Abstract
Phosphoinositide 3-kinases (PI3Ks) comprise a family of kinases that transfer the terminal phosphate of adenosine triphosphate to phosphoinositides at the 3-hydroxyl of the inositol ring to form phosphoinositide (3,4,5) triphosphate (PIP3). The PI3Ks have been shown to play key roles in cell growth, motility, morphology, and survival and thus are of interest as targets in anti-inflammatory and anti-oncogenic drug development. To facilitate identification of novel and selective inhibitors of PI3Ks, we have developed a TR-FRET assay that uses directly labeled reagents. The assay makes use of the high affinity binding of phosphoinositides to a Pleckstrin homology (PH) domain in the general receptor for phosphoinositides 1 (Grp1) protein. It monitors PIP3 produced from the enzymatic reaction by measuring its competition with Bodipy®-FL-labeled PIP3 for binding to Terbium chelate-labeled Grp1. By using directly labeled reagents, this assay configuration offers higher sensitivity and faster binding/dissociation kinetics than existing non-radioactive assays, which are critical for competitive assay formats. The assay is homogenous, robust (Z = 0.88), and simple and, thus, compatible with high throughput screening (HTS) processes.
Keywords: TR-FRET, phosphoinositide-3, PH domain, Grp1, PI3 Kinase
Combinatorial Chemistry & High Throughput Screening
Title: A Directly Labeled TR-FRET Assay for Monitoring Phosphoinositide-3-Kinase Activity
Volume: 9 Issue: 7
Author(s): Xiaoke Yang, Puyao Li, Larry Feldberg, Steven C. Kim, Michael Bowman, Irwin Hollander, Robert Mallon and Stanley F. Wolf
Affiliation:
Keywords: TR-FRET, phosphoinositide-3, PH domain, Grp1, PI3 Kinase
Abstract: Phosphoinositide 3-kinases (PI3Ks) comprise a family of kinases that transfer the terminal phosphate of adenosine triphosphate to phosphoinositides at the 3-hydroxyl of the inositol ring to form phosphoinositide (3,4,5) triphosphate (PIP3). The PI3Ks have been shown to play key roles in cell growth, motility, morphology, and survival and thus are of interest as targets in anti-inflammatory and anti-oncogenic drug development. To facilitate identification of novel and selective inhibitors of PI3Ks, we have developed a TR-FRET assay that uses directly labeled reagents. The assay makes use of the high affinity binding of phosphoinositides to a Pleckstrin homology (PH) domain in the general receptor for phosphoinositides 1 (Grp1) protein. It monitors PIP3 produced from the enzymatic reaction by measuring its competition with Bodipy®-FL-labeled PIP3 for binding to Terbium chelate-labeled Grp1. By using directly labeled reagents, this assay configuration offers higher sensitivity and faster binding/dissociation kinetics than existing non-radioactive assays, which are critical for competitive assay formats. The assay is homogenous, robust (Z = 0.88), and simple and, thus, compatible with high throughput screening (HTS) processes.
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Cite this article as:
Yang Xiaoke, Li Puyao, Feldberg Larry, Kim C. Steven, Bowman Michael, Hollander Irwin, Mallon Robert and Wolf F. Stanley, A Directly Labeled TR-FRET Assay for Monitoring Phosphoinositide-3-Kinase Activity, Combinatorial Chemistry & High Throughput Screening 2006; 9 (7) . https://dx.doi.org/10.2174/138620706777935360
DOI https://dx.doi.org/10.2174/138620706777935360 |
Print ISSN 1386-2073 |
Publisher Name Bentham Science Publisher |
Online ISSN 1875-5402 |
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