Current Chemical Genomics and Translational Medicine

2008, 1 : 54-64
Published online 2008 May 23. DOI: 10.2174/1875397300801010054
Publisher ID: CCGTM-1-54

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

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

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.