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BioAssay: AID 1467

qHTS Assay for Inhibitors of Human alpha-Galactosidase at pH 4.5.

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(226901)
 
 
Inactive(224842)
 
 
Inconclusive(2095)
 
 
 Tested Substances
 Tested Substances
All(229459)
 
 
Inactive(227350)
 
 
Inconclusive(2109)
 
 
AID: 1467
Data Source: NCGC (AGAL005)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2008-12-30
Modify Date: 2013-01-04

Data Table ( Complete ):           All
Target
Tested Compounds:
Depositor Specified Assays
AIDNameTypeProbeComment
1473Quantitative High-Throughput Screen for Inhibitors and Activators of Human alpha-Glucosidase as a Potential Chaperone Treatment of Pompe Disease: Summarysummary1 Summary AID
992Counterscreen for Glucocerebrosidase Inhibitors: qHTS Assay for Human alpha-Galactosidase at pH 4.5confirmatory
1472Quantitative High-Throughput Screen for Inhibitors of Human alpha-Galactosidase at pH 4.5: Summarysummary
2107qHTS Assay for Inhibitors and Activators of Human alpha-Galactosidase From Spleen Homogenateconfirmatory
2108Confirmation of Inhibitors of Human alpha-Galactosidase Using Spleen Homogenateconfirmatory
2109Confirmation of Inhibitors and Activators of Purified Human alpha-Galactosidaseconfirmatory
2115Confirmation of Inhibitors and Activators of Purified Human alpha-Glucosidaseconfirmatory
2242qHTS Assay for Activators of Human alpha-Glucosidase as a Potential Chaperone Treatment of Pompe Diseaseconfirmatory
Description:
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Production centers Network [MLPCN]

MLPCN Grant: 1R03MH084842-01
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 the genetic mutations may account for a significant numbers of Fabry patients. 1-deoxygalactonojirimycin (DGJ), an inhibitor of alpha-galactosidase, was reported to exhibit the pharmacological chaperone activity which significant increased the mutant enzyme activity in cells. We optimized this alpha-galactosidase assay in 1536-well plate format for identifying the novel small molecule inhibitors with the structures other than the sugar analogs in order to develop the new pharmacological chaperones. Although this assay has the same protocol as PubChem AID 992, this is a separate project and, therefore, is published as a separate assay
Protocol
NCGC Assay Protocol Summary:

This is a fluorogenic enzyme assay with 4-Methylumbelliferyl alpha-D-galactopyranoside as the substrate and human alpha-galactolucosidase as the enzyme preparation. Upon the hydrolysis of this fluorogenic substrate, the resulting product, 1. 4-Methyllumbelliferone, can be excited at 365 nm and emits at 440 nm. This fluorescence which can be detected by a standard fluorescence plate reader. Data were normalized to the controls for basal activity (without enzyme) and 100% activity (with enzyme). In 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 4.5), 0.005% Tween-20, pH 4.5. (pH 4.5 is an optimal condition for this enzyme assay)


1536-well assay protocol for the human alpha-galactosidase:
(1) Add 2 ul/well of enzyme (12 nM final)
(2) Add 23 nL compounds in DMSO solution. The final titration was 0.7 nM to 77 uM.
(3) Add 1 ul of substrate (400 uM final)
(4) Incubate at room temperature for 20 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: MLSMR, MLPCN, NIH Roadmap, qHTS, NCGC, : Alpha-galactosidase, Fabry Disease, pharmacological chaperone, chaperone therapy
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.
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 signficant, 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 0.169 uM (0.168566μM**)% Activity at given concentration.Float%
15Activity at 0.843 uM (0.842831μM**)% Activity at given concentration.Float%
16Activity at 4.214 uM (4.21415μM**)% Activity at given concentration.Float%
17Activity at 21.07 uM (21.0708μM**)% Activity at given concentration.Float%
18Activity at 105.4 uM (105.354μM**)% Activity at given concentration.Float%
19Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1R03MH084842-01

Data Table (Concise)
Classification
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