title: A homogeneous, high-throughput assay for phosphatidylinositol 5-phosphate 4-kinase with a novel, rapid substrate preparation.
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 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.
authors: National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland, United States of America.
authors: Mindy I
authors: MI
authors: Davis
authors: Atsuo T
authors: AT
authors: Sasaki
authors: Min
authors: M
authors: Shen
authors: Brooke M
authors: BM
authors: Emerling
authors: Natasha
authors: N
authors: Thorne
authors: Sam
authors: S
authors: Michael
authors: Rajan
authors: R
authors: Pragani
authors: Matthew
authors: M
authors: Boxer
authors: Kazutaka
authors: K
authors: Sumita
authors: Koh
authors: K
authors: Takeuchi
authors: Douglas S
authors: DS
authors: Auld
authors: Zhuyin
authors: Z
authors: Li
authors: Lewis C
authors: LC
authors: Cantley
authors: Anton
authors: A
authors: Simeonov
mesh_terms: D019870:1-Phosphatidylinositol 4-Kinase / Q000032:analysis / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000737:chemistry; D002396:Catechols / Q000737:chemistry; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D018129:Phosphatidylinositol Phosphates / Q000737:chemistry*; D010759:Phosphorus Isotopes; D013379:Substrate Specificity; D020032:Tyrphostins / Q000737:chemistry
chemical_list: C419770:AG 538; D002396:Catechols; D018129:Phosphatidylinositol Phosphates; D010759:Phosphorus Isotopes; D020032:Tyrphostins; C113567:phosphatidylinositol 5-phosphate; D000255:Adenosine Triphosphate; D019870:1-Phosphatidylinositol 4-Kinase