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.
Original language | English |
---|---|
Pages (from-to) | 17-28 |
Number of pages | 12 |
Journal | Journal of Biomolecular Screening |
Volume | 13 |
Issue number | 1 |
DOIs | |
State | Published - Feb 1 2008 |
Externally published | Yes |
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Keywords
- High-throughput screening
- Homogeneous time-resolved fluorescence
- HTS
- Kinase assays
- Kinase Glo
- Luminescence
- MLSCN
- NIH Roadmap initiative
- Rho-associated kinase II
- rhoK2
- ROCK-II
- rock2
ASJC Scopus subject areas
- Analytical Chemistry
- Clinical Biochemistry
- Biotechnology
- Biochemistry
- Molecular Biology
Cite this
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). / Schröter, Thomas; Minond, Dmitriy; Weiser, Amiee; Dao, Chinh; Habel, Jeff; Spicer, Timothy; Chase, Peter; Baillargeon, Pierre; Scampavia, Louis; Schuerer, Stephan C; Chung, Caty; Mader, Chris; Southern, Mark; Tsinoremas, Nicholas; LoGrasso, Philip; Hodder, Peter.
In: Journal of Biomolecular Screening, Vol. 13, No. 1, 01.02.2008, p. 17-28.Research output: Contribution to journal › Article
}
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 -