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BioAssay: AID 2115

Confirmation of Inhibitors and Activators of Purified Human alpha-Glucosidase

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(331)
 
 
Active(16)
 
 
Inactive(303)
 
 
Inconclusive(13)
 
 
 Tested Substances
 Tested Substances
All(338)
 
 
Active(19)
 
 
Inactive(306)
 
 
Inconclusive(13)
 
 
AID: 2115
Data Source: NCGC (AGLU008)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2009-11-06
Modify Date: 2010-03-30

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 16
Depositor Specified Assays
AIDNameTypeComment
1467qHTS Assay for Inhibitors of Human alpha-Galactosidase at pH 4.5.confirmatory
1472Quantitative High-Throughput Screen for Inhibitors of Human alpha-Galactosidase at pH 4.5: Summarysummary
1473Quantitative High-Throughput Screen for Inhibitors and Activators of Human alpha-Glucosidase as a Potential Chaperone Treatment of Pompe Disease: Summarysummary
2293Direct Measure of the Activation of Acid alpha-Glucosidase Catalytic Rateother
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 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. These results comprise confirmation of hits from primary screening in a purified enzyme assay.
Protocol
NCGC Assay Protocol Summary:

This is a fluorogenic enzyme assay with 4-methylumbelliferyl-alpha-D-pyranoside 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). 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), 0.005% Tween-20, pH 5.0. (pH 5.0 is an optimal condition for this enzyme assay)


1536-well assay protocol for the human alpha-glucosidase:
(1) Add 2 ul/well of enzyme (4 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: 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.
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 0.0000095173 uM (9.51735e-06μM**)% Activity at given concentration.Float%
15Activity at 0.0000225082 uM (2.25082e-05μM**)% Activity at given concentration.Float%
16Activity at 0.0000441891 uM (4.41891e-05μM**)% Activity at given concentration.Float%
17Activity at 0.0000883783 uM (8.83783e-05μM**)% Activity at given concentration.Float%
18Activity at 0.0001716108 uM (0.000171611μM**)% Activity at given concentration.Float%
19Activity at 0.0003401556 uM (0.000340156μM**)% Activity at given concentration.Float%
20Activity at 0.0006236150 uM (0.000623615μM**)% Activity at given concentration.Float%
21Activity at 0.00125 uM (0.001245μM**)% Activity at given concentration.Float%
22Activity at 0.00287 uM (0.00286978μM**)% Activity at given concentration.Float%
23Activity at 0.00518 uM (0.00518439μM**)% Activity at given concentration.Float%
24Activity at 0.011 uM (0.0106591μM**)% Activity at given concentration.Float%
25Activity at 0.024 uM (0.0242312μM**)% Activity at given concentration.Float%
26Activity at 0.043 uM (0.0431635μM**)% Activity at given concentration.Float%
27Activity at 0.091 uM (0.0912575μM**)% Activity at given concentration.Float%
28Activity at 0.205 uM (0.204599μM**)% Activity at given concentration.Float%
29Activity at 0.359 uM (0.359365μM**)% Activity at given concentration.Float%
30Activity at 0.718 uM (0.718003μM**)% Activity at given concentration.Float%
31Activity at 1.306 uM (1.30566μM**)% Activity at given concentration.Float%
32Activity at 2.648 uM (2.64804μM**)% Activity at given concentration.Float%
33Activity at 6.064 uM (6.06382μM**)% Activity at given concentration.Float%
34Activity at 10.87 uM (10.867μM**)% Activity at given concentration.Float%
35Activity at 22.67 uM (22.6711μM**)% Activity at given concentration.Float%
36Activity at 51.14 uM (51.1414μM**)% Activity at given concentration.Float%
37Activity at 76.25 uM (76.2454μM**)% Activity at given concentration.Float%
38Activity at 143.7 uM (143.678μM**)% Activity at given concentration.Float%
39Activity at 287.4 uM (287.356μM**)% 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)
Classification
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