Abstract
We describe the development and implementation of competitive fluorescence polarization (FP) based assays for determining activity of phosphoinositide 3-kinase (PI 3-K) and the type-II SH2-domaincontaining inositol 5-phosphatase (SHIP2). These assays are based on the interaction of specific phosphoinositide binding proteins with fluorophore-labeled phosphoinositide and inositol phosphate tracers. Enzyme reaction products are detected by their ability to compete with the fluorescent tracers for protein binding, leading to an increase in the amount of free tracer and a decrease in polarization (mP) values. A variety of fluorophore-labeled tracers were evaluated, and assay sensitivity and specificity for products of PI 3-K and SHIP2 activity was determined. Assay performance was evaluated using recombinant PI 3-Kα and SHIP2 with diC8-PI(4,5)P2 and diC8-PI(3,4,5)P3 as respective substrates. IC50 values for previously characterized PI 3-K inhibitors were within expected ranges. These assays are homogeneous, sensitive, and rapid, and suitable for HTS applications, and will facilitate screening for novel inhibitors of phosphoinositide kinases and phosphatases in drug development.
Keywords: competitive fluorescence, polarization assays, phosphoinositide 3-kinase, sh2-domaincontaining inositol 5-phosphatase
Combinatorial Chemistry & High Throughput Screening
Title: Competitive Fluorescence Polarization Assays for the Detection of Phosphoinositide Kinase and Phosphatase Activity
Volume: 6 Issue: 4
Author(s): Beth E. Drees, Amber Weipert, Heather Hudson, Colin G. Ferguson, Leena Chakravarty and Glenn D. Prestwich
Affiliation:
Keywords: competitive fluorescence, polarization assays, phosphoinositide 3-kinase, sh2-domaincontaining inositol 5-phosphatase
Abstract: We describe the development and implementation of competitive fluorescence polarization (FP) based assays for determining activity of phosphoinositide 3-kinase (PI 3-K) and the type-II SH2-domaincontaining inositol 5-phosphatase (SHIP2). These assays are based on the interaction of specific phosphoinositide binding proteins with fluorophore-labeled phosphoinositide and inositol phosphate tracers. Enzyme reaction products are detected by their ability to compete with the fluorescent tracers for protein binding, leading to an increase in the amount of free tracer and a decrease in polarization (mP) values. A variety of fluorophore-labeled tracers were evaluated, and assay sensitivity and specificity for products of PI 3-K and SHIP2 activity was determined. Assay performance was evaluated using recombinant PI 3-Kα and SHIP2 with diC8-PI(4,5)P2 and diC8-PI(3,4,5)P3 as respective substrates. IC50 values for previously characterized PI 3-K inhibitors were within expected ranges. These assays are homogeneous, sensitive, and rapid, and suitable for HTS applications, and will facilitate screening for novel inhibitors of phosphoinositide kinases and phosphatases in drug development.
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Cite this article as:
Drees E. Beth, Weipert Amber, Hudson Heather, Ferguson G. Colin, Chakravarty Leena and Prestwich D. Glenn, Competitive Fluorescence Polarization Assays for the Detection of Phosphoinositide Kinase and Phosphatase Activity, Combinatorial Chemistry & High Throughput Screening 2003; 6 (4) . https://dx.doi.org/10.2174/138620703106298572
DOI https://dx.doi.org/10.2174/138620703106298572 |
Print ISSN 1386-2073 |
Publisher Name Bentham Science Publisher |
Online ISSN 1875-5402 |
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