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BioAssay: AID 2502

Confirmatory Assay for Inhibitors of Human Galactokinase (GALK): Phenol-HRP redox assay counterscreen for probe SAR

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(38)
 
 
Inactive(36)
 
 
Inconclusive(2)
 
 
 Tested Substances
 Tested Substances
All(43)
 
 
Inactive(41)
 
 
Inconclusive(2)
 
 
 Related BioAssays
 Related BioAssays
AID: 2502
Data Source: NCGC (GALK640h)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-03-09
Hold-until Date: 2010-09-22
Modify Date: 2010-09-22

Data Table ( Complete ):           All
Tested Compounds:
Depositor Specified Assays
AIDNameTypeProbeComment
1379Counterscreen for Luciferase (Kinase-Glo TM) Inhibitionconfirmatory
1868qHTS Assay for Inhibitors of Human Galactokinase (GALK)confirmatory
2114qHTS Assay for Inhibitors of Human Galactokinase (GALK): Summarysummary1
2547Secondary Assay for Inhibitors of Human Galactokinase (GALK): HEK-293 Cell Viability Assayconfirmatory
493188Confirmation Assay for Inhibitors of Human Galactokinase (GALK): SAR round 2confirmatory
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:
One mM dithiothreitol (DTT) was used as a reducing agent to maintain the galactokinase (GALK) enzyme in its active form in the GALK qHTS screen (AID: 1868). However, it has been shown that in the presence of DTT a number of compounds also undergo redox recyling, generating a significant amount of H2O2 that can oxidize crucial cysteine residues in the enzyme, leading to and a false-positive result [1].
To evaluate and flag redox recycling compounds for compounds re-acquired from the qHTS screen, an endpoint colorimetric assay developed by Johnston et al. [1] was adopted and optimized for a 1536-well plate format. In brief, the assay detects the presence of H2O2 by following the change in absorbance at 600 nm due to oxidation of phenol red (Sigma, cat# 114537) catalyzed by horseradish peroxidase (HRP; Invitrogen). A stop reagent, 1N NaOH was added prior to reading the absorbance. DMSO and different concentrations of hydrogen peroxide (Sigma, cat# 216763) in the GALK buffer were used as negative and positive controls respectively.
Protocol
NCGC Assay Protocol Summary:
Two uL/well of GALK buffer (20mM HEPES pH8.0, 5 mM MgCl2, 60 mM NaCl, 1 mM DTT, 0.01% BSA final concentration) was dispensed into a 1536-well assay plates (Greiner, black clear bottom plates) with Aurora Discovery BioRAPTR Flying Reagent Dispenser (FRD; Beckton-Dickenson). Compound and control solutions (23 nL each) were transferred to the assay plate in their respective wells using Kalypsys pin tool equipped with a 1536-pin tool head and incubated at room temperature for 30 min. Presence of H2O2 produced by redox cycling compounds was detected by adding 2 uL/well phenol red - HRP detection reagent (100 uL/ml phenol red in 1x Hanks Balanced Salt Solution, 25 U/mL HRP final concentrations). After 15 min of room temperature incubation, the reaction was stopped by adding 2 uL/well 1N NaOH and incubated for an additional 1 hr at room temperature. The absorbance at 600 nm was measured using the ViewLux plate reader (Perkin Elmer, Waltham, MA) with 6000 light energy, 2 sec exposure, and 2x binning instrument settings.
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.
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 0.0000058904 uM (5.8904e-06μM**)% Activity at given concentration.Float%
15Activity at 0.0000270958 uM (2.70958e-05μM**)% Activity at given concentration.Float%
16Activity at 0.0001246408 uM (0.000124641μM**)% Activity at given concentration.Float%
17Activity at 0.0004350127 uM (0.000435013μM**)% Activity at given concentration.Float%
18Activity at 0.0006488539 uM (0.000648854μM**)% Activity at given concentration.Float%
19Activity at 0.00190 uM (0.00190303μM**)% Activity at given concentration.Float%
20Activity at 0.00368 uM (0.00368337μM**)% Activity at given concentration.Float%
21Activity at 0.00584 uM (0.00583969μM**)% Activity at given concentration.Float%
22Activity at 0.017 uM (0.0170962μM**)% Activity at given concentration.Float%
23Activity at 0.034 uM (0.0342764μM**)% Activity at given concentration.Float%
24Activity at 0.053 uM (0.0525747μM**)% Activity at given concentration.Float%
25Activity at 0.103 uM (0.102829μM**)% Activity at given concentration.Float%
26Activity at 0.158 uM (0.158099μM**)% Activity at given concentration.Float%
27Activity at 0.462 uM (0.462437μM**)% Activity at given concentration.Float%
28Activity at 0.925 uM (0.925463μM**)% Activity at given concentration.Float%
29Activity at 1.418 uM (1.4176μM**)% Activity at given concentration.Float%
30Activity at 2.776 uM (2.77639μM**)% Activity at given concentration.Float%
31Activity at 4.263 uM (4.2629μM**)% Activity at given concentration.Float%
32Activity at 12.49 uM (12.4858μM**)% Activity at given concentration.Float%
33Activity at 24.99 uM (24.9875μM**)% Activity at given concentration.Float%
34Activity at 38.31 uM (38.3142μM**)% Activity at given concentration.Float%
35Activity at 114.9 uM (114.943μM**)% 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)
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