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Automated high content screening for phosphoinositide kinase 3 inhibition using an AKT1 redistribution assay

Wolff, M., Haasen, D., Merk, S., Kroner, M., Maier, U., Bordel, S., Wiedenmann, J., Nienhaus, G.U., Valler, M. and Heilker, R. (2006) Automated high content screening for phosphoinositide kinase 3 inhibition using an AKT1 redistribution assay Combinatorial Chemistry & High Throughput Screening, 9, (5), pp. 339-350.

Record type: Article


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.

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Published date: June 2006


Local EPrints ID: 51182
ISSN: 1386-2073
PURE UUID: 0654c469-d41a-436a-95a9-56ce650edb2e
ORCID for J. Wiedenmann: ORCID iD

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Date deposited: 08 May 2008
Last modified: 17 Jul 2017 14:49

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Author: M. Wolff
Author: D. Haasen
Author: S. Merk
Author: M. Kroner
Author: U. Maier
Author: S. Bordel
Author: J. Wiedenmann ORCID iD
Author: G.U. Nienhaus
Author: M. Valler
Author: R. Heilker

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