Abstract
Inhibitors of kinase activities can be mechanistically diverse, genomically selective, and pathway sensitive. This potential has made these biological targets the focus of a number of drug discovery and development programs in the pharmaceutical industry. To this end, the high-throughput screening of kinase targets against diverse chemical libraries or focused compound collections is at the forefront of the drug discovery process. Thus, the platform technology used to screen such libraries must be flexible and produce reliable and comparable data. The Caliper HTS microfluidic platform provides a direct determination of a peptidic substrate and phosphorylated product through the electrophoretic separation of the two species. The resulting data are reliable and comparable among screens and cover a broad range of biological targets, provided there is a definable peptide substrate that permits separation. Here we present a method for the high-throughput screening of the cyclic AMP-dependent protein kinase (PKA) as an example of the simplicity of this microfluidic platform.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
O'Neill, L.A. (2006) Targeting signal transduction as a strategy to treat inflammatory diseases. Nat. Rev. Drug Discov. 5(7), 549–563.
Hennessy, B.T., Smith, D.L., Ram, P.T., Lu, Y., Mills, G.B. (2005) Exploiting the PI3K/AKT pathway for cancer drug discovery. Nat. Rev. Drug Discov. 4(12), 988–1004.
Vlahos, C.J., McDowell, S.A., Clerk, A. (2003) Kinases as therapeutic targets for heart failure. Nat. Rev. Drug Discov. 2(2), 99–113.
Druker, B.J., Tamura, S., Buchdunger, E., Ohno, S., Segal, G.M., Fanning, S., Zimmermann, J., Lydon, N.B. (1996) Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nature Med. 2(5), 561–566.
Warner, G., Illy, C., Pedro, L., Roby, P., Bosse, R. (2004) AlphaScreen kinase HTS platforms. Curr. Med. Chem. 11(6), 721–730.
Sportsman, J.R., Gaudet, E.A., Boge, A. (2004) Immobilized metal ion affinity-based fluorescence polarization (IMAP): advances in kinase screening. Assay Drug Dev. Technol. 2(2), 205–214.
Koresawa, M., Okabe, T. (2004) High-throughput screening with quantitation of ATP consumption: a universal non-radioisotope, homogeneous assay for protein kinase. Assay Drug Dev. Technol. 2(2), 153–160.
Dunne, J., Reardon, H., Trinh, V., Li, E., Farinas, J. (2004) Comparison of on-chip and off-chip microfluidic kinase assay formats. Assay Drug Dev. Technol. 2(2), 121–129.
Cheng, H.C., Kemp, B.E., Pearson, R.B., Smith, A.J., Misconi, L., Van Patten, S.M., Walsh, D.A. (1986) A potent synthetic peptide inhibitor of the cAMP-dependent protein kinase. J. Biol. Chem. 261(3), 989–992.
Janzen, W., Bernasconi, P., Cheatham, L., Mansky, P., Popa-Burke, I., Williams, K., Worley, J., Hodge, N. (2004) Optimizing the Chemical Genomics Process. In: Darvas, F., Guttman, A., and Dorman, G. (eds.), Chemical Genomics. Marcel Dekker, New York, pp. 59–100.
Zhang, J.H., Chung, T.D., 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.
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
Blackwell, L.J. et al. (2009). High-Throughput Screening of the Cyclic AMP-Dependent Protein Kinase (PKA) Using the Caliper Microfluidic Platform. 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_11
Download citation
DOI: https://doi.org/10.1007/978-1-60327-258-2_11
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60327-257-5
Online ISBN: 978-1-60327-258-2
eBook Packages: Springer Protocols