Abstract
Lipid kinases are important regulators of a variety of cellular processes and their dysregulation causes diseases such as cancer and metabolic diseases. Distinct lipid kinases regulate the seven different phosphorylated forms of phosphatidylinositol (PtdIns). Some lipid kinases utilize long-chain lipid substrates that have limited solubility in aqueous solutions, which can lead to difficulties in developing a robust and miniaturizable biochemical assay. The ability to prepare the lipid substrate and develop assays to identify modulators of lipid kinases is important and is the focus of this methods chapter. Herein, we describe a method to prepare a DMSO-based lipid mixture that enables the 1536-well screening of the lipid kinase phosphatidylinositol-5-phosphate 4-kinase α (PI5P4Kα) utilizing the d-myo-di16-PtIns(5)P substrate in quantitative high-throughput screening (qHTS) format using the ADP-Glo™ technology to couple the production of ADP to a bioluminescent readout.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Wymann MP, Schneiter R (2008) Lipid signalling in disease. Nat Rev Mol Cell Biol 9(2):162–176. doi:10.1038/nrm2335
Rameh LE, Tolias KF, Duckworth BC, Cantley LC (1997) A new pathway for synthesis of phosphatidylinositol-4,5-bisphosphate. Nature 390(6656):192–196. doi:10.1038/36621
Emerling BM, Hurov JB, Poulogiannis G, Tsukazawa KS, Choo-Wing R, Wulf GM, Bell EL, Shim HS, Lamia KA, Rameh LE, Bellinger G, Sasaki AT, Asara JM, Yuan X, Bullock A, Denicola GM, Song J, Brown V, Signoretti S, Cantley LC (2013) Depletion of a putatively druggable class of phosphatidylinositol kinases inhibits growth of p53-null tumors. Cell 155(4):844–857. doi:10.1016/j.cell.2013.09.057
Jude JG, Spencer GJ, Huang X, Somerville TD, Jones DR, Divecha N, Somervaille TC (2014) A targeted knockdown screen of genes coding for phosphoinositide modulators identifies PIP4K2A as required for acute myeloid leukemia cell proliferation and survival. Oncogene. doi:10.1038/onc.2014.77
Tanega C, Shen M, Mott BT, Thomas CJ, MacArthur R, Inglese J, Auld DS (2009) Comparison of bioluminescent kinase assays using substrate depletion and product formation. Assay Drug Dev Technol 7(6):606–614. doi:10.1089/adt.2009.0230
Harbert C, Marshall J, Soh S, Steger K (2008) Development of a HTRF kinase assay for determination of Syk activity. Curr Chem Genomics 1:20–26. doi:10.2174/1875397300801010020
Hastie CJ, McLauchlan HJ, Cohen P (2006) Assay of protein kinases using radiolabeled ATP: a protocol. Nat Protoc 1(2):968–971
Kleman-Leyer KM, Klink TA, Kopp AL, Westermeyer TA, Koeff MD, Larson BR, Worzella TJ, Pinchard CA, van de Kar SA, Zaman GJ, Hornberg JJ, Lowery RG (2009) Characterization and optimization of a red-shifted fluorescence polarization ADP detection assay. Assay Drug Dev Technol 7(1):56–67. doi:10.1089/adt.2008.175
Szoka F Jr, Papahadjopoulos D (1980) Comparative properties and methods of preparation of lipid vesicles (liposomes). Annu Rev Biophys Bioeng 9:467–508. doi:10.1146/annurev.bb.09.060180.002343
Demian DJ, Clugston SL, Foster MM, Rameh L, Sarkes D, Townson SA, Yang L, Zhang M, Charlton ME (2009) High-throughput, cell-free, liposome-based approach for assessing in vitro activity of lipid kinases. J Biomol Screen 14(7):838–844. doi:10.1177/1087057109339205
Voss MD, Czechtizky W, Li Z, Rudolph C, Petry S, Brummerhop H, Langer T, Schiffer A, Schaefer HL (2014) Discovery and pharmacological characterization of a novel small molecule inhibitor of phosphatidylinositol-5-phosphate 4-kinase, type II, beta. Biochem Biophys Res Commun 449(3):327–331. doi:10.1016/j.bbrc.2014.05.024
Vidugiriene J, Zegzouti H, Goueli SA (2009) Evaluating the utility of a bioluminescent ADP-detecting assay for lipid kinases. Assay Drug Dev Technol 7(6):585–597. doi:10.1089/adt.2009.0223
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, Simeonov A (2013) A homogeneous, high-throughput assay for phosphatidylinositol 5-phosphate 4-kinase with a novel, rapid substrate preparation. PLoS One 8(1):e54127. doi:10.1371/journal.pone.0054127
Copeland RA (2005) Evaluation of enzyme inhibitors in drug discovery. A guide for medicinal chemists and pharmacologists. Methods Biochem Anal 46:1–265
Yasgar A, Shinn P, Jadhav A, Auld D, Michael S, Zheng W, Austin CP, Inglese J, Simeonov A (2008) Compound management for quantitative high-throughput screening. JALA Charlottesv Va 13(2):79–89. doi:10.1016/j.jala.2007.12.004
Auld DS, Southall NT, Jadhav A, Johnson RL, Diller DJ, Simeonov A, Austin CP, Inglese J (2008) Characterization of chemical libraries for luciferase inhibitory activity. J Med Chem 51(8):2372–2386. doi:10.1021/jm701302v
Thorne N, Shen M, Lea WA, Simeonov A, Lovell S, Auld DS, Inglese J (2012) Firefly luciferase in chemical biology: a compendium of inhibitors, mechanistic evaluation of chemotypes, and suggested use as a reporter. Chem Biol 19(8):1060–1072. doi:10.1016/j.chembiol.2012.07.015
Acknowledgement
This work was supported by the Molecular Libraries Common Fund Program of the National Institutes of Health. The content of this publication does not necessarily reflect the views of policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Davis, M.I., Sasaki, A.T., Simeonov, A. (2016). Method for Assaying the Lipid Kinase Phosphatidylinositol-5-phosphate 4-kinase α in Quantitative High-Throughput Screening (qHTS) Bioluminescent Format. In: Waugh, M. (eds) Lipid Signaling Protocols. Methods in Molecular Biology, vol 1376. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3170-5_1
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3170-5_1
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3169-9
Online ISBN: 978-1-4939-3170-5
eBook Packages: Springer Protocols