Discussion

Fabienne Charrier-Savournin1, Iris Pribilla1, Benjamin Roux1, Alexandre Jean1, Katrin Undisz2, Heidi Pruefer2, Xavier Scerri3, Manuela Beil-Peter2, Sabine Oberleitner1
1Cisbio Bioassays, Codolet, France
2CyBio AG, Jena, Germany
3CyBio AG, Savigny-le-Temple, France

Exploring the regulatory network within the cell is the basis for understanding disease pathologies and for finding appropriate therapies. Disease biology is complex, and so are the signaling pathways that drive the biological responses to therapies. Smart tools that facilitate and thereby accelerate such targeted research are the key to success in drug discovery and development.
Based on TR-FRET, Cisbio’s homogeneous and robust HTRF® technology has been assigned to a phospho-protein assay platform for the detection of endogenous phosphorylation, to help dissect cellular signaling cascades. This study illustrates the parallel implementation of several Cisbio HTRF® cellular phospho-protein assays on the CyBi®-FeliX, a compact and flexible liquid handling platform, including all steps from the serial dilution of compounds to the detection of their effects on different levels within a signaling pathway. The CyBi®-FeliX, with its 12 deck positions on two levels, was used as a standalone fully automated system to perform all HTRF®-assay specific liquid handling tasks with one pipetting head. These include serial dilutions, multi-channel pipetting in columns and rows with 8-, 12- and 96-channel adapters, respectively, and transfers from 96-well cell culture plates to 384-well assay plates.
The study validates the automated testing of compound potency on significant nodes in the PI3K/AKT- translational control pathway by the pharmacological response to several well-known kinase inhibitors at different steps of the pathway. The phosphorylation levels of AKT (Ser473, Thr308, versus total), mTor (Ser2448), S6RP (Ser235/236), p70S6K (Thr389), EIF4E (Ser209) and 4E-BP1 (Thr37/46) were assessed in parallel dose-response experiments. The IC50s of the reference inhibitors were determined for each of these pathway steps and corresponded well to the individual inhibitors’ known mechanisms of action. The complex PI3K/AKT-pathway was thus dissected into individual measurable steps. The results demonstrate that Cisbio phospho-protein assays and CyBio liquid handling can be a perfect match to simplify pathway dissection and mechanism of action studies.