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)

TY - JOUR

T1 - 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)

AU - Schröter, Thomas

AU - Minond, Dmitriy

AU - Weiser, Amiee

AU - Dao, Chinh

AU - Habel, Jeff

AU - Spicer, Timothy

AU - Chase, Peter

AU - Baillargeon, Pierre

AU - Scampavia, Louis

AU - Schuerer, Stephan C

AU - Chung, Caty

AU - Mader, Chris

AU - Southern, Mark

AU - Tsinoremas, Nicholas

AU - LoGrasso, Philip

AU - Hodder, Peter

PY - 2008/2/1

Y1 - 2008/2/1

N2 - 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.

AB - 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.

KW - High-throughput screening

KW - Homogeneous time-resolved fluorescence

KW - HTS

KW - Kinase assays

KW - Kinase Glo

KW - Luminescence

KW - MLSCN

KW - NIH Roadmap initiative

KW - Rho-associated kinase II

KW - rhoK2

KW - ROCK-II

KW - rock2

UR - http://www.scopus.com/inward/record.url?scp=37749050446&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=37749050446&partnerID=8YFLogxK

U2 - 10.1177/1087057107310806

DO - 10.1177/1087057107310806

M3 - Article

C2 - 18227223

AN - SCOPUS:37749050446

VL - 13

SP - 17

EP - 28

JO - Journal of Biomolecular Screening

JF - Journal of Biomolecular Screening

SN - 1087-0571

IS - 1

ER -