AID	Name	Type	Comment
1466	qHTS Assay for Inhibitors of Human alpha-Glucosidase as a Potential Chaperone Treatment of Pompe Disease	confirmatory	Primary screening assay
1473	Quantitative High-Throughput Screen for Inhibitors and Activators of Human alpha-Glucosidase as a Potential Chaperone Treatment of Pompe Disease: Summary	summary	Summary assay
2112	qHTS Assay for Inhibitors and Activators of Human alpha-Glucosidase From Spleen Homogenate	confirmatory	Alternate primary screening assay
2100	qHTS Assay for Inhibitors and Activators of Human alpha-Glucosidase Cleavage of Glycogen	confirmatory	Another alternate primary screening assay

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

MLPCN Grant: MH084841-01
Assay Submitter (PI): Wei Zheng

NCGC Assay Overview:

Alpha-glucosidase is responsible for hydrolysis of terminal, non-reducing 1,4-linked alpha-D-glucose residues with release of alpha-D-glucose. It is a lysosomal hydrolase that is required for the degradation of a small percentage (1-3%) of cellular glycogen in human. Deficiency of this enzyme results in glycogen-storage disease type II (GSDII), also referred to as Pompe disease, an autosomal recessive disorder. Structurally normal glycogen is accumulated in lysosomes and cytoplasm in affected patients, primarily in muscle tissues. Excessive glycogen storage within lysosomes may interrupt normal functioning of other organelles and leads to cellular injury. In turn, this leads to enlargement and dysfunction of the entire organ involved (eg, cardiomyopathy and muscle weakness).

It has reported that the improper folding and trafficking of alpha-glucosidase resulting from the genetic mutations may account for a significant number of Pompe patients. N-butyl-deoxynojirimycin, an inhibitor of alpha-glucosidase, was reported to exhibit the pharmacological chaperone activity, which significantly increases the mutant enzyme activity in cells. We optimized this alpha-glucosidase assay in 1536-well plate format for identifying the novel small molecule inhibitors with the structures other than the sugar analogs in order to develop the new pharmacological chaperones.

This assay involves heating purified alpha-glucosidase (Myozyme) in the presence of inhibitors to observe potential stabilization of the enzyme. This binding assay is an indirect measure of chaperone activity, as stabilization may imply proper folding and trafficking of the enzyme to its functional site. Alpha-glucosidase was pre-incubated with only DMSO or 50x IC50 of compound before being exposed to 68#C heat inactivation. The heating was measured over time, and it was observed that untreated alpha-glucosidase lost more of its activity over time than enzyme treated with compounds. These results demonstrate that compound binding stabilizes alpha-glucosidase against thermal denaturation, and imply that these compounds may help promote folding and trafficking of the enzyme to the lysosomes.

Temperature Denature Protocol for alpha-Glucosidase

Assay buffer: 50 mM citric acid (titrated with potassium phosphate to pH 5.0), 0.01% Tween-20

1. Prepare 80X solution alpha-glucosidase solution (3.2 nM*80=255 nM) in assay buffer.
2. Divide solution into separate 1.5 ml tubes.
3. For each compound, add 50X IC50:
50x IC50 1-DNJ = 2.5 uM
50x IC50 of the test compounds = 50 uM
4. Incubate 10 min RT after compound addition.
5. Distribute 10 ul from each tube into PCR tubes.
6. Time course of heating: Heat solutions at 68C for 60 min, taking a set of tubes out at 10, 20, 30, 45, and 60 min, while keeping one set on ice whole time.
7. After heating, take out tubes and place on ice for a minimum of 5 min.
8. Transfer 10 ul solution to 790 ul (1:80 dilution) buffer in 1.5 ml tube.
9. Vortex and take three 20 ul aliquots and put in 384 plate.
10. Add 20 ul substrate solution to each well (100 uM 4-methylumbelliferyl alpha-D-glucopyranoside).
11. Incubate 20 min.
12. Add 25 ul stop solution (1 M NaOH, 1 M glycine pH 10).
13. Detect the assay plate in a ViewLux plate reader (PerkinElmer) with Ex=365 nm and Em=440nm.

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: MH084841-01