Method for Assaying the Lipid Kinase Phosphatidylinositol-5-phosphate 4-kinase α in Quantitative High-Throughput Screening (qHTS) Bioluminescent Format

Abstract

Summary ipid 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.

Inexpensive High-Throughput Screening of Kinase Inhibitors Using One-Step Enzyme-Coupled Fluorescence Assay for ADP Detection

“…[19][20][21][22][23][24] Among them, the adenosine diphosphate (ADP) quantitative assay may be the most commonly used platform for HTS nowadays, because ADP is the universal product of all kinases, in contrast to the phosphorylated products, which vary widely and are sometimes difficult to detect. These ADP assays include the ADP-Glo assay (Promega, Madison, WI), in which ADP is converted to ATP and the ATP is measured by means of luciferase/ luciferin reaction; [25][26][27] the Transcreener fluorescence polarization assay (Bellbrook Labs, Madison, WI) using anti-ADP antibody and far-red fluorescence-labeled ADP; [28][29][30] and the ADP-Hunter fluorescence assay (DiscoverX, Fremont, CA), which generates H 2 O 2 from ADP by enzyme coupling reaction followed by production of resorufin. 31,32 These generic ADP detection assay platforms do not employ radiolabeling and are homogeneous, so they are suitable for HTS, but their cost is quite high for large-scale screening, and this may be an issue especially in academia.…”