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BioAssay: AID 2499

Confirmation Assay for Inhibitors of Human Galactokinase (GALK): 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)
 
 
Active(20)
 
 
Inactive(12)
 
 
Inconclusive(7)
 
 
 Tested Substances
 Tested Substances
All(43)
 
 
Active(21)
 
 
Inactive(14)
 
 
Inconclusive(8)
 
 
AID: 2499
Data Source: NCGC (GALK536h)
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 ):           View Active Data    View All Data
Target
Sequence: galactokinase [Homo sapiens]
Description ..   
Protein Family: Galactokinase galactose-binding signature

Gene:GALK1     Related Protein 3D Structures     More BioActivity Data..
BioActive Compounds: 20
Related Experiments
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AIDNameTypeProbeComment
1379Counterscreen for Luciferase (Kinase-Glo TM) InhibitionConfirmatory depositor-specified cross reference
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
2547Secondary Assay for Inhibitors of Human Galactokinase (GALK): HEK-293 Cell Viability AssayConfirmatory 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
2502Confirmatory Assay for Inhibitors of Human Galactokinase (GALK): Phenol-HRP redox assay counterscreen for probe SARConfirmatory same project related to Summary assay
2506Confirmatory Assay for Inhibitors of Human Galactokinase (GALK): CDP-Me assay counterscreen for probe SARConfirmatory same project related to Summary assay
493189qHTS Validation Assay for Inhibitors of Human Galactokinase (GALK)Confirmatory 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
Inherited deficiency of galactose-1-phosphate uridyltransferase (GALT) can result in a potentially lethal disorder called Classic Galactosemia (OMIM 230400). Several lines of evidence indicate that an elevated level of galactose-1-phosphate (gal-1-p), the product of galactokinase (GALK), is a major, if not sole, pathogenic mechanism in patients with classic galactosemia. Therefore, finding inhibitors of GALK is a potential novel therapy for this inherent metabolic disease and is the long term goal of this project. Active compounds observed in the qHTS GALK assay (AID: 1868) were cherry picked and re-screened for validation.
GALK (provided by Kent Lai) was assayed using ATP and D-(+)-Galactose (Sigma-Aldrich cat# G0750) as substrates. Promega Kinase-Glo Plus (cat# V3774) technology was used to detect the residual ATP following kinetic reaction. Briefly, the Kinase-Glo Plus contains Ultra-Glo [1] luciferase and D-luciferin which generates a bioluminescence signal from the remaining ATP. Kinase reactions with and without GALK were used as negative and positive controls together with Protein Tyrosine Phosphatase CD45 inhibitor (EMD Bioscience cat# 540215) which is shown to inhibit GALK activity [2].
Protocol
NCGC Assay Protocol Summary:
Three uL/well of ATP-buffer solution (35 uM ATP, 20mM HEPES pH8.0, 5 mM MgCl2, 60 mM NaCl, 1 mM DTT, 0.01% BSA final concentration) was dispensed into 1536-well, assay plates (Greiner, solid white medium-binding plates) with Aurora Discovery BioRAPTR Flying Reagent Dispenser (FRD; Beckton-Dickenson). Compound and control solution (23 nL) was transferred to the assay plate using the Kalypsys 1536-pin tool apparatus. One uL/well GALK-galactose solution (5 nM GALK, 100 uM galactose, 20 mM HEPES pH8.0, 5 mM MgCl2, 60 mM NaCl, 1 mM DTT, 0.01% BSA final concentration) was then added using the FRD yielding a total kinase reaction volume of 4 uL/ well. After 1 hour of room temperature incubation, 4 uL Kinase-Glo Plus reagent was added for a final assay volume of 8 uL/well. Luminescence was detected with the ViewLux plate reader (Perkin Elmer, Waltham, MA) after 2 min incubation using a 1 sec exposure time and 2x binning.
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.
3. Compounds that interfere with the Ultra-Glo luciferase could interfere with this assay. PubChem AID: 1379 can be used as counter-screen for this [2]
Categorized Comment - additional comments and annotations
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 0.0000058904 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 0.0004329463 uM (0.000432946μM**)% Activity at given concentration.Float%
18Activity at 0.0006535811 uM (0.000653581μM**)% Activity at given concentration.Float%
19Activity at 0.00190 uM (0.0019037μM**)% Activity at given concentration.Float%
20Activity at 0.00370 uM (0.00370045μM**)% Activity at given concentration.Float%
21Activity at 0.00584 uM (0.00583969μM**)% Activity at given concentration.Float%
22Activity at 0.017 uM (0.0170315μM**)% Activity at given concentration.Float%
23Activity at 0.034 uM (0.0342573μM**)% Activity at given concentration.Float%
24Activity at 0.053 uM (0.0525744μM**)% Activity at given concentration.Float%
25Activity at 0.103 uM (0.102772μM**)% Activity at given concentration.Float%
26Activity at 0.158 uM (0.158092μM**)% Activity at given concentration.Float%
27Activity at 0.461 uM (0.460656μM**)% Activity at given concentration.Float%
28Activity at 0.925 uM (0.924948μM**)% Activity at given concentration.Float%
29Activity at 1.418 uM (1.41762μM**)% Activity at given concentration.Float%
30Activity at 2.775 uM (2.77484μM**)% Activity at given concentration.Float%
31Activity at 4.263 uM (4.2628μM**)% Activity at given concentration.Float%
32Activity at 12.44 uM (12.4377μM**)% Activity at given concentration.Float%
33Activity at 24.97 uM (24.9747μM**)% Activity at given concentration.Float%
34Activity at 38.31 uM (38.3142μM**)% Activity at given concentration.Float%
35Activity at 74.71 uM (74.7126μM**)% Activity at given concentration.Float%
36Activity at 114.9 uM (114.943μM**)% Activity at given concentration.Float%
37Compound 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
Classification
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