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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)

Department of Molecular Therapeutics and Drug Discovery, Scripps Florida, Jupiter, Florida

Dmitriy Minond

Lead Identification Department, Scripps Florida, Jupiter, Florida, dminond{at}scripps.edu

Amiee Weiser

Department of Molecular Therapeutics and Drug Discovery, Scripps Florida, Jupiter, Florida

Chinh Dao

Department of Molecular Therapeutics and Drug Discovery, Scripps Florida, Jupiter, Florida

Jeff Habel

Department of Molecular Therapeutics and Drug Discovery, Scripps Florida, Jupiter, Florida

Timothy Spicer

Lead Identification Department, Scripps Florida, Jupiter, Florida

Peter Chase

Lead Identification Department, Scripps Florida, Jupiter, Florida

Pierre Baillargeon

Lead Identification Department, Scripps Florida, Jupiter, Florida

Louis Scampavia

Lead Identification Department, Scripps Florida, Jupiter, Florida

Stephan Schürer

Informatics Department, Scripps Florida, Jupiter, Florida

Caty Chung

Informatics Department, Scripps Florida, Jupiter, Florida

Chris Mader

Informatics Department, Scripps Florida, Jupiter, Florida

Mark Southern

Informatics Department, Scripps Florida, Jupiter, Florida

Nick Tsinoremas

Informatics Department, Scripps Florida, Jupiter, Florida

Philip Lograsso

Department of Molecular Therapeutics and Drug Discovery, Scripps Florida, Jupiter, Florida, lograsso{at}scripps.edu

Peter Hodder

Lead Identification Department, Scripps Florida, Jupiter, Florida

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)

Key Words: ROCK-II • rock2 • rhoK2 • Rho-associated kinase II • homogeneous time-resolved fluorescence • high-throughput screening • kinase assays • Kinase Glo • luminescence • HTS • MLSCN • NIH Roadmap initiative

Journal of Biomolecular Screening, Vol. 13, No. 1, 17-28 (2008)
DOI: 10.1177/1087057107310806


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