This Article Full Text (PDF) References Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Saved Citations Download to citation manager Request Permissions Request Reprints Add to My Marked Citations Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Citing Articles via Scopus Google Scholar Articles by Hubert, C. L. Articles by Stancato, L. F. Search for Related Content PubMed PubMed Citation Articles by Hubert, C. L. Articles by Stancato, L. F. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Social Bookmarking                         What's this?

Data Concordance from a Comparison between Filter Binding and Fluorescence Polarization Assay Formats for Identification of ROCK-II Inhibitors

Eli Lilly and Company, Research Triangle Park, NC

Stacy E. Sherling

Eli Lilly and Company, Research Triangle Park, NC

Patricia A. Johnston

Eli Lilly and Company, Research Triangle Park, NC

Louis F. Stancato

Eli Lilly and Company, Research Triangle Park, NC

The Rho-associated coiled-coil-containing protein serine/threonine kinases ROCK-I and ROCK-II are thought to play a major role in cytoskeletal dynamics by serving as downstream effectors of the Rho/Rac family of cytokine- and growth factor-activated small GTPases. As such, the ROCK family members are attractive intervention targets for a variety of pathologies, including cancer and cardiovascular disease. The authors developed a high-throughput screen to identify ROCK-II inhibitors and report results from a direct comparison of 2 screening campaigns for ROCK-II inhibitors using fluorescence polarization(FP) and filter binding (FB). Screening protocolsto identify inhibitors of ROCK-II were developed in FB and FP formats under similar assay and kinetic conditions. A 30,000-member compound library was screened using FB (³³P) and FP detection systems, and compounds that were active in either assay were retested in 5-point curve confirmation assays. Analysis of these data showed an approximate 95% agreement of compounds identified as active in both assay formats. Also, compound potency determinations from FB and FP had a high degree of correlation and were considered equivalent. These data suggest that the assay methodology has little impact on the quality and productivity of the screen, provided that the assays are developed to standardize kinetic conditions.

Key Words: filtration • comparison • format • Rho kinase • ROCK inhibitors • fluorescence polarization • kinase assays

Journal of Biomolecular Screening, Vol. 8, No. 4, 399-409 (2003)
DOI: 10.1177/1087057103255071


CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    Twitter    What's this?

This article has been cited by other articles:

				T. Schroter, D. Minond, A. Weiser, C. Dao, J. Habel, T. Spicer, P. Chase, P. Baillargeon, L. Scampavia, S. Schurer, et al. 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) J Biomol Screen, January 1, 2008; 13(1): 17 - 28. [Abstract] [PDF]

				A. K. Quercia, W. A. Lamarr, J. Myung, C. C. Ozbal, J. A. Landro, and K. J. Lumb High-Throughput Screening by Mass Spectrometry: Comparison with the Scintillation Proximity Assay with a Focused-File Screen of AKT1/PKB{alpha} J Biomol Screen, June 1, 2007; 12(4): 473 - 480. [Abstract] [PDF]

				M. A. Kashem, R. M. Nelson, J. D. Yingling, S. S. Pullen, A. S. Prokopowicz III, J. W. Jones, J. P. Wolak, G. R. Rogers, M. M. Morelock, R. J. Snow, et al. Three Mechanistically Distinct Kinase Assays Compared: Measurement of Intrinsic ATPase Activity Identified the Most Comprehensive Set of ITK Inhibitors J Biomol Screen, February 1, 2007; 12(1): 70 - 83. [Abstract] [PDF]

				O. Von Ahsen, A. Schmidt, M. Klotz, and K. Parczyk Assay Concordance between SPA and TR-FRET in High-Throughput Screening J Biomol Screen, September 1, 2006; 11(6): 606 - 616. [Abstract] [PDF]

				B. J. Eastwood, M. W. Farmen, P. W. Iversen, T. J. Craft, J. K. Smallwood, K. E. Garbison, N. W. Delapp, and G. F. Smith The Minimum Significant Ratio: A Statistical Parameter to Characterize the Reproducibility of Potency Estimates from Concentration-Response Assays and Estimation by Replicate-Experiment Studies J Biomol Screen, April 1, 2006; 11(3): 253 - 261. [Abstract] [PDF]

				J.-h. Zhang, X. Wu, and M. A. Sills Probing the Primary Screening Efficiency by Multiple Replicate Testing: A Quantitative Analysis of Hit Confirmation and False Screening Results of a Biochemical Assay J Biomol Screen, October 1, 2005; 10(7): 695 - 704. [Abstract] [PDF]

				D. Sun, A. Whitty, J. Papadatos, M. Newman, J. Donnelly, S. Bowes, and S. Josiah Adopting a Practical Statistical Approach for Evaluating Assay Agreement in Drug Discovery J Biomol Screen, August 1, 2005; 10(5): 508 - 516. [Abstract] [PDF]