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