Development of an in vitro screening assay for PIP5K1α lipid kinase and identification of potent inhibitors

Katja Strätker; Samer Haidar; Ángel Amesty; Ehab El-Awaad; Claudia Götz; Ana Estévez-Braun; Joachim Jose

doi:10.1111/febs.15194

Development of an in vitro screening assay for PIP5K1α lipid kinase and identification of potent inhibitors

FEBS J. 2020 Jul;287(14):3042-3064. doi: 10.1111/febs.15194. Epub 2020 Jan 13.

Authors

Katja Strätker¹, Samer Haidar^{1

2}, Ángel Amesty³, Ehab El-Awaad^{1

4}, Claudia Götz⁵, Ana Estévez-Braun³, Joachim Jose¹

Affiliations

¹ Institut für Pharmazeutische und Medizinische Chemie, Westfälische Wilhelms-Universität Münster, Germany.
² Faculty of Pharmacy, Damascus University, Syria.
³ Departamento de Química Orgánica, Instituto Universitario de Bio-Orgánica Antonio González (CIBICAN), Universidad de La Laguna, Spain.
⁴ Department of Pharmacology, Faculty of Medicine, Assiut University, Egypt.
⁵ Universität des Saarlandes Medizinische Biochemie und Molekularbiologie Geb, Homburg, Germany.

PMID: 31876381
DOI: 10.1111/febs.15194

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 K_m value of 21.6 µm and V_max of 0.65 pmol·min^-1 for the cosubstrate ATP. The average Z' value was determined to be 0.86, indicating a high reliability of the assay. An in silico 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 50 µm. 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 1.55 µm, 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.

Keywords: PIP5K1α; benzoquinone; capillary electrophoresis; lipid kinase; prostate cancer.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Apoptosis
Cell Proliferation
Dose-Response Relationship, Drug
Drug Discovery*
High-Throughput Screening Assays
Humans
Male
Phosphotransferases (Alcohol Group Acceptor) / antagonists & inhibitors*
Prostatic Neoplasms / drug therapy*
Prostatic Neoplasms / pathology*
Protein Kinase Inhibitors / pharmacology*
Tumor Cells, Cultured

Substances

Protein Kinase Inhibitors
Phosphotransferases (Alcohol Group Acceptor)
1-phosphatidylinositol-4-phosphate 5-kinase