High-throughput screening with immobilized metal ion affinity-based fluorescence polarization detection, a homogeneous assay for protein kinases.
Protein kinases are one of the most important target classes in high-throughput screening today. The use of generic assay technologies facilitates assay development for new targets and decreases the time needed for implementation of assays in robotic screening. For tyrosine kinases, several generic assay technology platforms are available. These technologies make use of high-affinity antibodies that discriminate between phosphorylated tyrosines and non-phosphorylated tyrosines. Similar generic antibodies specific for phosphoserine or phosphothreonine are lacking. Recently, a non-antibody-based fluorescence polarization assay for protein kinases has become available, called IMAP (Molecular Devices, Sunnyvale, CA). In this assay, a fluorescently labeled peptide substrate that is phosphorylated by kinase is captured on metal-derivatized nanoparticles. We have evaluated IMAP in high-throughput screening, and compared this technology with a competition fluorescence polarization immunoassay based on an antibody specific for a phosphorylated peptide substrate. A random collection of >250000 compounds was screened with the two assays. Fluorescent library compounds were identified by calculation of fluorescence intensity values from the screening data, and by assaying in the absence of fluorescent reagents. Fluorescence polarization artifacts were filtered out further by testing in an ELISA-based kinase assay. Our data show that IMAP is a robust technology for high-throughput screening of kinase targets, and suggest that it is less susceptible to fluorescence polarization artifacts than the competition fluorescence polarization immunoassay.
Loomans
Elma E M G
EE
Lead Discovery Unit, N.V. Organon, Oss, The Netherlands. elma.loomans@organon.com
van Doornmalen
Antoon M
AM
Wat
Jesse W Y
JW
Zaman
Guido J R
GJ
D016477:Artifacts; D004797:Enzyme-Linked Immunosorbent Assay; D005454:Fluorescence Polarization / Q000379:methods*; D016231:Fluorescence Polarization Immunoassay; D005456:Fluorescent Dyes; D007477:Ions; D008670:Metals / Q000737:chemistry*; D036103:Nanotechnology; D010316:Particle Size; D010455:Peptides / Q000737:chemistry; D011494:Protein Kinases / Q000032:analysis*; D015203:Reproducibility of Results
D005456:Fluorescent Dyes; D007477:Ions; D008670:Metals; D010455:Peptides; D011494:Protein Kinases