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BioAssay: AID 743285

qHTS for Inhibitors of PI5P4K: ADP-Glo counter 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(1124)
 
 
Active(2)
 
 
Inactive(1116)
 
 
Inconclusive(6)
 
 
 Tested Substances
 Tested Substances
All(1124)
 
 
Active(2)
 
 
Inactive(1116)
 
 
Inconclusive(6)
 
 
 Related BioAssays
 Related BioAssays
AID: 743285
Data Source: NCGC (pi5p4k-counter-f11)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2014-02-19
Modify Date: 2014-02-26

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 2
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)Confirmatorydepositor-specified cross reference
743286qHTS for Inhibitors of PI5P4K: Confirmation in Primary AssayConfirmatorysame project related to Summary assay
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]. For this assay, an ATP/ADP mix of 80% ATP and 20% ADP was used to mimic the PI5P4K-alpha enzyme reaction. For a compound that does not interfere with the detection system, there should be no inhibition of the luminescence signal. Therefore, true inhibitors of PI5P4K-alpha should have no activity in this assay.

[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
Two micoliter of assay buffer (0.038 M HEPES pH 7.4, 0.28 mM EGTA, 0.1% CHAPS, 7.5% DMSO) was dispensed into Greiner white solid bottom 1536-well assay plates. Compounds were then transferred via Kalypsys pin tool equipped with 1536-pin array (10 nL slotted pins, V&P Scientific, San Diego, CA). Next, 1 uL of ATP / ADP mix (4 uM ATP / 1 uM ADP final concentrations in 20 mM HEPES pH 7.4, 60 mM MgCl2, and 0.1% CHAPS) was added for a total reaction volume of 3 uL. The plates were incubated at room temperature for 60 minutes. To detect the activity, 2 uL of ADP-Glo reagent 1 was added to each well followed by 45 min room temperature incubation. Then, 4 uL of ADP-Glo reagent 2 was added to each well and the plates were incubated further for 30 min at room temperature. Finally, luminescence signal was detected using the PerkinElmer ViewLux plate reader (30 sec exposure). The data was normalized to control wells with 20% ADP / 80% ATP and wells with 100% ATP.
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.
Categorized Comment - additional comments and annotations
From BioAssay Depositor:
Counterscreen Assay:
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1PhenotypeIndicates type of activity observed: inhibitor, activator, fluorescent, cytotoxic, inactive, or inconclusive.String
2Potency*Concentration at which compound exhibits half-maximal efficacy, AC50. Extrapolated AC50s also include the highest efficacy observed and the concentration of compound at which it was observed.FloatμM
3EfficacyMaximal efficacy of compound, reported as a percentage of control. These values are estimated based on fits of the Hill equation to the dose-response curves.String
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 uM (0.00492μM**)% activity at a given concentrationFloat%
16Activity at 0.025 uM (0.025μM**)% activity at a given concentrationFloat%
17Activity at 0.123 uM (0.123μM**)% activity at a given concentrationFloat%
18Activity at 0.615 uM (0.615μM**)% activity at a given concentrationFloat%
19Activity at 3.080 uM (3.08μM**)% activity at a given concentrationFloat%
20Activity at 15.40 uM (15.4μM**)% activity at a given concentrationFloat%
21Activity at 76.90 uM (76.9μM**)% activity at a given concentrationFloat%
22Compound QCString

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: MH096575

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