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

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© 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 (, 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:
# These authors contributed equally to this work.


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.