qHTS for Inhibitors of PI5P4K: PI5P4K-alpha 32P-ATP TLC Secondary Assay for Probe SAR
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 ..
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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.
A qHTS PI5P4K-alpha ADP-Glo assay was developed to identify specific modulators of human PI5P4K, phosphatidylinositol-5-phosphate 4-kinase (Type II PIPK) . To confirm that the active compounds and their analogs were true and selective inhibitors, a secondary Thin-layer Chromatography (TLC) assay against recombinant PI5P4K-alpha, PI4P5K-alpha or PI5P4K-beta that utilize a radioactively labeled ATP were developed [1,2]. The data herein are the result of the PI5P4Kalpha TLC assay. Selective inhibitors of the PI5P4Kalpha enzyme would be active against this assay and have no significant activity or lower potency in the PI4P5K-alpha and PI5P4K-beta TLC assay.
 Davis et al., PLoS One. 2013; 8(1):e54127.
 Rameh et al. Nature. 1997 Nov 13; 390(6656):192-6.
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
The kinase reaction was carried out in a total of 50 microL of kinase buffer (50 mM HEPES pH 7.4 and 10 mM MgCl2) containing 20 microM of the lipids that were suspended by sonication. Non-radiolabeled ATP, [gamma-32P] ATP (Perkin Elmer) were incubated (10 microM unless otherwise noted) with 1 microg of recombinant PI5P4K-alpha, PI4P5K-alpha or PI5P4K-beta enzyme for 10 min at room temperature. Unless otherwise indicated, 1 microg of PI5P or PI4P was used with 1 microg of phosphatidylserine as the basal lipid. The reaction was terminated by adding 20 microL of 4 N HCl. Phosphoinositides were extracted by adding 140 microL of methanol/chloroform (1:1, vol:vol) mix and subjected to the TLC (thin-layer chromatography) assay using heat-activated 2% oxaloacetate-coated silica gel 60 plates (20 cm x 20 cm, EMD Chemicals Inc., Billerica, MA, USA). As for the solvent, 1-propanol/ 2 M Acetic acid (65:35, vol:vol) was used. The radiolabeled product, PI(4,5)P2, was quantified with a phosphorimager (Molecular Dynamics, STORM840, GE Healthcare, Waukesha, WI, USA).
* Activity Concentration. ** Test Concentration.
Data Table (Concise)