qHTS for Inhibitors of PI5P4K: PI5P4K-alpha Confirmatory 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 ..
BioActive Compounds: 18
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 PI5P4Kalpha ADP-Glo assay was developed to identify specific modulators of human PI5P4K, phosphatidylinositol-5-phosphate 4-kinase (Type II PIPK) . This assay was screened against the MLSMR containing ~400,000 small molecules (PubChem AID 652105). The same assay was used to validate subsequent Medicinal Chemistry efforts on the active compounds and its analogs. Inhibition of the enzyme activity (signal decrease) is the desired outcome for this assay.
 Davis et al., PLoS One. 2013; 8(1):e54127.
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
Two microliter of PI5P4Kalpha/substrate buffer (columns 1-2, 5-48) was dispensed into Greiner white solid bottom 1536-well assay plates; column 3 received only PI5P4Kalpha buffer as no substrate control; column 4 receives only substrate buffer as no 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 were then transferred via Kalypsys pin tool equipped with 1536-pin array. Following addition of compound, 1 uL of ATP (5 uM final concentration) was added to initiate the reaction. ATP was diluted in an assay buffer consisting of 0.02 M HEPES pH 7.4, 60 mM MgCl2, 15 uM ATP, and 0.1% CHAPS. The plates were then incubated at room temperature for 60 minutes. ADP-Glo reagent 1 was added and plates incubated for 45 minutes. ADP-Glo reagent 2 was added and the plates were incubated for 30 minutes. The plates were transferred to a ViewLux high-throughput CCD imager (PerkinElmer) wherein single end-point measurements of luminescence were acquired using a clear filter.
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)