scholarly journals High-Throughput Examination of Fluorescence Resonance Energy Transfer-Detected Metal-Ion Response in Mammalian Cells

2012 ◽  
Vol 134 (5) ◽  
pp. 2488-2491 ◽  
Author(s):  
Hairong Ma ◽  
Emily A. Gibson ◽  
Philip J. Dittmer ◽  
Ralph Jimenez ◽  
Amy E. Palmer
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
High Throughput ◽  
Mammalian Cells ◽  
Metal Ion ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Fluorescence Resonance

scholarly journals A High-Throughput Fluorescence Resonance Energy Transfer-Based Assay for DNA Ligase

2011 ◽  
Vol 16 (5) ◽  
pp. 486-493 ◽  
Author(s):  
Adam B. Shapiro ◽  
Ann E. Eakin ◽  
Grant K. Walkup ◽  
Olga Rivin
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
High Throughput ◽  
High Throughput Screening ◽  
Pathogenic Bacteria ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Dna Ligase ◽  
Phosphodiester Bond ◽  
Fluorescence Resonance

DNA ligase is the enzyme that catalyzes the formation of the backbone phosphodiester bond between the 5′-PO4 and 3′-OH of adjacent DNA nucleotides at single-stranded nicks. These nicks occur between Okazaki fragments during replication of the lagging strand of the DNA as well as during DNA repair and recombination. As essential enzymes for DNA replication, the NAD+-dependent DNA ligases of pathogenic bacteria are potential targets for the development of antibacterial drugs. For the purposes of drug discovery, a high-throughput assay for DNA ligase activity is invaluable. This article describes a straightforward, fluorescence resonance energy transfer–based DNA ligase assay that is well suited for high-throughput screening for DNA ligase inhibitors as well as for use in enzyme kinetics studies. Its use is demonstrated for measurement of the steady-state kinetic constants of Haemophilus influenzae NAD+-dependent DNA ligase and for measurement of the potency of an inhibitor of this enzyme.

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)

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