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BioAssay: AID 2113

Confirmation of Inhibitors and Activators of Human alpha-Glucosidase From Spleen Homogenate

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(414)
 
 
Active(53)
 
 
Inactive(331)
 
 
Inconclusive(32)
 
 
 Tested Substances
 Tested Substances
All(423)
 
 
Active(57)
 
 
Inactive(334)
 
 
Inconclusive(32)
 
 
AID: 2113
Data Source: NCGC (AGLU009)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2009-11-06
Modify Date: 2010-10-19

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 53
Related Experiments
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AIDNameTypeProbeComment
1466qHTS Assay for Inhibitors of Human alpha-Glucosidase as a Potential Chaperone Treatment of Pompe DiseaseConfirmatory 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
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
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: 1R03MH084841-01
Assay Submitter (PI): Wei Zheng

NCGC Assay Overview:

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 resultinged from the genetic mutations may account for a significant number of Pomrope patients. N-butyl-deoxynojirimycin, an inhibitor of alpha-glucosidase, was reported to exhibit the pharmacological chaperone activity, which significant increasesd 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. In particular, this assay is directed towards discovering compounds active in a more natural setting, tissue homogenate, that may have been missed by screens of the purified enzyme under more stringent conditions. This assay attempted to confirm hits from the primary screening.
Protocol
NCGC Assay Protocol Summary:

This is a fluorogenic enzyme assay with 4-methylumbelliferyl-alpha-D-glucopyranoside as the substrate and human spleen homogenate containing alpha-glucosidase 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-glucosidase 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 37 C 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-glucosidase, Pompe Disease, pharmacological chaperone, chaperone therapy, high throughput screening, alpha-glucosidase 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 activators are ranked higher than compounds that showed apparent inhibition.
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
Assay Cell Type: NULL
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 0.0000067553 uM (6.75526e-06μM**)% Activity at given concentration.Float%
15Activity at 0.0000148413 uM (1.48413e-05μM**)% Activity at given concentration.Float%
16Activity at 0.0000300197 uM (3.00197e-05μM**)% Activity at given concentration.Float%
17Activity at 0.0000602903 uM (6.02903e-05μM**)% Activity at given concentration.Float%
18Activity at 0.0001281403 uM (0.00012814μM**)% Activity at given concentration.Float%
19Activity at 0.0002666522 uM (0.000266652μM**)% Activity at given concentration.Float%
20Activity at 0.0004928285 uM (0.000492828μM**)% Activity at given concentration.Float%
21Activity at 0.00103 uM (0.00102768μM**)% Activity at given concentration.Float%
22Activity at 0.00220 uM (0.00220397μM**)% Activity at given concentration.Float%
23Activity at 0.00486 uM (0.00486279μM**)% Activity at given concentration.Float%
24Activity at 0.011 uM (0.0111957μM**)% Activity at given concentration.Float%
25Activity at 0.025 uM (0.0245034μM**)% Activity at given concentration.Float%
26Activity at 0.047 uM (0.0470946μM**)% Activity at given concentration.Float%
27Activity at 0.094 uM (0.0944564μM**)% Activity at given concentration.Float%
28Activity at 0.208 uM (0.207829μM**)% Activity at given concentration.Float%
29Activity at 0.428 uM (0.42825μM**)% Activity at given concentration.Float%
30Activity at 0.901 uM (0.900612μM**)% Activity at given concentration.Float%
31Activity at 1.969 uM (1.96867μM**)% Activity at given concentration.Float%
32Activity at 3.792 uM (3.79222μM**)% Activity at given concentration.Float%
33Activity at 7.644 uM (7.644μM**)% Activity at given concentration.Float%
34Activity at 16.60 uM (16.6032μM**)% Activity at given concentration.Float%
35Activity at 31.48 uM (31.4845μM**)% Activity at given concentration.Float%
36Activity at 60.53 uM (60.5316μM**)% Activity at given concentration.Float%
37Activity at 131.9 uM (131.851μM**)% Activity at given concentration.Float%
38Activity at 286.5 uM (286.472μM**)% Activity at given concentration.Float%
39Activity at 460.1 uM (460.123μM**)% Activity at given concentration.Float%
40Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: MH084841-01

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