qHTS for Inhibitors of PI5P4K: BT474 Cell Proliferation 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 Compound: 1
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 further confirm inhibitors of PI4P5K, cell proliferation assays against BT474 cells were performed. This invasive ductal (breast) carcinoma cell line was shown to have high levels of PI5P4K and knockdown of PI5P4K (both alpha and beta) by shRNA abrogates cell proliferation and impaired tumor growth . Therefore PI5P4K inhibitors would also cause a decrease in BT474 proliferation. Inhibition is the desired outcome for this assay.
 Davis et al., PLoS One. 2013; 8(1):e54127.
 Emerling et al., Cell. 2013 Nov 7; 155(4):844-57.
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
BT474 cells were cultured in DMEM with 10% FBS and plated in 1536-well white solid-bottom tissue-culture treated plates at a density of 500 cells per 5 microL well. After allowing the cells to adhere for 4 hours, 23 nL of compound was transferred to the plate with a pintool (Kalpysys). After 72 hours of incubation at 37 degrees C 5% CO2 the level of ATo was measured using the Cell Titer Glo luciferase-coupled ATP detection system from Promega. This assays monitors cells by quantitating the amount of ATP present by luciferase, which in the presence of ATP converts pro-luciferin into the luminescent luciferin product. A compound that inhibits cell proliferation would result in a decrease in luminescence. The data were normalized to control columns representing maximum signal (cells with DMSO) and background (no cells with DMSO). The luminescence was read on a Viewlux (Perkin Elmer) plate reader.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)