Bookmark and Share
BioAssay: AID 2587

qHTS 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 Compound

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 ..
_
   
 Tested Compounds
 Tested Compounds
All(4)
 
 
Active(4)
 
 
 Tested Substances
 Tested Substances
All(4)
 
 
Active(4)
 
 
AID: 2587
Data Source: NCGC (GCNS626)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-03-17
Hold-until Date: 2010-10-01
Modify Date: 2010-10-01

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 4
Depositor Specified Assays
AIDNameTypeComment
2101qHTS Assay for Inhibitors and Activators of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Diseaseconfirmatory
2593qHTS Assay for Inhibitors and Activators of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Summarysummary
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
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.

This assay attempts to quantitate translocated glucocerebrosidase protein in patient-derived fibroblasts following extended compound incubation. The fibroblasts tested in this experiment were homozygous forN370S glucocerebrosidase.
Protocol
NCGC Assay Protocol Summary:

1. Seeded 3000 cells/well in 96-well plates using DMEM (10% FBS), with only medium in outer wells to eliminate the edge effect.
2. Next day, replaced medium with OptiMem (2% FBS) and spiked in various concentrations of compound.
3. 2 days later, replaced with fresh OptiMem, and again spiked each well the same as before.
4. 3 days later, washed 1X with PBS.
5. Incubated with 3% Paraformaldehyde for 15 min, replaced with PBS.
6. Aspirated PBS and added block solution (5% goat serum + 0.1% saposin + 15 mg/ml glycine in PBS) and incubated at RT for 40 min.
7. Aspirated block solution and added r386 antibody solution to stain glucocerebrosidase (20 ul ab in 19 ml of 5% goat serum, 0.1% saposin in PBS) (~1:1000 dilution).
8. Left at 4C overnight.
9. Next day, washed 3x with block solution (no glycine). Waited 10 min between washes.
10. Added mix of secondary antibody solution (1:100 dilution of Cy-3 for GC, 1:100 dilution of FITC for LAMP2, 1:5000 dilution of Hoechst) in 5% goat serum solution and incubated 1 hr.
11. Rinsed 3X with PBS, waiting 5 minutes between washes.
12. Visualized using a fluorescence microscope and the intensity of fluorescence in the selected areas were calculated.

Keywords: Glucocerebrosidase, beta-glucosidase, Gaucher Disease, pharmacological chaperone, chaperone therapy, high throughput screening, glucocerebrosidase 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 20 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 19. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Result Definitions
Show more
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
5Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
6Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
7Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
8Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
9Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
10Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
11Excluded_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
12Activity at 0.01000 uM (0.01μM**)% Activity at given concentration.Float%
13Activity at 0.100 uM (0.1μM**)% Activity at given concentration.Float%
14Activity at 1.000 uM (1μM**)% Activity at given concentration.Float%
15Activity at 10.000 uM (10μM**)% Activity at given concentration.Float%
16Activity at 50.00 uM (50μM**)% Activity at given concentration.Float%
17Activity at 100.00 uM (100μM**)% Activity at given concentration.Float%
18Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: MH086442-01

Data Table (Concise)
Classification
PageFrom: