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

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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Citation

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.

More information

Published date: June 2006

Identifiers

Local EPrints ID: 51182
URI: http://eprints.soton.ac.uk/id/eprint/51182
ISSN: 1386-2073
PURE UUID: 0654c469-d41a-436a-95a9-56ce650edb2e
ORCID for J. Wiedenmann: ORCID iD orcid.org/0000-0003-2128-2943

Catalogue record

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

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Contributors

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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