title: High-throughput, cell-free, liposome-based approach for assessing in vitro activity of lipid kinases.
abstract: 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 [gamma-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 (PIP4KIIbeta and alpha) 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 IIbeta 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.
authors.affiliation: Research Technology Center, Pfizer, Inc., Cambridge, Massachusetts 02139, USA.
authors.forename: Douglas J
authors.initials: DJ
authors.lastname: Demian
authors.forename: Susan L
authors.initials: SL
authors.lastname: Clugston
authors.forename: Meta M
authors.initials: MM
authors.lastname: Foster
authors.forename: Lucia
authors.initials: L
authors.lastname: Rameh
authors.forename: Deborah
authors.initials: D
authors.lastname: Sarkes
authors.forename: Sharon A
authors.initials: SA
authors.lastname: Townson
authors.forename: Lily
authors.initials: L
authors.lastname: Yang
authors.forename: Melvin
authors.initials: M
authors.lastname: Zhang
authors.forename: Maura E
authors.initials: ME
authors.lastname: Charlton
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D002151:Calorimetry; D002474:Cell-Free System; D002855:Chromatography, Thin Layer; D018360:Crystallography, X-Ray; D003720:Densitometry; D057166:High-Throughput Screening Assays / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D020128:Inhibitory Concentration 50; D007700:Kinetics; D050356:Lipid Metabolism / Q000187:drug effects; D008081:Liposomes / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D010770:Phosphotransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology; D015203:Reproducibility of Results; D013379:Substrate Specificity / Q000187:drug effects
chemical_list: D008081:Liposomes; D010761:Phosphorus Radioisotopes; D047428:Protein Kinase Inhibitors; D000255:Adenosine Triphosphate; D010770:Phosphotransferases