title: 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).
abstract: 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.
authors.affiliation: Department of Molecular Therapeutics and Drug Discovery, Scripps Florida, Jupiter, Florida 33458, USA.
authors.forename: Thomas
authors.initials: T
authors.lastname: Schröter
authors.forename: Dmitriy
authors.initials: D
authors.lastname: Minond
authors.forename: Amiee
authors.initials: A
authors.lastname: Weiser
authors.forename: Chinh
authors.initials: C
authors.lastname: Dao
authors.forename: Jeff
authors.initials: J
authors.lastname: Habel
authors.forename: Timothy
authors.initials: T
authors.lastname: Spicer
authors.forename: Peter
authors.initials: P
authors.lastname: Chase
authors.forename: Pierre
authors.initials: P
authors.lastname: Baillargeon
authors.forename: Louis
authors.initials: L
authors.lastname: Scampavia
authors.forename: Stephan
authors.initials: S
authors.lastname: Schürer
authors.forename: Caty
authors.initials: C
authors.lastname: Chung
authors.forename: Chris
authors.initials: C
authors.lastname: Mader
authors.forename: Mark
authors.initials: M
authors.lastname: Southern
authors.forename: Nick
authors.initials: N
authors.lastname: Tsinoremas
authors.forename: Philip
authors.initials: P
authors.lastname: LoGrasso
authors.forename: Peter
authors.initials: P
authors.lastname: Hodder
mesh_terms: D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D066298:In Vitro Techniques; D008156:Luciferases; D008163:Luminescent Measurements / Q000379:methods*; D008904:Miniaturization; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics
chemical_list: D017931:DNA Primers; D047428:Protein Kinase Inhibitors; D011994:Recombinant Proteins; D008156:Luciferases; C515852:ROCK2 protein, human; D054460:rho-Associated Kinases