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BioAssay: AID 992

Counterscreen for Glucocerebrosidase Inhibitors: qHTS Assay for 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(1243)
 
 
Inactive(1243)
 
 
 Tested Substances
 Tested Substances
All(1284)
 
 
Inactive(1284)
 
 
AID: 992
Data Source: NCGC (AGAL240)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2007-12-27
Modify Date: 2010-07-12

Data Table ( Complete ):           View All Data
Target
Tested Compounds:
Related Experiments
AIDNameTypeComment
348Glucocerebrosidase-p2Confirmatorydepositor-specified cross reference
360GlucocerebrosidaseConfirmatorydepositor-specified cross reference
1382Confirmation Concentration-Response Assay for Glucocerebrosidase InhibitorsConfirmatorydepositor-specified cross reference
1393Quantitative High-Throughput Screen for Inhibitors of Glucocerebrosidase: SummarySummarydepositor-specified cross reference
1467qHTS Assay for Inhibitors of Human alpha-Galactosidase at pH 4.5.Confirmatorydepositor-specified cross reference
957Counterscreen for Glucocerebrosidase Inhibitors: qHTS Assay for Coffee Bean alpha-Galactosidase at pH 4.5Confirmatorysame project related to Summary assay
997Counterscreen for Glucocerebrosidase Inhibitors: qHTS Assay for Rice alpha-Glucosidase at pH 5.0Confirmatorysame project related to Summary assay
998Counterscreen for Glucocerebrosidase Inhibitors: qHTS Assay for Coffee Bean alpha-Galactosidase at pH 5.9Confirmatorysame project related to Summary assay
Description:
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Screening Centers Network [MLSCN]

MLSCN Grant: 1 X01MH78932-01
PI Name: Zheng, Wei [NIH]

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 reported that the improper folding and trafficking of alpha-galactosidase resulted 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. This is a counterscreen for the glucocerebrosidase project that includes assays AID 360 and 348.
Protocol
NCGC Assay Protocol Summary:

This is a fluorogenic enzyme assay with 4-Methylumbelliferyl alpha-D-galactopyranoside as the substrate and human alpha-glucosidase 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 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, 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 (2 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 (80 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: 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.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
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 0.0009853846 uM (0.000985385μM**)% Activity at given concentration.Float%
15Activity at 0.00220 uM (0.00220231μM**)% Activity at given concentration.Float%
16Activity at 0.00492 uM (0.00492462μM**)% Activity at given concentration.Float%
17Activity at 0.011 uM (0.0110123μM**)% Activity at given concentration.Float%
18Activity at 0.025 uM (0.0246231μM**)% Activity at given concentration.Float%
19Activity at 0.055 uM (0.0550569μM**)% Activity at given concentration.Float%
20Activity at 0.123 uM (0.123107μM**)% Activity at given concentration.Float%
21Activity at 0.275 uM (0.275267μM**)% Activity at given concentration.Float%
22Activity at 0.615 uM (0.615497μM**)% Activity at given concentration.Float%
23Activity at 1.376 uM (1.37625μM**)% Activity at given concentration.Float%
24Activity at 3.077 uM (3.0773μM**)% Activity at given concentration.Float%
25Activity at 6.881 uM (6.88084μM**)% Activity at given concentration.Float%
26Activity at 15.39 uM (15.3856μM**)% Activity at given concentration.Float%
27Activity at 34.40 uM (34.4021μM**)% Activity at given concentration.Float%
28Activity at 76.92 uM (76.9231μM**)% Activity at given concentration.Float%
29Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.

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