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

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

Journal Name: Combinatorial Chemistry & High Throughput Screening

Volume 9 , Issue 5 , 2006


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.

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

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Article Details

Year: 2006
Page: [339 - 350]
Pages: 12
DOI: 10.2174/138620706777452447
Price: $58

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