Confirmation of Inhibitors and Activators of Purified Human alpha-Galactosidase
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 Compounds: 3
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 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. These results comprise confirmation of hits from primary screening in a purified enzyme assay.
NCGC Assay Protocol Summary:
This is a fluorogenic enzyme assay with 4-methylumbelliferyl-alpha-D-pyranoside as the substrate and human alpha-glucosidase as the enzyme preparation. Upon the hydrolysis of this fluorogenic substrate, the resulting product, 1. 4-methyllumbelliferone, can be excited at 365 nm and emits at 440 nm which can be detected by a standard fluorescence plate reader. Data were normalized to the controls for basal activity (without enzyme) and 100% activity (with enzyme). In 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), 0.005% Tween-20, pH 5.0. (pH 5.0 is an optimal condition for this enzyme assay)
1536-well assay protocol for the human alpha-glucosidase:
(1) Add 2 ul/well of enzyme (4 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 substrate (400 uM final)
(4) Incubate at room temperature for 20 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
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.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)