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BioAssay: AID 2547

Secondary Assay for Inhibitors of Human Galactokinase (GALK): HEK-293 Cell Viability Assay

Assay Submitter (PI): Kent Lai (University of Utah School of Medicine, 50 N Mario Capecchi Drive, Salt Lake City, UT 84132) ..more
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 Tested Compounds
 Tested Compounds
All(171)
 
 
Active(19)
 
 
Inactive(133)
 
 
Inconclusive(20)
 
 
 Tested Substances
 Tested Substances
All(174)
 
 
Active(19)
 
 
Inactive(135)
 
 
Inconclusive(20)
 
 
 Related BioAssays
 Related BioAssays
AID: 2547
Data Source: NCGC (GALK602)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-03-13
Hold-until Date: 2010-09-22
Modify Date: 2010-09-22

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 19
Related Experiments
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AIDNameTypeProbeComment
1868qHTS Assay for Inhibitors of Human Galactokinase (GALK)Confirmatory depositor-specified cross reference
2114qHTS Assay for Inhibitors of Human Galactokinase (GALK): SummarySummary1 depositor-specified cross reference
2499Confirmation Assay for Inhibitors of Human Galactokinase (GALK): probe SARConfirmatory depositor-specified cross reference
2502Confirmatory Assay for Inhibitors of Human Galactokinase (GALK): Phenol-HRP redox assay counterscreen for probe SARConfirmatory depositor-specified cross reference
2506Confirmatory Assay for Inhibitors of Human Galactokinase (GALK): CDP-Me assay counterscreen for probe SARConfirmatory depositor-specified cross reference
493188Confirmation Assay for Inhibitors of Human Galactokinase (GALK): SAR round 2Confirmatory depositor-specified cross reference
2015Confirmation Assay for Inhibitors of Human Galactokinase (GALK)Confirmatory same project related to Summary assay
2035Confirmatory Assay for Inhibitors of Human Galactokinase (GALK): Phenol-HRP redox assay counterscreenConfirmatory same project related to Summary assay
493189qHTS Validation Assay for Inhibitors of Human Galactokinase (GALK)Confirmatory same project related to Summary assay
720664qHTS for Inhibitors of Human Galactokinase (GALK): SAR round 3Other same project related to Summary assay
Description:
NIH Molecular Libraries Probe Production Centers Network [MLPCN]
NIH Chemical Genomics Center [NCGC]
MLPCN Grant: R03 MH085689-01
Assay Submitter (PI): Kent Lai (University of Utah School of Medicine, 50 N Mario Capecchi Drive, Salt Lake City, UT 84132)

NCGC Assay Overview
Cytotoxicity of the active compounds in the GALK qHTS screen (AID 1868) was assessed using the CellTiter-Glo Luminescent Cell Viability Assay kit (Promega, USA) in the HEK-293 (ATCC) cell line. CellTiter-Glo measures the intracellular ATP present in metabolically active cells using Ultra-Glo as the luciferase to detect ATP levels. In brief, the luciferase in the buffer catalyzes the oxidation of luciferin to oxyluciferin and light which is dependent on ATP. Hence, luminescence is proportional to the amount of ATP present in the cell lysis, and this can be used to measure the number of viable HEK-293 cells [1]. Wells treated with various concentrations of cycloheximide (Sigma) and DMSO, and wells with media alone were used as positive and negative controls, respectively. Compounds that do not decrease the luminescence signal are flagged and prioritized.

[1] http://www.promega.com/tbs/tb288/tb288.pdf
Protocol
NCGC Assay Protocol Summary:
HEK-293 cells were grown in a flask up to 80% confluence. Cells were trypsinized, washed, and resuspended in MEM Eagle + 10% FBS + 1% pen-strep media at a concentration of 120 cells/uL. Five uL/well of cell solution was dispensed into a 1536-well assay plates (Greiner, solid white medium-binding plates) with Aurora Discovery BioRAPTR Flying Reagent Dispenser (FRD; Beckton-Dickenson). Cells were pre-incubated at 37 degree Celsius, 5% CO2 for four hours. Compound and control solutions (23nL) were then transferred to the assay plate using the Kalypsys 1536-pin tool. Assay plates were re- incubated at 37 degree Celsius, 5% CO2. After 24 or 48 hours, five uL of CellTiter Glo detection reagent was added. Luminescence signal was read with Perkin Elmer ViewLux plate reader after 30 min room temperature incubation.
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".
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 PubChem:
Assay Cell Type: HEK293
From ChEMBL:
Assay Type: Functional
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
5Curve_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
6Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
7Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
8Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
9Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
10Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
11Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
12Excluded_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
13Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
14Activity at 0.0000023616 uM (2.36159e-06μM**)% Activity at given concentration.Float%
15Activity at 0.0000108633 uM (1.08633e-05μM**)% Activity at given concentration.Float%
16Activity at 0.0000499712 uM (4.99712e-05μM**)% Activity at given concentration.Float%
17Activity at 0.0001744060 uM (0.000174406μM**)% Activity at given concentration.Float%
18Activity at 0.0002601396 uM (0.00026014μM**)% Activity at given concentration.Float%
19Activity at 0.0007627823 uM (0.000762782μM**)% Activity at given concentration.Float%
20Activity at 0.00148 uM (0.00147674μM**)% Activity at given concentration.Float%
21Activity at 0.00234 uM (0.00234126μM**)% Activity at given concentration.Float%
22Activity at 0.00685 uM (0.00685247μM**)% Activity at given concentration.Float%
23Activity at 0.014 uM (0.0137422μM**)% Activity at given concentration.Float%
24Activity at 0.021 uM (0.0210783μM**)% Activity at given concentration.Float%
25Activity at 0.041 uM (0.0412265μM**)% Activity at given concentration.Float%
26Activity at 0.063 uM (0.0633844μM**)% Activity at given concentration.Float%
27Activity at 0.185 uM (0.185423μM**)% Activity at given concentration.Float%
28Activity at 0.371 uM (0.371038μM**)% Activity at given concentration.Float%
29Activity at 0.568 uM (0.568348μM**)% Activity at given concentration.Float%
30Activity at 1.113 uM (1.11311μM**)% Activity at given concentration.Float%
31Activity at 1.709 uM (1.70908μM**)% Activity at given concentration.Float%
32Activity at 5.006 uM (5.00642μM**)% Activity at given concentration.Float%
33Activity at 10.02 uM (10.018μM**)% Activity at given concentration.Float%
34Activity at 15.36 uM (15.361μM**)% Activity at given concentration.Float%
35Activity at 46.08 uM (46.0829μM**)% Activity at given concentration.Float%
36Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: R03 MH085689-01

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