Cellular Ser/Thr-Kinase Assays Using Generic Peptide Substrates
Deanna G Adams#, 1, Yu Wang#, 1, Puiying A Mak1, Jason Chyba1, Orzala Shalizi1, Jason Matzen1, Paul Anderson1, Tim R Smith1, Michael Garcia1, Genevieve L Welch1, Emmanuel J Claret2, Michel Fink2, Anthony P Orth1, Jeremy S Caldwell1, Achim Brinker*, 1
Identifiers and Pagination:Year: 2008
First Page: 54
Last Page: 64
Publisher Id: CCGTM-1-54
Article History:Received Date: 14/2/2008
Revision Received Date: 31/3/2008
Acceptance Date: 4/4/2008
Electronic publication date: 23/5/2008
Collection year: 2008
open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.5/), which permits unrestrictive use, distribution, and reproduction in any medium, provided the original work is properly cited.
High-throughput cellular profiling has successfully stimulated early drug discovery pipelines by facilitating targeted as well as opportunistic lead finding, hit annotation and SAR analysis. While automation-friendly universal assay formats exist to address most established drug target classes like GPCRs, NHRs, ion channels or Tyr-kinases, no such cellular assay technology is currently enabling an equally broad and rapid interrogation of the Ser/Thr-kinase space. Here we present the foundation of an emerging cellular Ser/Thr-kinase platform that involves a) coexpression of targeted kinases with promiscuous peptide substrates and b) quantification of intracellular substrate phosphorylation by homogeneous TR-FRET. Proof-of-concept data is provided for cellular AKT, B-RAF and CamK2δ assays. Importantly, comparable activity profiles were found for well characterized B-Raf inhibitors in TR-FRET assays relying on either promiscuous peptide substrates or a MEK1(WT) protein substrate respectively. Moreover, IC50-values correlated strongly between cellular TR-FRET assays and a gold standard Ba/F3 proliferation assay for B-Raf activity. Finally, we expanded our initial assay panel by screening a kinase-focused cDNA library and identified starting points for >20 cellular Ser/Thr-kinase assays.