Cell-based kinase assays in HTS – potential and limitations for primary and secondary screening

Benjamin Bader


Bayer Healthcare, Berlin, Germany

5th HTRF Symposium, Avignon, France

Kinases represent an important target class for pharmaceutical research. Recent drug approvals of novel chemical entities have proven the value of targeting kinases for cancer treatment. Today, the toolbox of assays and reagents to measure kinase activity with purified proteins in a biochemical assay offers plenty of opportunities. Nevertheless, almost every screening program for kinase inhibitors relies on cellular assays at a certain point in time. Cell-based activity of hit compounds constitutes a critical parameter in the selection of candidate drugs for in vivo testing. Still, the development of sensitive, robust and predictive cellular kinase assays remains a challenge in many projects and often relies on the availability and quality of antibodies for phosphorylated proteins. In this presentation I will discuss the development of two TR-FRET based cellular kinase assays for measuring activity of the PI3K-Akt-mTOR pathway. We used tumor cells infected with Baculovirus expressing GFP-Tagged PRAS40 to measure its phosphorylation status by the LanthaScreen™ technology. In comparison, an HTRF® assay was employed detecting endogenous phosphorylated Akt in lysates of tumor cell lines. Data will be presented on the optimization of the assays to suit the requirements of our HTS environment, including sensitivity, robustness, frozen cells and fit to the 1536 assay-ready plate format.

Kinases, Phosphorylated proteins