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 primary screen 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 characterizes compound activity using purified, mutant enzyme cleavage of the native substrate for the enzyme instead of mutant enzyme from tissue homogenate. Compound activity differences may be due to the absence of endogenous cofactors found in tissue homogenate as well as inherent differences in enzyme stability and catalytic activity using the difference substrates.

NCGC Assay Protocol Summary:

This is an enzyme assay using glucosylceramide from soy (Avanti Polar Lipids Catalog #: 131304P) as the substrate and recombinant human glucocerebrosidase as the enzyme preparation. Upon hydrolysis of the substrate, the glucose product can be detected using the Amplex Red Glucose Oxidase Assay Kit (Invitrogen catalog #: A22189). The product of this reaction can be read with a fluorescence plate reader with an excitation at 573 nm and an emission at 610 nm. Data were normalized to the controls for basal activity (without enzyme) and 100% activity (with enzyme). The AC50 values were determined from concentration-response data modeled with the standard Hill equation.

Assay buffer for enzyme reaction: 50 mM citric acid (titrated with potassium phosphate to pH 5.9), 0.01% Tween-20 (pH 5.9 is an optimal condition for this enzyme assay)

Assay buffer for Amplex Red reaction: Tris-HCl, pH 7.5

1536-well assay protocol for the glucocerebrosidase assay:
(1) Add 2 ul/well glucocerebrosidase enzyme solution (5 nM final)
(2) Add 23 nL compounds in DMSO solution. The final titration was 0.7 nM to 77 uM.
(3) Add 1 ul of glucosylceramide solution (50 uM final)
(4) Incubate at 37C for 40 min
(5) Add 2 ul tris-HCl buffer with Amplex Red reagents
(6) Incubate 45 min at room temperature.
(7) Detect the assay plate in a ViewLux plate reader (PerkinElmer) with Ex=573 nm and Em=610nm.

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 inhibitors are ranked higher than compounds that showed apparent activation.
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 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.

Grant Number: MH086442-01