Cellular Ser/Thr-kinase assays using generic peptide substrates.
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 CamK2delta 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, IC(50)-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.
Adams
Deanna G
DG
Genomics Institute of the Novartis Research Foundation, San Diego, California, USA.
Wang
Yu
Y
Mak
Puiying A
PA
Chyba
Jason
J
Shalizi
Orzala
O
Matzen
Jason
J
Anderson
Paul
P
Smith
Tim R
TR
Garcia
Michael
M
Welch
Genevieve L
GL
Claret
Emmanuel J
EJ
Fink
Michel
M
Orth
Anthony P
AP
Caldwell
Jeremy S
JS
Brinker
Achim
A