alteration of RNA Splicing by Small-Molecule Inhibitors of the Interaction between NHP2L1 and U4

2017 ◽  
Vol 23 (2) ◽  
pp. 164-173 ◽  
Author(s):  
Barthelemy Diouf ◽  
Wenwei Lin ◽  
Asli Goktug ◽  
Christy R. R. Grace ◽  
Michael Brett Waddell ◽  
...  
Keyword(s):  
Small Molecules ◽  
Rna Splicing ◽  
High Throughput Screening ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Therapeutic Strategies ◽  
Biological Processes ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
U4 Rna

Splicing is an important eukaryotic mechanism for expanding the transcriptome and proteome, influencing a number of biological processes. Understanding its regulation and identifying small molecules that modulate this process remain a challenge. We developed an assay based on time-resolved fluorescence resonance energy transfer (TR-FRET) to detect the interaction between the protein NHP2L1 and U4 RNA, which are two key components of the spliceosome. We used this assay to identify small molecules that interfere with this interaction in a high-throughput screening (HTS) campaign. Topotecan and other camptothecin derivatives were among the top hits. We confirmed that topotecan disrupts the interaction between NHP2L1 and U4 by binding to U4 and inhibits RNA splicing. Our data reveal new functions of known drugs that could facilitate the development of therapeutic strategies to modify splicing and alter gene function.

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

2008 ◽  
Vol 13 (10) ◽  
pp. 1025-1034 ◽  
Author(s):  
Debasis Patnaik ◽  
Jun Xian ◽  
Marcie A. Glicksman ◽  
Gregory D. Cuny ◽  
Ross L. Stein ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
High Throughput ◽  
Histone H3 ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Kinase Assay ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Fluorescence Resonance

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 Km 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. ( Journal of Biomolecular Screening 2008:1025-1034)

scholarly journals Comparison of Miniaturized Time-Resolved Fluorescence Resonance Energy Transfer and Enzyme-Coupled Luciferase High-Throughput Screening Assays to Discover Inhibitors of Rho-Kinase II (ROCK-II)

2007 ◽  
Vol 13 (1) ◽  
pp. 17-28 ◽  
Author(s):  
Thomas Schröter ◽  
Dmitriy Minond ◽  
Amiee Weiser ◽  
Chinh Dao ◽  
Jeff Habel ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
High Throughput ◽  
High Throughput Screening ◽  
Resonance Energy Transfer ◽  
Rho Kinase ◽  
Resonance Energy ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Fluorescence Resonance

Kinases are important drug discovery targets for a wide variety of therapeutic indications; consequently, the measurement of kinase activity remains a common high-throughput screening (HTS) application. Recently, enzyme-coupled luciferase-kinase (LK) format assays have been introduced. This format measures luminescence resulting from metabolism of adenosine triphosphate (ATP) via a luciferin/luciferase-coupled reaction. In the research presented here, 1536-well format time-resolved fluorescence resonance energy transfer (TR-FRET) and LK assays were created to identify novel Rho-associated kinase II (ROCK-II) inhibitors. HTS campaigns for both assays were conducted in this miniaturized format. It was found that both assays were able to consistently reproduce the expected pharmacology of inhibitors known to be specific to ROCK-II (fasudil IC50: 283 ± 27 nM and 336 ± 54 nM for TR-FRET and LK assays, respectively; Y-27632 IC50: 133 ± 7.8 nM and 150 ± 22 nM for TR-FRET and LK assays, respectively). In addition, both assays proved robust for HTS efforts, demonstrating excellent plate Z′ values during the HTS campaign (0.84 ± 0.03; 0.72 ± 0.05 for LK and TR-FRET campaigns, respectively). Both formats identified scaffolds of known and novel ROCK-II inhibitors with similar sensitivity. A comparison of the performance of these 2 assay formats in an HTS campaign was enabled by the existence of a subset of 25,000 compounds found in both our institutional and the Molecular Library Screening Center Network screening files. Analysis of the HTS campaign results based on this subset of common compounds showed that both formats had comparable total hit rates, hit distributions, amount of hit clusters, and format-specific artifact. It can be concluded that both assay formats are suitable for the discovery of ROCK-II inhibitors, and the choice of assay format depends on reagents and/or screening technology available. ( Journal of Biomolecular Screening 2008:17-28)

Multiplexing Terbium- and Europium-Based TR-FRET Readouts to Increase Kinase Assay Capacity

2010 ◽  
Vol 15 (8) ◽  
pp. 1008-1015 ◽  
Author(s):  
Robert A. Horton ◽  
Kurt W. Vogel
Keyword(s):  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Kinase Assay ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Lipid Kinase ◽  
Identification And Characterization

Identification and characterization of kinase inhibitor potency and selectivity is often an iterative process in which a library of compounds is first screened against a single kinase, and hits from that screen are then profiled against other kinases to determine specificity. By developing kinase assays that employ either a terbium- or a europium-based time-resolved fluorescence resonance energy transfer (TR-FRET) readout, one can take advantage of the distinct emission properties of these labels to develop assays for 2 kinases that can be performed simultaneously in the same well. This not only increases the information content provided per assay well but can immediately provide information on compound specificity. The authors have applied this strategy to the development of multiplexed assays for 2 examples systems: EGFR and IKKβ, as well as lipid kinase family members mTOR and PIK3C3. They demonstrate the ability of these multiplexed assays to characterize selective kinase inhibitors in a dose-response mode, with no difference in results obtained from traditional single kinase assays performed separately.

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

2002 ◽  
Vol 7 (3) ◽  
pp. 191-214 ◽  
Author(s):  
Matthew A. Sills ◽  
Donna Weiss ◽  
Quynhchi Pham ◽  
Robert Schweitzer ◽  
Xiang Wu ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
High Throughput ◽  
High Throughput Screening ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Small Degree ◽  
Kinase Assay ◽  
Active Compounds ◽  
Time Resolved ◽  
Lead Finding

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 IC50 values obtained in the HTR-FRET and FP assays, poor correlations were obtained with the IC50 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.

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