qHTS for Activators of Human alpha-Glucosidase as a Potential Chaperone Treatment of Pompe Disease: Aqueous Solubility
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 more ..
BioActive Compound: 1
Depositor Specified Assays
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 bioassay provides profiling data on aqueous kinetic solubility (PBS @ pH 7.4) of one of the lead compounds developed in the Alpha-Glu activators project.
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Production Centers Network [MLPCN]
MLPCN Grant: MH084841
Assay Submitter (PI): Wei Zheng
1. 8ul of reference and test compound stock solution were added to 392ul 100mM phosphate buffer (pH 7.4)
2. Sample tubes were shaken for 1 hr (1000 rpm) at room temperature
3. Samples were centrifuged (10 min at 12000 rpm) to precipitate un-dissolved particles
4. Supernatants were transferred to new tubes
5. Concentration of the supernatant was determined by LC-UV or LC-MS/MS
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.
Compounds are "active" if solubility is greater than 20; "inconclusive" if solubility is between 10 and 20; "inactive" if solubility is less than 10.
PUBCHEM_ACTIVITY_SCORE is the solubility rounded to the closet whole number. If solubility is >100 uM, it is assigned a score of 100.
Data Table (Concise)