Phosphoinositides are a family of tiny cellular lipids formed through a series of phosphorylation processes catalyzed by enzymes termed phosphatidylinositol-phosphate kinases (PIPKs). The phosphatidylinositol 4-phosphate 5-kinase type 1 α (PIP5K1α) is the main isoform responsible for generating membrane pools of phosphatidylinositol-4,5-bisphosphate (PIP2), which, in turn, serves as a substrate for the well-established cancer-relevant target, phosphatidylinositol 3-kinases (PI3Ks). Recent studies provide strong evidence for a key role of PIP5K1α isoform in the development of prostate and breast cancers indicating that targeting this kinase could offer an effective therapeutic strategy in certain types of cancer.

To identify small molecules that can directly inhibit the catalytic activity of PIP5K1α, we developed a robust nonradiometric assay to determine the activity of recombinantly expressed human PIP5K1α in bacterial cell lysates. This assay is based on the separation of a fluorescently labelled PIP5K1α substrate and its corresponding enzymatically phosphorylated product by capillary electrophoresis (CE).

Here, a compound with a 2‐amino‐3‐cyano‐4H‐pyranobenzoquinone scaffold is presented as an example of potent inhibitors of human PIP5K1α identified recently in our lab using the developed CE-based assay. This compound exhibited potent inhibitory effect on PIP5K1α activity with an IC₅₀ value of 1.55 µM, in a substrate‐competitive mode of action. Furthermore, its ability to induce anticancer effects in 2D and 3D cell culture experiments was evaluated. The identified compound may provide the basis for developing highly potent and selective inhibitors of PIP5K1α in cancer cells.