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BioAssay: AID 743381

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 ..
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 Tested Compounds
 Tested Compounds
All(23)
 
 
Active(18)
 
 
Inactive(1)
 
 
Inconclusive(5)
 
 
 Tested Substances
 Tested Substances
All(24)
 
 
Active(18)
 
 
Inactive(1)
 
 
Inconclusive(5)
 
 
AID: 743381
Data Source: NCGC (pi5p4k-SAR-f3458-13-28)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2014-04-07
Hold-until Date: 2015-04-04
Modify Date: 2015-04-08

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 18
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
743285qHTS for Inhibitors of PI5P4K: ADP-Glo counter assayConfirmatorysame project related to Summary assay
743286qHTS for Inhibitors of PI5P4K: Confirmation in Primary AssayConfirmatorysame project related to Summary assay
743379qHTS for Inhibitors of PI5P4K: PI5P4K-beta 32P-ATP TLC Secondary Assay for Probe SARConfirmatorysame project related to Summary assay
743380qHTS for Inhibitors of PI5P4K: PI4P5K-alpha 32P-ATP TLC Secondary Assay for Probe SARConfirmatorysame project related to Summary assay
743382qHTS for Inhibitors of PI5P4K: BT474 Cell Proliferation Assay for Probe SARConfirmatorysame project related to Summary assay
743383qHTS for Inhibitors of PI5P4K: PI5P4K-alpha 32P-ATP TLC Secondary Assay for Probe SARConfirmatorysame 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 PI5P4Kalpha ADP-Glo assay was developed to identify specific modulators of human PI5P4K, phosphatidylinositol-5-phosphate 4-kinase (Type II PIPK) [1]. 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.

[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 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.
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:
Confirmatory screen for Probe SAR:
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.Float%
4Analysis CommentAnnotation/notes on a particular compound's data or its analysis.String
5Activity_ScoreActivity score.Integer
6Curve_DescriptionA description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically significant, but below 80% of control.String
7Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
8Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
9Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
10Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
11Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
12Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
13Excluded_PointsWhich dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.String
14Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
15Activity at 0.00130 uM (0.00130269μM**)% Activity at given concentration.Float%
16Activity at 0.00252 uM (0.00251643μM**)% Activity at given concentration.Float%
17Activity at 0.00391 uM (0.00391033μM**)% Activity at given concentration.Float%
18Activity at 0.00782 uM (0.00781623μM**)% Activity at given concentration.Float%
19Activity at 0.012 uM (0.0117161μM**)% Activity at given concentration.Float%
20Activity at 0.019 uM (0.0187801μM**)% Activity at given concentration.Float%
21Activity at 0.023 uM (0.0234486μM**)% Activity at given concentration.Float%
22Activity at 0.035 uM (0.0351801μM**)% Activity at given concentration.Float%
23Activity at 0.072 uM (0.0719026μM**)% Activity at given concentration.Float%
24Activity at 0.106 uM (0.105519μM**)% Activity at given concentration.Float%
25Activity at 0.188 uM (0.188438μM**)% Activity at given concentration.Float%
26Activity at 0.317 uM (0.316504μM**)% Activity at given concentration.Float%
27Activity at 0.622 uM (0.622208μM**)% Activity at given concentration.Float%
28Activity at 0.950 uM (0.950371μM**)% Activity at given concentration.Float%
29Activity at 1.899 uM (1.89934μM**)% Activity at given concentration.Float%
30Activity at 2.847 uM (2.84748μM**)% Activity at given concentration.Float%
31Activity at 5.384 uM (5.38428μM**)% Activity at given concentration.Float%
32Activity at 8.547 uM (8.54701μM**)% Activity at given concentration.Float%
33Activity at 9.615 uM (9.61538μM**)% Activity at given concentration.Float%
34Activity at 17.78 uM (17.7785μM**)% Activity at given concentration.Float%
35Activity at 25.64 uM (25.641μM**)% Activity at given concentration.Float%
36Activity at 46.59 uM (46.5928μM**)% Activity at given concentration.Float%
37Activity at 76.92 uM (76.9231μM**)% Activity at given concentration.Float%
38Activity at 153.8 uM (153.846μM**)% Activity at given concentration.Float%
39Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

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

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