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BioAssay: AID 2593

qHTS Assay for Inhibitors and Activators of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Summary

Glucocerebrosidase (GC) 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 more ..
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 Tested Compounds
 Tested Compounds
All(2)
 
 
Probe(2)
 
 
Active(2)
 
 
 Tested Substances
 Tested Substances
All(2)
 
 
Probe(2)
 
 
Active(2)
 
 
AID: 2593
Data Source: NCGC (GCNS634)
BioAssay Type: Summary, Candidate Probes/Leads with Supporting Evidence
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-03-17
Modify Date: 2010-11-03

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: Chemical Probe: 2    Active: 2
Depositor Specified Assays
Show more
AIDNameTypeComment
2101qHTS Assay for Inhibitors and Activators of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher DiseaseconfirmatoryPrimary screen against N370S GC from tissue homogenate.
2590qHTS Assay for Inhibitors and Activators of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Primary Screen ConfirmationconfirmatoryConfirmation screen against N370S GC from tissue homogenate.
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 SubstrateconfirmatoryConfirmation screen against N370S GC from tissue homogenate using red probe.
2592qHTS Assay for Inhibitors and Activators of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Activity in Non-Mutant Spleen HomogenateconfirmatoryConfirmation screen against wildtype GC from tissue homogenate.
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 SubstrateconfirmatoryConfirmation screen against wildtype GC from tissue homogenate using red probe.
2595qHTS Assay for Inhibitors and Activators of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Purified Non-mutant GlucocerebrosidaseconfirmatoryConfirmation screen against purified, wildtype GC.
2597qHTS Assay for Inhibitors and Activators of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Purified N370S GlucocerebrosidaseconfirmatoryConfirmation screen against purified, N370S GC.
2596qHTS Assay for Inhibitors and Activators of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Purified N370S Glucocerebrosidase Cleavage of GlucosylceramideconfirmatoryConfirmation screen against purified, N370S GC using natural substrate.
2577qHTS Assay for Inhibitors and Activators of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Alpha-Glucosidase CounterscreenconfirmatoryCounterscreen against alpha-glucosidase.
2578qHTS Assay for Inhibitors and Activators of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Alpha-Galactosidase CounterscreenconfirmatoryCounterscreen against alpha-galactosidase.
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 CompoundconfirmatoryImmunostaining of N370S GC containing fibroblast lysosomes.
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 CompoundconfirmatoryImmunostaining of wildtype GC containing fibroblast lysosomes.
488834qHTS Assay for Inhibitors and Activators of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Alpha-Glucosidase Counterscreen for Probe SARconfirmatoryCounterscreen against alpha-glucosidase.
488845qHTS Assay for Inhibitors and Activators of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Primary Screen Confirmation Using LC/MSconfirmatoryConfirmation screen against N370S GC from tissue homogenate with alternate LC/MS detection/readout.
488846qHTS Assay for Inhibitors and Activators of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Alpha-Galactosidase Counterscreen for Probe SARconfirmatoryCounterscreen against alpha-galactosidase.
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 SARconfirmatoryConfirmation screen against wildtype GC from tissue homogenate.
488850qHTS Assay for Inhibitors and Activators of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Purified N370S Glucocerebrosidase Confirmation for Probe SARconfirmatoryConfirmation screen against purified, N370S GC.
488851qHTS Assay for Inhibitors and Activators of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Purified Non-mutant Confirmation for Probe SARconfirmatoryConfirmation screen against purified, wildtype GC.
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 SARconfirmatoryConfirmation screen against N370S GC from tissue homogenate using red probe.
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 SARconfirmatoryConfirmation screen against wildtype GC from tissue homogenate using red probe.
488854qHTS Assay for Inhibitors and Activators of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Confirmation for Probe SARconfirmatoryConfirmation screen against N370S GC from tissue homogenate.
588853qHTS for Activators of Human Glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Fibroblast Translocationother
504746Inhibitors of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Metabolic Stability Profile with NADPHother
504745Inhibitors of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Efflux Ratio Profilingother
504748Inhibitors of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Metabolic Stability Profileother
504747Inhibitors of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Caco-2 Permeabilityother
2671qHTS Assay for Inhibitors and Activators of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Hit Validationconfirmatory
Description:
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Production Centers Network [MLPCN]

MLPCN Grant: MH086442-01
Assay Submitter (PI): Wei Zheng

Glucocerebrosidase (GC) 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 GC inhibitors have been shown to act as chemical chaperones, stabilizing the conformation of mutant proteins and thus restoring their function. These inhibitors include iminosugar analogs and three non-iminosugar inhibitors identified from a previous HTS of 62,000 compounds with the wild type recombinant enzyme. 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. While activators for GC have not yet been identified, 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.

We have optimized a new assay using N370S mutant GC derived from the spleen of a Gaucher patient. The new assay differs significantly from the previous screen because the N370S mutant enzyme is used instead of wildtype GC and an enzyme preparation derived from patient tissue instead of the purified recombinant GC that should have the physiologically relevant subunit/subunits and cofactors. In addition, the MLPCN compound library has expanded, which increases the chance of finding new and better probes.
Protocol
See non-summary AIDS for detailed assay protocols.
Comment
This summary is written for the purposes of summarizing the probe activities from the project. MLSCN probes are given a score of 100. Molecules in the prior art are given a score of 80. Other, less active molecules in the same chemical series as the probe molecules are given a score of 50. Inactive analogues from these series are given a score of 0. ML155 (SID 85267237) and ML156 (SID 89449177) have been declared as probes on this project.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1ActivityTextual description of type of activity demonstrated by compound.String
2Glucocerebrosidase PotencyConcentration of compound demonstrating half-maximal activity of compound.FloatμM
3Alpha-Glucosidase PotencyConcentration of compound demonstrating half-maximal activity of compound.FloatμM
4Alpha-Galactosidase PotencyConcentration of compound demonstrating half-maximal activity of compound.FloatμM
5Gaucher Fibroblast PotencyConcentration of compound demonstrating increased translocation of glucocerebrosidase to the lysosome of patient-derived (N370S GC) fibroblasts.FloatμM
Additional Information
Grant Number: MH086442-01

Data Table (Concise)
Classification
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