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-32P-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 IC50 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.

Development of a High-Throughput Screening Assay to Identify Inhibitors of the Lipid Kinase PIP5K1C

“…Unlike the PIP4K2s, there are no crystal structures of any of the PIP5K1s making structure-guided drug design impractical 11 ; however, standard structure-activity relationship (SAR) studies are feasible and required. As we previously reported 9 , UNC3230 has limited aqueous solubility, highlighting the need for medicinal chemistry optimization to increase solubility while maintaining potency and selectivity.…”

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

“…Protein was expressed and purified as described in [13]. To make this reagent, 500 μL of lipid mix described in 2.1 was added to 6130 μL of buffer (43 mM Hepes pH 7.4, 0.27 mM EGTA, 0.108% CHAPS), and the mixture was sonicated and mixed by vortexing.…”

“…Transfer 23 nL of each library compound dissolved in DMSO from a 1536-well clear compound plate arrayed in columns 5-48 and 23 nL of control compounds (DMSO in column 1, 3-4, and Tyrphostin AG82 [13] in duplicate 16-pt dose response in column 2) from a 1536-well compound plate arrayed in columns 1-4 into the assay plate using the pintool (see note 7).…”

Evolution of a strategy for preparing bioactive small molecules by sequential multicomponent assembly processes, cyclizations, and diversification

“…62 The PI4P5Ks regulate AKT signaling and tumor cell growth, 63 and recent findings have illuminated PI4P5K's potential involvement in oncogenesis, suggesting that modulating its activity may be an effective therapeutic approach to treating cancers.…”