High-throughput, cell-free, liposome-based approach for assessing in vitro activity of lipid kinases.
J Biomol Screen
; 14(7): 838-44, 2009 Aug.
Article
em En
| MEDLINE
| ID: mdl-19641220
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Fosfotransferases
/
Metabolismo dos Lipídeos
/
Ensaios de Triagem em Larga Escala
/
Lipossomos
Tipo de estudo:
Prognostic_studies
Limite:
Humans
Idioma:
En
Revista:
J Biomol Screen
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2009
Tipo de documento:
Article
País de afiliação:
Estados Unidos