A high-throughput screening-compatible homogeneous time-resolved fluorescence assay measuring the glycohydrolase activity of human poly(ADP-ribose) glycohydrolase

2016 ◽  
Vol 503 ◽  
pp. 58-64 ◽  
Author(s):  
Alexandra I.J. Stowell ◽  
Dominic I. James ◽  
Ian D. Waddell ◽  
Neil Bennett ◽  
Caroline Truman ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Fluorescence Assay ◽  
Time Resolved Fluorescence ◽  
Time Resolved

scholarly journals High-Throughput Screening for Linear Ubiquitin Chain Assembly Complex (LUBAC) Selective Inhibitors Using Homogenous Time-Resolved Fluorescence (HTRF)-Based Assay System

2018 ◽  
Vol 23 (10) ◽  
pp. 1018-1029 ◽  
Author(s):  
Ken Katsuya ◽  
Yuji Hori ◽  
Daisuke Oikawa ◽  
Tomohisa Yamamoto ◽  
Kayo Umetani ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Inflammatory Responses ◽  
Mass Spectrometry Analysis ◽  
Regulatory Subunit ◽  
Luciferase Reporter ◽  
Time Resolved Fluorescence ◽  
Ubiquitin Chain ◽  
Time Resolved ◽  
Gene Assay

The nuclear factor κB (NF-κB) pathway is critical for regulating immune and inflammatory responses, and uncontrolled NF-κB activation is closely associated with various inflammatory diseases and malignant tumors. The Met1-linked linear ubiquitin chain, which is generated by linear ubiquitin chain assembly complex (LUBAC), is important for regulating NF-κB activation. This process occurs through the linear ubiquitination of NF-κB essential modulator, a regulatory subunit of the canonical inhibitor of the NF-κB kinase complex. In this study, we have established a robust and efficient high-throughput screening (HTS) platform to explore LUBAC inhibitors, which may be used as tool compounds to elucidate the pathophysiological role of LUBAC. The HTS platform consisted of both cell-free and cell-based assays: (1) cell-free LUBAC-mediated linear ubiquitination assay using homogenous time-resolved fluorescence technology and (2) cell-based LUBAC assay using the NF-κB luciferase reporter gene assay. By using the HTS platform, we performed a high-throughput chemical library screen and identified several hit compounds with selectivity against a counterassay. Liquid chromatography–mass spectrometry analysis revealed that these compounds contain a chemically reactive lactone structure, which is transformed to give reactive α,β-unsaturated carbonyl compounds. Further investigation revealed that the reactive group of these compounds is essential for the inhibition of LUBAC activity.

P4-342 High throughput screening of β-amyloid secretion inhibitors using homogenous time-resolved fluorescence

2004 ◽  
Vol 25 ◽  
pp. S572
Author(s):  
Bruno P. Imbimbo ◽  
Hugo Albrecht ◽  
Peter Zbinden ◽  
Andrea Rizzi ◽  
Ginno Villetti ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Β Amyloid

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)

High Throughput Screening of β-Amyloid Secretion Inhibitors Using Homogenous Time-Resolved Fluorescence

2004 ◽  
Vol 7 (8) ◽  
pp. 745-756 ◽  
Author(s):  
Hugo Albrecht ◽  
Peter Zbinden ◽  
Andrea Rizzi ◽  
Gino Villetti ◽  
Benedetta Riccardi ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Time Resolved Fluorescence ◽  
Time Resolved
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