RESEARCH ARTICLE


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
1 Genomics Institute of the Novartis Research Foundation, San Diego, California, USA
2 Cisbio International, HTRF/Bioassays, Bagnols-sure-Ceze, France


Article Metrics

CrossRef Citations:
0
Total Statistics:

Full-Text HTML Views: 597
Abstract HTML Views: 365
PDF Downloads: 193
Total Views/Downloads: 1155
Unique Statistics:

Full-Text HTML Views: 374
Abstract HTML Views: 171
PDF Downloads: 131
Total Views/Downloads: 676



© Adams et al.; Licensee Bentham Open.

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.

* Address correspondence to this author at the Genomics Institute of the Novartis Research Foundation, San Diego, USA; E-mail: abrinker@gnf.org
# These authors contributed equally to this work.


Abstract

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.