Evaluation of compound interference in immobilized metal ion affinity-based fluorescence polarization detection with a four million member compound collection.
IMAP is a non-separation-based, antibody-independent, FP assay that can be applied to many types of protein kinases and phosphatases. This technology is currently being used in many high-throughput screening campaigns throughout the industry. In this technology, a fluorescently labeled peptide substrate is phosphorylated and then captured on immobilized metal (M(III)) nanoparticles, an interaction that is enhanced at low pH (pH 5.5). The binding of the phosphorylated peptide to the nanoparticles is detected using FP. IMAP differs from other FP formats in that the polarization signal is antibody-independent and involves metal coordination complexes detected at low pH. Here, this technology is evaluated against a 4000000-member compound collection using a 1536-well assay design that is devoid of enzymes so that only interference of the compounds with the detection system is measured. Miniaturization of the assay to 1536-well plates is discussed. Compound interference due to inhibition of phosphopeptide binding to the M(III) nanoparticles is not observed. Additionally, it is concluded that the level of fluorescence compound interference is similar to typical FP formats for the majority of the compound collection.
Beasley
James R
JR
Pharmacopeia, Inc, Princeton, NJ 08512, USA.
Dunn
David A
DA
Walker
Tiffany L
TL
Parlato
Susan M
SM
Lehrach
John M
JM
Auld
Douglas S
DS
D020650:Combinatorial Chemistry Techniques; D016208:Databases, Factual; D005454:Fluorescence Polarization / Q000379:methods*; D007477:Ions; D008670:Metals / Q000032:analysis*; D008904:Miniaturization
D007477:Ions; D008670:Metals