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A High-Throughput Liposome Substrate Assay with Automated Lipid Extraction Process for PI 3-Kinase

Millennium Pharmaceuticals, Inc., Cambridge, Massachusetts

John Donovan

Millennium Pharmaceuticals, Inc., Cambridge, Massachusetts

Zhi Li

Millennium Pharmaceuticals, Inc., Cambridge, Massachusetts

Ping Li

Millennium Pharmaceuticals, Inc., Cambridge, Massachusetts

Amanda Doucette

Millennium Pharmaceuticals, Inc., Cambridge, Massachusetts

Sean Harrison

Millennium Pharmaceuticals, Inc., Cambridge, Massachusetts

Jeffrey A. Ecsedy

Millennium Pharmaceuticals, Inc., Cambridge, Massachusetts

Lenny Dang

Millennium Pharmaceuticals, Inc., Cambridge, Massachusetts

Wenhai Zhang

Millennium Pharmaceuticals, Inc., Cambridge, Massachusetts, wenhai.zhang{at}mpi.com

The signaling pathways involving lipid kinase class I phosphatidylinositol 3-kinases (PI 3-kinases) regulate cell growth, proliferation, and survival. Class I PI 3-kinases catalyze the conversion of PI (4,5)P₂ to PI (3,4,5)P₃, which acts as a lipid second messenger to activate mitogenic signaling cascades. Recently, p110, a class IA PI 3-kinase, was found to be mutated frequently in many human cancers. Therefore, it is increasingly studied as an anticancer drug target. Traditionally, PI 3-kinase activities have been studied using liposome substrates. This method, however, is hampered significantly by the labor-intensive manual lipid extraction followed by a low-throughput thin-layer chromatography analysis. The authors describe a high-throughput liposome substrate-based assay based on an automated lipid extraction method that allows them to study PI 3-kinase enzyme mechanism and quantitatively measure inhibitor activity using liposome substrates in a high-throughput mode. This improved assay format can easily be extended to study other classes of phosphoinositide lipid kinases. (Journal of Biomolecular Screening 2008:906-911)

Key Words: PI 3-kinase p110 • liposome • lipid extraction • high-throughput screening

This version was published on October 1, 2008

Journal of Biomolecular Screening, Vol. 13, No. 9, 906-911 (2008)
DOI: 10.1177/1087057108324498


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