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BioAssay: AID 588853

qHTS for Activators of Human Glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Fibroblast Translocation

Glucocerebrosidase (GCase) catalyzes the hydrolysis of beta-glucocerebroside to glucose and ceramide in lysosomes. Mutations in the glucocerebrosidase gene result in Gaucher disease, an autosomal recessive lysosomal storage disorder. Many of the mutations encountered in patients with Gaucher disease are missense alterations that may cause misfolding, decreased stability and/or mistrafficking of more ..
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 Tested Compounds
 Tested Compounds
All(2)
 
 
Active(2)
 
 
 Tested Substances
 Tested Substances
All(2)
 
 
Active(2)
 
 
AID: 588853
Data Source: NCGC (GCNS756)
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-12-06
Hold-until Date: 2012-12-05
Modify Date: 2012-12-05

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 2
Related Experiments
Show more
AIDNameTypeProbeComment
2593qHTS Assay for Inhibitors and Activators of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: SummarySummary2 depositor-specified cross reference
2101qHTS Assay for Inhibitors and Activators of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher DiseaseConfirmatory same project related to Summary assay
2577qHTS Assay for Inhibitors and Activators of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Alpha-Glucosidase CounterscreenConfirmatory same project related to Summary assay
2578qHTS Assay for Inhibitors and Activators of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Alpha-Galactosidase CounterscreenConfirmatory same project related to Summary assay
2587qHTS Assay for Inhibitors and Activators of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Chaperone Activity in Gauche Fibroblasts After Multi-day Incubation with CompoundConfirmatory same project related to Summary assay
2588qHTS Assay for Inhibitors and Activators of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Activity in Non-Mutant Spleen Homogenate Using a Red Fluorescent SubstrateConfirmatory same project related to Summary assay
2589qHTS Assay for Inhibitors and Activators of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Chaperone Activity in Non-Gauche Fibroblasts After Multi-day Incubation with CompoundConfirmatory same project related to Summary assay
2590qHTS Assay for Inhibitors and Activators of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Primary Screen ConfirmationConfirmatory same project related to Summary assay
2592qHTS Assay for Inhibitors and Activators of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Activity in Non-Mutant Spleen HomogenateConfirmatory same project related to Summary assay
2595qHTS Assay for Inhibitors and Activators of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Purified Non-mutant GlucocerebrosidaseConfirmatory same project related to Summary assay
2596qHTS Assay for Inhibitors and Activators of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Purified N370S Glucocerebrosidase Cleavage of GlucosylceramideConfirmatory same project related to Summary assay
2597qHTS Assay for Inhibitors and Activators of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Purified N370S GlucocerebrosidaseConfirmatory same project related to Summary assay
2613qHTS Assay for Inhibitors and Activators of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Activity in N370S Spleen Homogenate Using a Red Fluorescent SubstrateConfirmatory same project related to Summary assay
2671qHTS Assay for Inhibitors and Activators of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Hit ValidationConfirmatory same project related to Summary assay
488834qHTS Assay for Inhibitors and Activators of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Alpha-Glucosidase Counterscreen for Probe SARConfirmatory same project related to Summary assay
488845qHTS Assay for Inhibitors and Activators of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Primary Screen Confirmation Using LC/MSConfirmatory same project related to Summary assay
488846qHTS Assay for Inhibitors and Activators of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Alpha-Galactosidase Counterscreen for Probe SARConfirmatory same project related to Summary assay
488849qHTS Assay for Inhibitors and Activators of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Activity in Non-Mutant Spleen Confirmation for Probe SARConfirmatory same project related to Summary assay
488850qHTS Assay for Inhibitors and Activators of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Purified N370S Glucocerebrosidase Confirmation for Probe SARConfirmatory same project related to Summary assay
488851qHTS Assay for Inhibitors and Activators of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Purified Non-mutant Confirmation for Probe SARConfirmatory same project related to Summary assay
488852qHTS Assay for Inhibitors and Activators of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Activity in N370S Spleen Homogenate Confirmation Using Alternate Substrate for Probe SARConfirmatory same project related to Summary assay
488853qHTS Assay for Inhibitors and Activators of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Activity in Non-Mutant Spleen Using Alternate Substrate Confirmation for Probe SARConfirmatory same project related to Summary assay
488854qHTS Assay for Inhibitors and Activators of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Confirmation for Probe SARConfirmatory same project related to Summary assay
504745Inhibitors of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Efflux Ratio ProfilingOther same project related to Summary assay
504746Inhibitors of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Metabolic Stability Profile with NADPHOther same project related to Summary assay
504747Inhibitors of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Caco-2 PermeabilityOther same project related to Summary assay
504748Inhibitors of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Metabolic Stability ProfileOther same project related to Summary assay
Description:
Glucocerebrosidase (GCase) catalyzes the hydrolysis of beta-glucocerebroside to glucose and ceramide in lysosomes. Mutations in the glucocerebrosidase gene result in Gaucher disease, an autosomal recessive lysosomal storage disorder. Many of the mutations encountered in patients with Gaucher disease are missense alterations that may cause misfolding, decreased stability and/or mistrafficking of this lysosomal protein. Some GCase inhibitors have been shown to act as chemical chaperones, stabilizing the conformation of mutant proteins and thus restoring their function. However, the enhancement of enzyme activity by the chaperone action of an enzyme inhibitor must be balanced against the direct inhibition of the enzyme. An enzyme activator could also function as a chaperone by binding to the enzyme and helping to correct its misfolding and mistrafficking. Activators for GCase have not yet been identified, and they may have better therapeutic potential than inhibitors. Therefore the discovery and development of chemical activators may provide a new strategy for the chaperone therapy.

The probe that has been identified was tested in a chaperone translocation experiment in human fibroblasts. This assay attempts to quantitate translocated glucocerebrosidase protein in patient-derived fibroblasts following extended compound incubation. The fibroblasts tested in this experiment were homozygous either for N370S glucocerebrosidase or wildtype GCase.

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

MLPCN Grant: MH086442-01
Assay Submitter (PI): Wei Zheng
Protocol
Primary dermal fibroblasts derived from skin biopsies from two previously described N370S/N370S Gaucher patients and a control were seeded in Lab-Tek 4 chamber slides (Fisher Scientific, Pittsburgh, PA). After compound 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 microM glycine, 0.1% BSA and 2% donkey serum. This is followed by an incubation with mouse monoclonal anti-LAMP1 or LAMP-2 (1:100, Developmental Studies Hybridoma bank, University of Iowa, Iowa City, IA) and the rabbit polyclonal anti-GCase R386 antibody (1:500); the cells were washed and incubated with secondary donkey anti-mouse or anti-rabbit antibodies conjugated to ALEXA-488 or ALEXA-555, respectively (Invitrogen, Carlsbad, CA), 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). Low and high magnification images were acquired using a Plan-Apochromat 20X/0.75 objective and a Plan-Apochromat 100x/1.4 oil DIC objective, respectively. Images were taken with the same laser settings and all the images shown are collapsed z-stacks.
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 5 uMWas translocation observed at 5 uM? Yes, No, or Maybe?String
2Compound QCString
Additional Information
Grant Number: MH086442

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