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BioAssay: AID 2293

Direct Measure of the Activation of Acid alpha-Glucosidase Catalytic Rate

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(1)
 
 
Active(1)
 
 
 Tested Substances
 Tested Substances
All(1)
 
 
Active(1)
 
 
AID: 2293
Data Source: NCGC (AGLU012)
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-01-25

Data Table ( Complete ):           Active    All
Target
BioActive Compound: 1
Depositor Specified Assays
Show more
AIDNameTypeProbeComment
1473Quantitative High-Throughput Screen for Inhibitors and Activators of Human alpha-Glucosidase as a Potential Chaperone Treatment of Pompe Disease: Summarysummary1
2242qHTS Assay for Activators of Human alpha-Glucosidase as a Potential Chaperone Treatment of Pompe Diseaseconfirmatory
2115Confirmation of Inhibitors and Activators of Purified Human alpha-Glucosidaseconfirmatory
2113Confirmation of Inhibitors and Activators of Human alpha-Glucosidase From Spleen Homogenateconfirmatory
2112qHTS Assay for Inhibitors and Activators of Human alpha-Glucosidase From Spleen Homogenateconfirmatory
2111Confirmation of Inhibitors and Activators of Human alpha-Glucosidase From Spleen Homogenate Using an Alternate Red Fluorescent Susbtrateconfirmatory
2110Confirmation of Inhibitors and Activators of Purified Human alpha-Glucosidase Using an Alternate Red Fluorescent Susbtrateconfirmatory
2109Confirmation of Inhibitors and Activators of Purified Human alpha-Galactosidaseconfirmatory
2108Confirmation of Inhibitors of Human alpha-Galactosidase Using Spleen Homogenateconfirmatory
2107qHTS Assay for Inhibitors and Activators of Human alpha-Galactosidase From Spleen Homogenateconfirmatory
2101qHTS Assay for Inhibitors and Activators of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Diseaseconfirmatory
2100qHTS Assay for Inhibitors and Activators of Human alpha-Glucosidase Cleavage of Glycogenconfirmatory
1466qHTS Assay for Inhibitors 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: 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.

This assay was performed to establish that the compounds tested increase substrate turnover by directly following the consumption of substrate and formation of product using LC-MS.
Protocol
The catalysis rate of the 4MU-alpha-Gluc substrate by acid alpha-glucosidase was measured using LC-MS. Various concentrations of compound were added to enzyme solution in tubes. This was incubated with substrate and then stopped with stop solution. LC-MS was performed on each solution to determine the amount of product. The formation of product showed a dose-dependent increase.

(1) Add 50 ul of enzyme to 1.5 ml tubes (4 nM final)
(2) Add 0.5 ul compounds in DMSO solution. The final titration was 0.7 nM to 77 uM.
(3) Add 50 ul of substrate (400 uM final)
(4) Incubate at room temperature for 20 min.
(5) Add 50 ul stop solution (1M NaOH and 1M Glycine mixture, pH 10)
(6) Run LC-MS and integrate peaks to quantitate amount of product
Comment
The one compound tested activated substrate cleavage, and was given a score of 100.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Percent Converstion without CompoundFloat%
2Percent Conversion at 5uM Compound (5μM**)Float%
3Percent Conversion at 37.5uM Compound (37.5μM**)Float%

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

Data Table (Concise)
Classification
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