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BioAssay: AID 2107

qHTS Assay for Inhibitors and Activators of Human alpha-Galactosidase From Spleen Homogenate

Alpha-galactosidase is a homodimeric glycoprotein that hydrolyzes the terminal alpha-galactosyl moieties from glycolipids and glycoproteins. Deficiency of this enzyme results in Fabry Disease with progressive accumulation of globotriaosylceramide and other glycosphingolipids in vascular endothelial cells that causes renal failure, myocardial infarction and stroke, and premature death in patients. more ..
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 Tested Compounds
 Tested Compounds
All(234910)
 
 
Active(19)
 
 
Inactive(232141)
 
 
Inconclusive(2816)
 
 
 Tested Substances
 Tested Substances
All(239498)
 
 
Active(19)
 
 
Inactive(236623)
 
 
Inconclusive(2856)
 
 
AID: 2107
Data Source: NCGC (AGAL007)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2009-11-05

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 19
Related Experiments
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AIDNameTypeProbeComment
1467qHTS Assay for Inhibitors of Human alpha-Galactosidase at pH 4.5.Confirmatory depositor-specified cross reference
1472Quantitative High-Throughput Screen for Inhibitors of Human alpha-Galactosidase at pH 4.5: SummarySummary depositor-specified cross reference
1473Quantitative High-Throughput Screen for Inhibitors and Activators of Human alpha-Glucosidase as a Potential Chaperone Treatment of Pompe Disease: SummarySummary1 depositor-specified cross reference
2293Direct Measure of the Activation of Acid alpha-Glucosidase Catalytic RateOther depositor-specified cross reference
2108Confirmation of Inhibitors of Human alpha-Galactosidase Using Spleen HomogenateConfirmatory same project related to Summary assay
2109Confirmation of Inhibitors and Activators of Purified Human alpha-GalactosidaseConfirmatory same project related to Summary assay
2115Confirmation of Inhibitors and Activators of Purified Human alpha-GlucosidaseConfirmatory same project related to Summary assay
1466qHTS Assay for Inhibitors of Human alpha-Glucosidase as a Potential Chaperone Treatment of Pompe DiseaseConfirmatory same project related to Summary assay
2100qHTS Assay for Inhibitors and Activators of Human alpha-Glucosidase Cleavage of GlycogenConfirmatory same project related to Summary assay
2101qHTS Assay for Inhibitors and Activators of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher DiseaseConfirmatory same project related to Summary assay
2108Confirmation of Inhibitors of Human alpha-Galactosidase Using Spleen HomogenateConfirmatory same project related to Summary assay
2109Confirmation of Inhibitors and Activators of Purified Human alpha-GalactosidaseConfirmatory same project related to Summary assay
2110Confirmation of Inhibitors and Activators of Purified Human alpha-Glucosidase Using an Alternate Red Fluorescent SusbtrateConfirmatory same project related to Summary assay
2111Confirmation of Inhibitors and Activators of Human alpha-Glucosidase From Spleen Homogenate Using an Alternate Red Fluorescent SusbtrateConfirmatory same project related to Summary assay
2112qHTS Assay for Inhibitors and Activators of Human alpha-Glucosidase From Spleen HomogenateConfirmatory same project related to Summary assay
2113Confirmation of Inhibitors and Activators of Human alpha-Glucosidase From Spleen HomogenateConfirmatory same project related to Summary assay
2115Confirmation of Inhibitors and Activators of Purified Human alpha-GlucosidaseConfirmatory same project related to Summary assay
2242qHTS Assay for Activators of Human alpha-Glucosidase as a Potential Chaperone Treatment of Pompe DiseaseConfirmatory same project related to Summary assay
2641qHTS Assay for Inhibitors of Human alpha-Glucosidase as a Potential Chaperone Treatment of Pompe Disease: Stabilizers of Alpha-Glucosidase Under Thermal Denaturation ConditionsOther same project related to Summary assay
504681Inhibitors of Human alpha-Glucosidase: PBS Stability ProfilingOther same project related to Summary assay
504686Inhibitors of Human alpha-Glucosidase: Caco-2 Cell Permeability ProfilingOther same project related to Summary assay
504688Inhibitors of Human alpha-Glucosidase: Caco-2 Efflux Ratio ProfilingOther same project related to Summary assay
540341Activators of Human alpha-Glucosidase as a Potential Chaperone Treatment of Pompe Disease: Glucocerebrosidase Counter ScreenConfirmatory same project related to Summary assay
540361Activators of Human alpha-Glucosidase as a Potential Chaperone Treatment of Pompe Disease: Alpha-Galactosidase Counter ScreenConfirmatory same project related to Summary assay
602122qHTS for Activators of Human alpha-Glucosidase as a Potential Chaperone Treatment of Pompe Disease: Fibroblast TranslocationOther same project related to Summary assay
602237qHTS for Activators of Human alpha-Glucosidase as a Potential Chaperone Treatment of Pompe Disease: Aqueous SolubilityOther same project related to Summary assay
602238qHTS for Activators of Human alpha-Glucosidase as a Potential Chaperone Treatment of Pompe Disease: Metabolic StabilityOther same project related to Summary assay
602239qHTS for Activators of Human alpha-Glucosidase as a Potential Chaperone Treatment of Pompe Disease: Metabolic Stability in presence of NADPHOther same project related to Summary assay
Description:
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Production Centers Network [MLPCN]

MLPCN Grant: R03 MH084842-01
Assay Submitter (PI): Wei Zheng

NCGC Assay Overview:

Alpha-galactosidase is a homodimeric glycoprotein that hydrolyzes the terminal alpha-galactosyl moieties from glycolipids and glycoproteins. Deficiency of this enzyme results in Fabry Disease with progressive accumulation of globotriaosylceramide and other glycosphingolipids in vascular endothelial cells that causes renal failure, myocardial infarction and stroke, and premature death in patients. It has been reported that the improper folding and trafficking of alpha-galactosidase resulting from genetic mutations may account for a significant number of Fabry patients. 1-deoxygalactonojirimycin (DGJ), an inhibitor of alpha-galactosidase, was reported to exhibit pharmacological chaperone activity which significantly increased the mutant enzyme activity in cells. We optimized this alpha-galactosidase assay in 1536-well plate format for identification of novel small molecule inhibitors or activators with structures other than the sugar analogs in order to develop new pharmacological chaperones. In particular, this assay is directed towards discovering compounds active in more natural setting, tissue homogenate, that may have been missed by screens of the purified enzyme under more stringent conditions.
Protocol
NCGC Assay Protocol Summary:
This is a fluorogenic enzyme assay with 4-methylumbelliferyl-alpha-D-galactopyranoside as the substrate and human spleen homogenate containing alpha-galactosidase as the enzyme preparation. Upon hydrolysis of this fluorogenic substrate, the resulting product, 4-methylumbelliferone (which excites at 365 nm and emits at 440 nm) can be detected by a fluorescence plate reader. Data were normalized to the controls for basal activity (without enzyme) and 100% activity (with enzyme). The AC50 values were determined from concentration-response data modeled with the standard Hill equation.
Assay buffer: 50 mM citric acid (titrated with potassium phosphate to pH 5.0), 0.01% Tween-20 (pH 5.0 is an optimal condition for this enzyme assay)
1536-well assay protocol for the alpha-galactosidase from human spleen homogenate:
(1) Add 2 ul/well spleen homogenate (1 ug)
(2) Add 23 nL compounds in DMSO solution. The final titration was 0.5 nM to 58 uM.
(3) Add 2 ul of substrate (1 mM final)
(4) Incubate at 37C for 40 min
(5) Add 2 ul stop solution (1M NaOH and 1M Glycine mixture, pH 10)
(6) Detect the assay plate in a ViewLux plate reader (PerkinElmer) with Ex=365 nm and Em=440nm.
Keywords: Alpha-galactosidase, Fabry Disease, pharmacological chaperone, chaperone therapy, high throughput screening, alpha- galactosidase inhibitor, MLSMR, MLSCN, NIH Roadmap, qHTS and NCGC
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 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 0.00220 uM (0.0022μM**)% Activity at given concentration.Float%
15Activity at 0.00492 uM (0.00492308μM**)% Activity at given concentration.Float%
16Activity at 0.011 uM (0.011μM**)% Activity at given concentration.Float%
17Activity at 0.025 uM (0.0246154μM**)% Activity at given concentration.Float%
18Activity at 0.055 uM (0.0550769μM**)% Activity at given concentration.Float%
19Activity at 0.125 uM (0.124757μM**)% Activity at given concentration.Float%
20Activity at 0.178 uM (0.178233μM**)% Activity at given concentration.Float%
21Activity at 0.270 uM (0.269986μM**)% Activity at given concentration.Float%
22Activity at 0.559 uM (0.558551μM**)% Activity at given concentration.Float%
23Activity at 0.685 uM (0.685382μM**)% Activity at given concentration.Float%
24Activity at 1.120 uM (1.12003μM**)% Activity at given concentration.Float%
25Activity at 1.722 uM (1.72172μM**)% Activity at given concentration.Float%
26Activity at 3.154 uM (3.1543μM**)% Activity at given concentration.Float%
27Activity at 4.599 uM (4.59867μM**)% Activity at given concentration.Float%
28Activity at 6.925 uM (6.92508μM**)% Activity at given concentration.Float%
29Activity at 14.29 uM (14.2912μM**)% Activity at given concentration.Float%
30Activity at 17.46 uM (17.4613μM**)% Activity at given concentration.Float%
31Activity at 29.07 uM (29.0689μM**)% Activity at given concentration.Float%
32Activity at 43.01 uM (43.0148μM**)% Activity at given concentration.Float%
33Activity at 80.00 uM (80.0011μM**)% Activity at given concentration.Float%
34Activity at 119.2 uM (119.229μM**)% Activity at given concentration.Float%
35Activity at 181.4 uM (181.412μM**)% Activity at given concentration.Float%
36Activity at 250.8 uM (250.769μM**)% 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: MH084842-01

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