Development of an invitro screening assay for PIP5K1α lipid kinase and identification of potent inhibitors.

Abstract

The human phosphatidylinositol 4-phosphate 5-kinase type I α (hPIP5K1α) participates in the phosphoinositide-3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway. Despite the evidence that hPIP5K1α plays a role in the development of prostate cancer (PCa), only one inhibitor is known to date. With the aim of identifying new inhibitors, a nonradiometric assay for measurement of the hPIP5K1α enzyme activity was developed. The assay is based on the separation of the fluorescently labeled substrate phosphatidylinositol-4-phosphate (PI(4)P) and the resulting product phosphatidylinositol-4,5-bisphosphate (PIP2 ) by capillary electrophoresis (CE). Furthermore, an inactive mutant K261A of hPIP5K1α was generated by site-directed mutagenesis and used as a control. Michaelis-Menten analysis revealed a Km value of 21.6µm and Vmax of 0.65pmol·min-1 for the cosubstrate ATP. The average Z' value was determined to be 0.86, indicating a high reliability of the assay. An insilico screening of an in-house compound library was performed employing the crystal structure of zebrafish PIP5K1α. By applying this strategy, three compounds with a 2-amino-3-cyano-4H-pyranobenzoquinone scaffold were identified and tested using the CE-based assay. These compounds inhibited hPIP5K1α to >90% at a concentration of 50µm. Subsequently, the inhibitory activity of all compounds with a pyranobenzoquinone scaffold (29) was tested on hPIP5K1α. Compound 4-(2-amino-3-cyano-6-hydroxy-5,8-dioxo-7-undecyl-5,8-dihydro-4H-chromen-4-yl)benzoic acid appeared to be the most potent inhibitor of hPIP5K1α identified so far with an IC50 value of 1.55µm, exhibiting a substrate-competitive mode of action. The effects of this compound on cell viability and the induction of apoptosis were investigated in LNCaP, DU145, and PC3 PCa cells.