AID	Name	Type	Comment
1466	qHTS Assay for Inhibitors of Human alpha-Glucosidase as a Potential Chaperone Treatment of Pompe Disease	confirmatory
1473	Quantitative High-Throughput Screen for Inhibitors and Activators of Human alpha-Glucosidase as a Potential Chaperone Treatment of Pompe Disease: Summary	summary
2293	Direct Measure of the Activation of Acid alpha-Glucosidase Catalytic Rate	other

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

MLPCN Grant: 1R03MH084841-01
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 resultinged from the genetic mutations may account for a significant number of Pomrope patients. N-butyl-deoxynojirimycin, an inhibitor of alpha-glucosidase, was reported to exhibit the pharmacological chaperone activity, which significant increasesd 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. In particular, this assay is directed towards discovering compounds active in a more natural setting, tissue homogenate, that may have been missed by screens of the purified enzyme under more stringent conditions. This assay attempted to confirm hits from the primary screening using an alternate fluor than the primary screen used.

NCGC Assay Protocol Summary:

This is a fluorogenic enzyme assay with Resorufin-alpha-D-glucopyranoside as the substrate and human spleen homogenate containing alpha-glucosidase as the enzyme preparation. Upon hydrolysis of this fluorogenic substrate, the resulting product, resorufin (which excites at 573 nm and emits at 610 nm) can be detected by a fluorescence plate reader. 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: 50 mM citric acid (titrated with potassium phosphate to pH 5.0), 0.01% Tween-20 (pH 5.0 is an optimal condition for this enzyme assay)


1536-well assay protocol for the alpha-glucosidase from human spleen homogenate:
(1) Add 2 ul/well spleen homogenate (1 ug)
(2) Add 23 nL compounds in DMSO solution. The final titration was 0.5 nM to 58 uM.
(3) Add 2 ul of substrate (1 mM final)
(4) Incubate at 37 C for 40 min
(5) Add 2 ul stop solution (1M NaOH and 1M Glycine mixture, pH 10)
(6) Detect the assay plate in a ViewLux plate reader (PerkinElmer) with Ex=365 nm and Em=440nm.

Keywords: Alpha-glucosidase, Pompe Disease, pharmacological chaperone, chaperone therapy, high throughput screening, alpha-glucosidase 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 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