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BioAssay: AID 743286

qHTS for Inhibitors of PI5P4K: Confirmation in Primary Assay

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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 Tested Compounds
 Tested Compounds
All(1126)
 
 
Active(343)
 
 
Inactive(332)
 
 
Inconclusive(451)
 
 
 Tested Substances
 Tested Substances
All(1126)
 
 
Active(343)
 
 
Inactive(332)
 
 
Inconclusive(451)
 
 
AID: 743286
Data Source: NCGC (pi5p4k-f10-cherrypick-dpi)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2014-02-19

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 343
Related Experiments
AIDNameTypeComment
652103qHTS for Inhibitors of phosphatidylinositol 5-phosphate 4-kinase (PI5P4K): SummarySummarydepositor-specified cross reference
652105qHTS for Inhibitors of phosphatidylinositol 5-phosphate 4-kinase (PI5P4K)Confirmatorysame project related to Summary assay
743285qHTS for Inhibitors of PI5P4K: ADP-Glo counter assayConfirmatorysame project related to Summary assay
743379On Hold
743380On Hold
743381On Hold
743382On Hold
743383On Hold
Description:
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). This assay was screened against the MLSMR containing ~400,000 small molecules (PubChem AID 652105). To confirm that the hits were not interacting with the detection enzyme system, a 1536-well counterscreen against the ADP-Glo kit (Promega) was utilized, as described in Davis et al., 2013 [1].

[1] 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
Protocol
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.
Comment
Compound Ranking:
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.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1PhenotypeString
2Potency*FloatμM
3EfficacyString
4Analysis CommentString
5Activity_ScoreString
6Curve_DescriptionString
7Fit_LogAC50String
8Fit_HillSlopeString
9Fit_R2String
10Fit_InfiniteActivityString
11Fit_ZeroActivityString
12Fit_CurveClassString
13Excluded_PointsString
14Max_ResponseString
15Activity at 0.00492 uMString
16Activity at 0.025 uMString
17Activity at 0.123 uMString
18Activity at 0.615 uMString
19Activity at 3.080 uMString
20Activity at 15.40 uMString
21Activity at 76.90 uMString
22Compound QCString
23Activity at 0.0001736932 uMString
24Activity at 0.0004343491 uMString
25Activity at 0.0006954697 uMString
26Activity at 0.00130 uMString
27Activity at 0.00246 uMString
28Activity at 0.00392 uMString
29Activity at 0.00759 uMString
30Activity at 0.012 uMString
31Activity at 0.022 uMString
32Activity at 0.035 uMString
33Activity at 0.070 uMString
34Activity at 0.106 uMString
35Activity at 0.218 uMString
36Activity at 0.317 uMString
37Activity at 0.695 uMString
38Activity at 0.971 uMString
39Activity at 2.006 uMString
40Activity at 3.025 uMString
41Activity at 5.609 uMString
42Activity at 8.716 uMString
43Activity at 23.86 uMString
44Activity at 31.57 uMString
45Activity at 76.25 uMString
46Activity at 105.2 uMString
47Activity at 162.8 uMString
48Activity at 251.0 uMString

* Activity Concentration.
Additional Information
Grant Number: MH096575

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
Classification
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