PUBLICATION
Development of an in vitro screening assay for PIP5K1α lipid kinase and identification of potent inhibitors
- Authors
- Strätker, K., Haidar, S., Amesty, Á., El-Awaad, E., Götz, C., Estévez-Braun, A., Jose, J.
- ID
- ZDB-PUB-191227-2
- Date
- 2019
- Source
- The FEBS journal 287(14): 3042-3064 (Journal)
- Registered Authors
- Keywords
- PIP5K1α, benzoquinone, capillary electrophoresis, lipid kinase, prostate cancer
- 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
- PubMed
- 31876381 Full text @ FEBS J.
Citation
Strätker, K., Haidar, S., Amesty, Á., El-Awaad, E., Götz, C., Estévez-Braun, A., Jose, J. (2019) Development of an in vitro screening assay for PIP5K1α lipid kinase and identification of potent inhibitors. The FEBS journal. 287(14):3042-3064.
Abstract
The human phosphatidylinositol 4-phosphate 5-kinase type I α (hPIP5K1α) participates in the PI3K/AKT/mTOR 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 non-radiometric 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 21.6 µM and Vmax of 0.65 pmol/min for the co-substrate 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 more than 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. Effects of this compound on cell viability and the induction of apoptosis were investigated in LNCaP, DU145, and PC3 prostate cancer cells.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping