Automated High Content Screening for Phosphoinositide 3 Kinase Inhibition Using an AKT1 Redistribution Assay

Title: Automated High Content Screening for Phosphoinositide 3 Kinase Inhibition Using an AKT1 Redistribution Assay

Volume: 9 Issue: 5

Author(s): Michael Wolff, Dorothea Haasen, Susanne Merk, Margareta Kroner, Udo Maier, Sandra Bordel, Jorg Wiedenmann, Gerd U. Nienhaus, Martin Valler and Ralf Heilker

Affiliation:

Keywords: High content screening, phosphoinositide 3 kinase, AKT1 redistribution, high-throughput screening

Abstract: High Content Screening (HCS), a combination of fluorescence microscopic imaging and automated image analysis, has become a frequently applied tool to study test compound effects in cellular disease-modelling systems. In this work, we established a medium to high throughput HCS assay in the 384-well format to measure cellular type I phosphoinositide 3 kinase (PI3K) activity. Type I PI3K is involved in several intracellular pathways such as cell survival, growth and differentiation as well as immunological responses. As a cellular model system we used Chinese Hamster Ovary (CHO) cells that had been stably transfected with human insulin receptor (hIR) and an AKT1-enhanced green fluorescent protein (EGFP) fusion construct. Upon stimulation of the hIR with insulin-like growth factor-1 (IGF-1), PI3K was activated to phosphorylate phosphatidylinositol (PtdIns)-4,5-bisphosphate at the 3-position, resulting in the recruitment of AKT1-EGFP to the plasma membrane. The AKT1-EGFP redistribution assay was robust and displayed little day-to-day variability, the quantification of the fluorescence intensity associated with plasma membrane spots delivered good Z ’ statistics. A novel format of compound dose-response testing was employed using serial dilutions of test compounds across consecutive microtiter plates (MTPs). The dose response testing of a PI3K inhibitor series provided reproducible IC50 values. The profiling of the redistribution assay with isoform-selective inhibitors indicates that PI3K?? is the main isoform activated in the CHO host cells after IGF- 1 stimulation. Toxic compound side effects could be determined using automated image analysis. We conclude that the AKT1-EGFP redistribution assay represents a solid medium/high throughput screening (MTS/HTS) format to determine the cellular activity of PI3K inhibitors under conditions of growth factor stimulation.

Cite this article as:

Wolff Michael, Haasen Dorothea, Merk Susanne, Kroner Margareta, Maier Udo, Bordel Sandra, Wiedenmann Jorg, Nienhaus U. Gerd, Valler Martin and Heilker Ralf, Automated High Content Screening for Phosphoinositide 3 Kinase Inhibition Using an AKT1 Redistribution Assay, Combinatorial Chemistry & High Throughput Screening 2006; 9 (5) . https://dx.doi.org/10.2174/138620706777452447