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A Homogeneous, High-Throughput Assay for Phosphatidylinositol 5-Phosphate 4-Kinase with a Novel, Rapid Substrate Preparation
Phosphoinositide kinases regulate diverse cellular functions and are important targets for therapeutic development for diseases, such as diabetes and cancer. Preparation of the lipid substrate is crucial for the development of a robust and miniaturizable lipid kinase assay. Enzymatic assays for phos...
Autores principales: | , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2013
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3542272/ https://www.ncbi.nlm.nih.gov/pubmed/23326584 http://dx.doi.org/10.1371/journal.pone.0054127 |
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author | Davis, Mindy I. Sasaki, Atsuo T. Shen, Min Emerling, Brooke M. Thorne, Natasha Michael, Sam Pragani, Rajan Boxer, Matthew Sumita, Kazutaka Takeuchi, Koh Auld, Douglas S. Li, Zhuyin Cantley, Lewis C. Simeonov, Anton |
author_facet | Davis, Mindy I. Sasaki, Atsuo T. Shen, Min Emerling, Brooke M. Thorne, Natasha Michael, Sam Pragani, Rajan Boxer, Matthew Sumita, Kazutaka Takeuchi, Koh Auld, Douglas S. Li, Zhuyin Cantley, Lewis C. Simeonov, Anton |
author_sort | Davis, Mindy I. |
collection | PubMed |
description | Phosphoinositide kinases regulate diverse cellular functions and are important targets for therapeutic development for diseases, such as diabetes and cancer. Preparation of the lipid substrate is crucial for the development of a robust and miniaturizable lipid kinase assay. Enzymatic assays for phosphoinositide kinases often use lipid substrates prepared from lyophilized lipid preparations by sonication, which result in variability in the liposome size from preparation to preparation. Herein, we report a homogeneous 1536-well luciferase-coupled bioluminescence assay for PI5P4Kα. The substrate preparation is novel and allows the rapid production of a DMSO-containing substrate solution without the need for lengthy liposome preparation protocols, thus enabling the scale-up of this traditionally difficult type of assay. The Z’-factor value was greater than 0.7 for the PI5P4Kα assay, indicating its suitability for high-throughput screening applications. Tyrphostin AG-82 had been identified as an inhibitor of PI5P4Kα by assessing the degree of phospho transfer of γ-(32)P-ATP to PI5P; its inhibitory activity against PI5P4Kα was confirmed in the present miniaturized assay. From a pilot screen of a library of bioactive compounds, another tyrphostin, I-OMe tyrphostin AG-538 (I-OMe-AG-538), was identified as an ATP-competitive inhibitor of PI5P4Kα with an IC(50) of 1 µM, affirming the suitability of the assay for inhibitor discovery campaigns. This homogeneous assay may apply to other lipid kinases and should help in the identification of leads for this class of enzymes by enabling high-throughput screening efforts. |
format | Online Article Text |
id | pubmed-3542272 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-35422722013-01-16 A Homogeneous, High-Throughput Assay for Phosphatidylinositol 5-Phosphate 4-Kinase with a Novel, Rapid Substrate Preparation Davis, Mindy I. Sasaki, Atsuo T. Shen, Min Emerling, Brooke M. Thorne, Natasha Michael, Sam Pragani, Rajan Boxer, Matthew Sumita, Kazutaka Takeuchi, Koh Auld, Douglas S. Li, Zhuyin Cantley, Lewis C. Simeonov, Anton PLoS One Research Article Phosphoinositide kinases regulate diverse cellular functions and are important targets for therapeutic development for diseases, such as diabetes and cancer. Preparation of the lipid substrate is crucial for the development of a robust and miniaturizable lipid kinase assay. Enzymatic assays for phosphoinositide kinases often use lipid substrates prepared from lyophilized lipid preparations by sonication, which result in variability in the liposome size from preparation to preparation. Herein, we report a homogeneous 1536-well luciferase-coupled bioluminescence assay for PI5P4Kα. The substrate preparation is novel and allows the rapid production of a DMSO-containing substrate solution without the need for lengthy liposome preparation protocols, thus enabling the scale-up of this traditionally difficult type of assay. The Z’-factor value was greater than 0.7 for the PI5P4Kα assay, indicating its suitability for high-throughput screening applications. Tyrphostin AG-82 had been identified as an inhibitor of PI5P4Kα by assessing the degree of phospho transfer of γ-(32)P-ATP to PI5P; its inhibitory activity against PI5P4Kα was confirmed in the present miniaturized assay. From a pilot screen of a library of bioactive compounds, another tyrphostin, I-OMe tyrphostin AG-538 (I-OMe-AG-538), was identified as an ATP-competitive inhibitor of PI5P4Kα with an IC(50) of 1 µM, affirming the suitability of the assay for inhibitor discovery campaigns. This homogeneous assay may apply to other lipid kinases and should help in the identification of leads for this class of enzymes by enabling high-throughput screening efforts. Public Library of Science 2013-01-10 /pmc/articles/PMC3542272/ /pubmed/23326584 http://dx.doi.org/10.1371/journal.pone.0054127 Text en https://creativecommons.org/publicdomain/zero/1.0/ This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration, which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. |
spellingShingle | Research Article Davis, Mindy I. Sasaki, Atsuo T. Shen, Min Emerling, Brooke M. Thorne, Natasha Michael, Sam Pragani, Rajan Boxer, Matthew Sumita, Kazutaka Takeuchi, Koh Auld, Douglas S. Li, Zhuyin Cantley, Lewis C. Simeonov, Anton A Homogeneous, High-Throughput Assay for Phosphatidylinositol 5-Phosphate 4-Kinase with a Novel, Rapid Substrate Preparation |
title | A Homogeneous, High-Throughput Assay for Phosphatidylinositol 5-Phosphate 4-Kinase with a Novel, Rapid Substrate Preparation |
title_full | A Homogeneous, High-Throughput Assay for Phosphatidylinositol 5-Phosphate 4-Kinase with a Novel, Rapid Substrate Preparation |
title_fullStr | A Homogeneous, High-Throughput Assay for Phosphatidylinositol 5-Phosphate 4-Kinase with a Novel, Rapid Substrate Preparation |
title_full_unstemmed | A Homogeneous, High-Throughput Assay for Phosphatidylinositol 5-Phosphate 4-Kinase with a Novel, Rapid Substrate Preparation |
title_short | A Homogeneous, High-Throughput Assay for Phosphatidylinositol 5-Phosphate 4-Kinase with a Novel, Rapid Substrate Preparation |
title_sort | homogeneous, high-throughput assay for phosphatidylinositol 5-phosphate 4-kinase with a novel, rapid substrate preparation |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3542272/ https://www.ncbi.nlm.nih.gov/pubmed/23326584 http://dx.doi.org/10.1371/journal.pone.0054127 |
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