Abstract
The integration of fluorescent microscopy imaging technologies and image analysis into high-content screening (HCS) has been applied throughout the drug discovery pipeline to identify, evaluate, and advance compounds from early lead generation through preclinical candidate selection. In this chapter we describe the development, validation, and implementation of an HCS assay to screen compounds from a kinase-focused small-molecule library to identify inhibitors of the p38 pathway using the GE InCell 3000 automated imaging platform. The assay utilized a genetically modified HeLa cell line stably expressing mitogen-activated, protein-activating protein kinase-2 fused to enhanced green fluorescent protein (MK2–EGFP) and measured the subcellular distribution of the MK2–EGFP as a direct readout of p38 activation. The MK2–EGFP translocation assay performed in 384-well glass bottom microtiter plates exhibited a robust Z-factor of 0.46 and reproducible EC50 and IC50 determinations for activators and inhibitors, respectively. A total of 32,891 compounds were screened in singlicate at 50 μM and 156 were confirmed as inhibitors of p38-mediated MK2–EGFP translocation in follow-up IC50 concentration response curves. Thirty-one compounds exhibited IC50s less than 1 μM, and at least one novel structural class of p38 inhibitor was identified using this HCA/HCS chemical biology screening approach.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Cowan, K. J. and Storey, K. B. (2003). Mitogen-activated protein kinases: new signaling pathways functioning in cellular responses to environmental stress. J. Exp. Biol. 206, 1107–1115.
Garrington, T. P. and Johnson, G. L. (1999). Organization and regulation of mitogen activated protein kinase signaling pathways. Curr. Opin. Cell Biol. 11, 211–218. Also Refs. (14, 15).
English, J. M. and Cobb, M. H. (2002). Pharmacological inhibitors of MAPK pathways. Trends Pharmacol. Sci. 23, 40–45.
Johnston, P. A. and Johnston, P. A. (2002). Cellular platforms for HTS: three case studies. Drug Discov. Today 7, 353–363.
Ono, K. and Han, J. (2000). The p38 signal transduction pathway, activation and function. Cell. Signal. 12, 1–13.
Noble, M. E. M., Endicott, J. A., and Johnson, L. N. (2004). Protein Kinase Inhibitors: insights into drug design and structure. Science 303, 1800–1805.
Regan, J., Breitfelder, S., Cirillo, P., Gilmore, T., Graham, A. G., Hickey, E., Klaus, B., Madwed, J., Moriak, M., Moss, N., Pargellis, C., Pav, S., Proto, A., Swinamer, A., Tong, L., and Torcellini, C. (2002). Pyrazole urea-based inhibitors of p38 MAP kinase: from lead compound to clinical candidate. J. Med. Chem. 45, 2994–3008.
Fabbro, D., Ruetz, S., Buchdunger, E., Cowan-Jacob, S. W., Fendrich, G., Liebetanz, J., Mestan, J., O'Reilly, T., Traxler, P., Chaudhuri, B., Fretz, H., Zimmermann, J., Meyer, T., Caravatti, G., Furet, P., and Manley, P. W. (2002). Protein kinases as targets for anticancer agents: from inhibitors to useful drugs. Pharmacol. Ther. 93, 79–98.
Zu, Y. L., Ai, Y., and Huang C. K. (1995). Characterization of an autoinhibitory domain in human mitogen-activated protein kinase-activated protein kinase 2. J. Biol. Chem. 270, 202–206.
Engel, K., Kotlyarov, A., and Gaestel, M. (1998). Leptomycin B-sensitive nuclear export of MAPKAP kinase 2 is regulated by phosphorylation. EMBO J. 17, 3363–3371.
Neininger, A., Thielemann, H., and Gaestel, M. (2001). FRET-based detection of different conformations of MK2. EMBO Rep. 2, 703–708.
Williams, R. G., Kandasamy, R., Nickischer, D., Trask, O. J., Jr., Laethem, C., Johnston, P. A., and Johnston, P. A. (2006). Generation and characterization of a stable MK2-EGFP cell line and subsequent development of a high-content imaging assay on the Cellomics ArrayScan platform to screen for p38 mitogen-activated protein kinase inhibitors. Methods Enzymol. 414, 364–388.
Trask, O. J., Jr., Baker, A., Williams, R. G., Kickischer, D., Kandasamy, R., Laethem, C., Johnston, P. A., and Johnston, P. A. (2006). Assay development and case history of a 32 K biased library high-content MK2-EGFP translocation screen to identify p38 MAPK inhibitors on the ArrayScan 3.1 imaging platform. Methods Enzymol. 414, 419–439.
Almholt D. L., Loechel, F., Nielsen, S. J., Krog-Jensen, C., Terry, R., Bjorn, S. P., Pedersen, H. C., Praestegaard, M., Moller, S., Heide, M., Pagliaro, L., Mason, A. J., Butcher, S., and Dahl, S.W. (2004). Nuclear export inhibitors and kinase inhibitors identified using a MAPK-activated protein kinase 2 redistribution screen. Assay Drug Dev. Technol. 2, 7–20.
Lundholt, B. K., Linde, V., Loechel, F., Pedersen, H. C., Moller, S., Praestegaard, M., Mikkelsen, I., Scudder, K., Bjorn, S. P., Heide, M., Arkhammar, P. O., Terry, R., and Nielsen, S. J. (2005). Identification of Akt pathway inhibitors using redistribution screening on the FLIPR and the InCell 3000 analyzer. J. Biomol. Screen. 10, 20–29.
Oakley, R. H., Hudson, C. C., Cruickshank, R. D., Meyers, D. M., Payne, R. E., Jr., Rhem, S. M., and Loomis, C. R. (2002). The cellular distribution of fluorescently labeled arrestins provides a robust, sensitive, and universal assay for screening G protein-coupled receptors. Assay Drug Dev. Technol. 1, 21–30.
Bennett, B. L., Sasaki, D. T., Murray, B. W., O'Leary, E. C., Sakata, S. T., Xu, W., Leisten, J. C., Motiwala, A., Pierce, S., Satoh, Y., Bhagwat, S. S., Manning, A. M., and Anderson, D. W. (2001). SP600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase. Proc. Natl. Acad. Sci. USA 98, 13681–13686.
Han, Z., Boyle, D. L., Chang, L., Bennett, B., Karin, M., Yang, L., Manning, A. M., and Firestein, G. S. (2001). c-Jun N-terminal kinase is required for metalloproteinase expression and joint destruction in inflammatory arthritis. J. Clin. Invest. 108, 73–81.
Zhang, J. H., Chung, T. D., and Oldenburg, K. R. (1999). A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J. Biomol. Screen. 4, 67–73.
Acknowledgments
We want to thank Tim Harris, Jennifer I. Colonell, and William J. Karsh formerly of GE Healthcare and Amersham Biosciences for the technical contributions for the work on the InCell 3000.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Trask, O.J. et al. (2009). High-Throughput Automated Confocal Microscopy Imaging Screen of a Kinase-Focused Library to Identify p38 Mitogen-Activated Protein Kinase Inhibitors Using the GE InCell 3000 Analyzer. In: Janzen, W., Bernasconi, P. (eds) High Throughput Screening. Methods in Molecular Biology, vol 565. Humana Press. https://doi.org/10.1007/978-1-60327-258-2_8
Download citation
DOI: https://doi.org/10.1007/978-1-60327-258-2_8
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60327-257-5
Online ISBN: 978-1-60327-258-2
eBook Packages: Springer Protocols