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BioAssay: AID 540341

Activators of Human alpha-Glucosidase as a Potential Chaperone Treatment of Pompe Disease: Glucocerebrosidase Counter Screen

Alpha-glucosidase is responsible for hydrolysis of terminal, non-reducing 1,4-linked alpha-D-glucose residues with release of alpha-D-glucose. It is a lysosomal hydrolase that is required for the degradation of a small percentage (1-3%) of cellular glycogen in human. Deficiency of this enzyme results in glycogen-storage disease type II (GSDII), also referred to as Pompe disease, an autosomal more ..
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 Tested Compounds
 Tested Compounds
All(29)
 
 
Inactive(26)
 
 
Inconclusive(3)
 
 
 Tested Substances
 Tested Substances
All(29)
 
 
Inactive(26)
 
 
Inconclusive(3)
 
 
AID: 540341
Data Source: NCGC (AGLU741)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2011-08-11
Modify Date: 2011-08-19

Data Table ( Complete ):           View All Data
Target
Tested Compounds:
Related Experiments
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AIDNameTypeProbeComment
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: Summary AID
1466qHTS Assay for Inhibitors of Human alpha-Glucosidase as a Potential Chaperone Treatment of Pompe DiseaseConfirmatory same project related to Summary assay
1467qHTS Assay for Inhibitors of Human alpha-Galactosidase at pH 4.5.Confirmatory 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
2107qHTS Assay for Inhibitors and Activators of Human alpha-Galactosidase From Spleen HomogenateConfirmatory 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
2293Direct Measure of the Activation of Acid alpha-Glucosidase Catalytic RateOther 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
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:
Alpha-glucosidase is responsible for hydrolysis of terminal, non-reducing 1,4-linked alpha-D-glucose residues with release of alpha-D-glucose. It is a lysosomal hydrolase that is required for the degradation of a small percentage (1-3%) of cellular glycogen in human. Deficiency of this enzyme results in glycogen-storage disease type II (GSDII), also referred to as Pompe disease, an autosomal recessive disorder. Structurally normal glycogen is accumulated in lysosomes and cytoplasm in affected patients, primarily in muscle tissues. Excessive glycogen storage within lysosomes may interrupt normal functioning of other organelles and leads to cellular injury. In turn, this leads to enlargement and dysfunction of the entire organ involved (eg, cardiomyopathy and muscle weakness).

It has reported that the improper folding and trafficking of alpha-glucosidase resulting from the genetic mutations may account for a significant number of Pompe patients. N-butyl-deoxynojirimycin, an inhibitor of alpha-glucosidase, was reported to exhibit the pharmacological chaperone activity, which significantly increases the mutant enzyme activity in cells. We optimized this alpha-glucosidase 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.

NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Production Centers Network [MLPCN]

MLPCN Grant: MH086442-01
Assay Submitter (PI): Wei Zheng
Protocol
This is a fluorogenic enzyme assay with 4-methylumbelliferyl-beta-D-glucopyranoside as the substrate and purified wildtype glucocerebrosidase as the enzyme preparation. Upon the hydrolysis of this fluorogenic substrate, the resulting product, 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). 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), 100 mM potassium chloride, 10 mM sodium chloride, 1 mM magnesium chloride, 0.01% Tween-20.


1536-well assay protocol:
(1) Add 2 ul/well of glucocerebrosidase enzyme solution (5 nM final)
(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: Glucocerebrosidase, beta-glucosidase, Gaucher Disease, pharmacological chaperone, chaperone therapy, high throughput screening, glucocerebrosidase 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.

This is a counter screen for alpha-glucosidase for specificity of the compounds, hence inactivity in this alpha-galactosidase screen is desirable.
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 0.0003144144 uM (0.000314414μM**)% Activity at given concentration.Float%
15Activity at 0.0009432432 uM (0.000943243μM**)% Activity at given concentration.Float%
16Activity at 0.00241 uM (0.0024111μM**)% Activity at given concentration.Float%
17Activity at 0.00290 uM (0.00289765μM**)% Activity at given concentration.Float%
18Activity at 0.00668 uM (0.00667853μM**)% Activity at given concentration.Float%
19Activity at 0.00863 uM (0.00863081μM**)% Activity at given concentration.Float%
20Activity at 0.018 uM (0.0182899μM**)% Activity at given concentration.Float%
21Activity at 0.026 uM (0.0257502μM**)% Activity at given concentration.Float%
22Activity at 0.076 uM (0.0764027μM**)% Activity at given concentration.Float%
23Activity at 0.229 uM (0.229208μM**)% Activity at given concentration.Float%
24Activity at 0.564 uM (0.564212μM**)% Activity at given concentration.Float%
25Activity at 0.702 uM (0.702353μM**)% Activity at given concentration.Float%
26Activity at 1.481 uM (1.48148μM**)% Activity at given concentration.Float%
27Activity at 2.086 uM (2.08576μM**)% Activity at given concentration.Float%
28Activity at 4.444 uM (4.44444μM**)% Activity at given concentration.Float%
29Activity at 6.257 uM (6.25729μM**)% Activity at given concentration.Float%
30Activity at 18.57 uM (18.5659μM**)% Activity at given concentration.Float%
31Activity at 48.75 uM (48.7509μM**)% Activity at given concentration.Float%
32Activity at 57.14 uM (57.1429μM**)% Activity at given concentration.Float%
33Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: MH086442

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