Davis, MI, Sasaki, AT, Shen, M, Emerling, BM, Thorne, N, Michael, S, Pragani, R, Boxer, M, Sumita, K, Takeuchi, K, Auld, DS, Li, Z, Cantley, LC and Simeonov, A (2013) A Homogeneous, High-Throughput Assay for Phosphatidylinositol 5-Phosphate 4-Kinase with a Novel, Rapid Substrate Preparation. PLoS One.

Abstract

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 PI5P4Kalpha. 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 PI5P4Kalpha assay, indicating its suitability for high-throughput screening applications. Tyrphostin AG-82 had been identified as an inhibitor of PI5P4Kalpha by assessing the degree of phospho transfer of -<sup>32</sup>P-ATP to PI5P; its inhibitory activity against PI5P4Kalpha 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 PI5P4Kalpha with an IC<sub>50</sub> of 1 muM, 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

Item Type:	Article
Additional Information:	pubid: 2 nvp_institute: NIBR contributor_address: (Davis, Shen, Thorne, Michael, Pragani, Boxer, Auld, Li, Simeonov) National Institutes of Health Chemical Genomics Center, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, United States (Sasaki, Emerling, Cantley) Beth Israel Deaconess Medical Center, Department of Medicine, Division of Signal Transduction, Department of Systems Biology, Harvard Medical School, Boston, MA, United States (Sasaki, Sumita) Division of Hematology and Oncology, Department of Internal Medicine, Neuroscience Institute: Brain Tumor Center, University of Cincinnati, College of Medicine, Cincinnati, OH, United States (Takeuchi) Biomedicinal Information Research Center, National Institute of Advanced Industrial Science and Technology, Koto, Tokyo, Japan (Auld) Center for Proteomic Chemistry, Novartis Institutes for Biomedical Research, Cambridge, MA, United States
Date Deposited:	13 Oct 2015 13:13
Last Modified:	13 Oct 2015 13:13
URI:	https://oak.novartis.com/id/eprint/21858