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High-Throughput, Cell-Free, Liposome-Based Approach for Assessing In Vitro Activity of Lipid KinasesResearch Technology Center, Pfizer, Inc., Cambridge, Massachusetts
Research Technology Center, Pfizer, Inc., Cambridge, Massachusetts
Research Technology Center, Pfizer, Inc., Cambridge, Massachusetts
Boston Biomedical Research Institute, Watertown, Massachusetts
Boston Biomedical Research Institute, Watertown, Massachusetts
Research Technology Center, Pfizer, Inc., Cambridge, Massachusetts
Research Technology Center, Pfizer, Inc., Cambridge, Massachusetts
Research Technology Center, Pfizer, Inc., Cambridge, Massachusetts
Research Technology Center, Pfizer, Inc., Cambridge, Massachusetts, maura.e.charlton{at}pfizer.com
Lipid kinases are central players in lipid signaling pathways involved in inflammation, tumorigenesis, and metabolic syndrome. A number of these kinase targets have proven difficult to investigate in higher throughput cell-free assay systems. This challenge is partially due to specific substrate interaction requirements for several of the lipid kinase family members and the resulting incompatibility of these substrates with most established, homogeneous assay formats. Traditional, cell-free in vitro investigational methods for members of the lipid kinase family typically involve substrate incorporation of [
Key Words: lipid kinase • PIP4KIIβ • liposome • luminescence • fluorescent polarization
This version was published on August
1, 2009 Journal of Biomolecular Screening, Vol. 14, No. 7,
838-844 (2009) |
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-32P] and resolution of signal by thin-layer chromatography (TLC) and autoradiograph densitometry. This approach, although highly sensitive, does not lend itself to high-throughput testing of large numbers of small molecules (100 s to 1 MM+). The authors present the development and implementation of a fully synthetic, liposome-based assay for assessing in vitro activity of phosphatidylinositol-5-phosphate-4-kinase isoforms (PIP4KIIβ and 
) in 2 commonly used homogeneous technologies. They have validated these assays through compound testing in both traditional TLC and radioactive filterplate approaches as well as binding validation using isothermic calorimetry. A directed library representing known kinase pharmacophores was screened against type IIβ phosphatidylinositol-phosphate kinase (PIPK) to identify small-molecule inhibitors. This assay system can be applied to other types and isoforms of PIPKs as well as a variety of other lipid kinase targets. (Journal of Biomolecular Screening 2009:838-844)