qHTS for Inhibitors of phosphatidylinositol 5-phosphate 4-kinase (PI5P4K)
Phosphatidylinositol (PI) signaling has been shown to impact a large and diverse set of cellular processes including proliferation, survival, and growth; their dysregulation is common in cancer and other diseases. Recently, PI5P has been shown to regulate the tumor suppressor ING2. In addition to finding PI5P, we discovered the type II phosphatidylinositol 5-phosphate 4-kinase (PI5P4K) family more ..
BioActive Compounds: 4076
Phosphatidylinositol (PI) signaling has been shown to impact a large and diverse set of cellular processes including proliferation, survival, and growth; their dysregulation is common in cancer and other diseases. Recently, PI5P has been shown to regulate the tumor suppressor ING2. In addition to finding PI5P, we discovered the type II phosphatidylinositol 5-phosphate 4-kinase (PI5P4K) family (alpha, beta, gamma isoforms) that catalyze the conversion of PI5P to PI(4,5)P2. Alternatively, PI(4,5)P2 can also be synthesized through phosphatidylinositol-4-phosphate (PI4P) by the type I phosphatidylinositol 4-phosphate 5-kinases (PI4P5K). These PI5P4K enzymes therefore represent one way by which cells can regulate endogenous PI5P levels and play important roles in insulin signaling and in stress responses. Furthermore, recent findings have demonstrated a critical role for PI5P4K in tumor cell growth and support a potential role in oncogenesis for PI5P4K.
This proposal aims to identify and optimize specific modulators of human PI5P4K, phosphatidylinositol-5-phosphate 4-kinase (Type II PIPK), a family of kinases that regulate AKT signaling and tumor cell growth. In this project we aim to identify inhibitors of human PI5P4K using a quantitative high-throughput screening approach against the MLSMR containing ~400,000 small molecules.
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]
MLPCN Grant: MH096575
Assay Submitter (PI): Atsuo Sasaki, Beth Israel Deaconess Medical Center
2 microL of PI5P4Kalpha/substrate buffer (columns 1-2, 5-48) are dispensed into Greiner white solid bottom 1536-well assay plates; column 3 receives only PI5P4Kalpha buffer as 0x substrate controls; column 4 receives only substrate buffer as 0x PI5P4Kalpha controls. Final assay buffer consists of 0.038 M HEPES pH 7.4, 0.28 mM EGTA, 0.1% CHAPS, and 5% DMSO (final concentration). Compounds are then transferred via Kalypsys pin tool equipped with 1536-pin array (10 nL slotted pins, V&P Scientific, San Diego, CA). Following addition of compound, 1 microL of ATP (5 microM final concentration, all columns) is added to initiate the reaction. ATP is diluted in an assay buffer consisting of 0.02 M HEPES pH 7.4, 60 mM MgCl2, 15 microM ATP, and 0.1% CHAPS. The plates are then incubated at room temperature for 60 minutes. ADP-Glo reagent 1 is added (2 microL) and plates incubated for 45 minutes, before ADP-Glo reagent 2 (4 microL) is added and plates incubated for 30 minutes. They are then transferred to a ViewLux high-throughput CCD imager (PerkinElmer) wherein single end-point measurements of luminescence are acquired using a clear filter.
Compounds whose "max activity" is <= -50 are considered "active" and assigned a score of 90; compounds where the "max activity" is > -50% and < -30% are consider "inconclusive" and are assigned a score of 50; for compounds with a "max activity" >= -30% they are considered "inactive" and are assigned a score of 10.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)