AID	Name	Type	Comment
2101	qHTS Assay for Inhibitors and Activators of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease	confirmatory
2593	qHTS Assay for Inhibitors and Activators of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Summary	summary
2613	qHTS 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 Substrate	confirmatory
2671	qHTS Assay for Inhibitors and Activators of N370S glucocerebrosidase as a Potential Chaperone Treatment of Gaucher Disease: Hit Validation	confirmatory

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.

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

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.

Grant Number: MH086442-01