AID	Name	Type	Comment
1473	Quantitative High-Throughput Screen for Inhibitors and Activators of Human alpha-Glucosidase as a Potential Chaperone Treatment of Pompe Disease: Summary	summary	Summary AID

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.

Validated hits were tested in this fibroblast translocation assay that measures fluorescence through immunostaining Pompe patient derived fibroblasts.

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

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

Wild type primary dermal human fibroblasts (ATCC) or Pompe fibroblasts were seeded in Lab-Tek 4 chamber slides (Fisher Scientific, Pittsburgh, PA). After chemical compound treatment for six days ( fresh medium with compound was given on day 3 of treatment), fibroblasts were fixed in 3% paraformaldehyde. The cells were permeabelized with 0.1 % Triton-X for 10 min. and blocked in PBS containing 0.1% saponin, 100 M glycine, 0.1% BSA and 2% donkey serum followed by incubation with mouse monoclonal anti-GAA (Abnova, 1:100) or goat polyclonal anti-cathepsin D (R&D Systems, Minneapolis, MN, 1:100). The cells were washed and incubated with secondary donkey anti-mouse or anti-goat antibodies conjugated to ALEXA-488 or ALEXA-555, respectively (Invitrogen, 1:300), washed again, and mounted in VectaShield with DAPI (Vector Laboratories, Burlingame, CA).

Cells were imaged with a Zeiss 510 META confocal laser-scanning microscope (Carl Zeiss, Microimaging Inc., Germany) using an Argon (458, 477, 488, 514 nm) 30 mW laser, a HeNe (543 nm) 1 mW laser, and a laser diode (405 nm). Images were acquired using a Plan-Apochromat 63x/1.4 Oil. Images were taken at the same laser settings.

This is a translocation in human fibroblast measuring immunostaining as observed through a microscope. If translocation was visually observed, the compounds are considered "active"; if no translocation was observed, compounds are considered "inactive"; if the results is unclear, compounds are considered "inconclusive".

Active compounds are assigned a score of 50, inactive compounds are assigned a score of 0, inconclusive compounds are assigned a score of 10.

Grant Number: MH084841