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.