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BioAssay: AID 602122

qHTS for Activators of Human alpha-Glucosidase as a Potential Chaperone Treatment of Pompe Disease: Fibroblast Translocation

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(2)
 
 
Active(2)
 
 
 Tested Substances
 Tested Substances
All(2)
 
 
Active(2)
 
 
AID: 602122
Data Source: NCGC (ALGU452)
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-12-10
Hold-until Date: 2012-12-07
Modify Date: 2012-12-07

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 2
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
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
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.

Validated hits were tested in this fibroblast translocation assay that measures fluorescence through immunostaining Pompe patient derived fibroblasts.

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

MLPCN Grant: MH084841
Assay Submitter (PI): Wei Zheng
Protocol
Wild type primary dermal human fibroblasts (ATCC) or Pompe fibroblasts were seeded in Lab-Tek 4 chamber slides (Fisher Scientific, Pittsburgh, PA). After chemical compound treatment for six days ( fresh medium with compound was given on day 3 of treatment), fibroblasts were fixed in 3% paraformaldehyde. The cells were permeabelized with 0.1 % Triton-X for 10 min. and blocked in PBS containing 0.1% saponin, 100 M glycine, 0.1% BSA and 2% donkey serum followed by incubation with mouse monoclonal anti-GAA (Abnova, 1:100) or goat polyclonal anti-cathepsin D (R&D Systems, Minneapolis, MN, 1:100). The cells were washed and incubated with secondary donkey anti-mouse or anti-goat antibodies conjugated to ALEXA-488 or ALEXA-555, respectively (Invitrogen, 1:300), washed again, and mounted in VectaShield with DAPI (Vector Laboratories, Burlingame, CA).
Cells were imaged with a Zeiss 510 META confocal laser-scanning microscope (Carl Zeiss, Microimaging Inc., Germany) using an Argon (458, 477, 488, 514 nm) 30 mW laser, a HeNe (543 nm) 1 mW laser, and a laser diode (405 nm). Images were acquired using a Plan-Apochromat 63x/1.4 Oil. Images were taken at the same laser settings.
Comment
This is a translocation in human fibroblast measuring immunostaining as observed through a microscope. If translocation was visually observed, the compounds are considered "active"; if no translocation was observed, compounds are considered "inactive"; if the results is unclear, compounds are considered "inconclusive".
Active compounds are assigned a score of 50, inactive compounds are assigned a score of 0, inconclusive compounds are assigned a score of 10.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Translocation at 20 uM Was translocation observed? Yes, No, or Maybe.String
2Compound QC String
Additional Information
Grant Number: MH084841

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