Counterscreen for Glucocerebrosidase Inhibitors: qHTS Assay for Human alpha-Galactosidase at pH 4.5
Alpha-galactosidase is a homodimeric glycoprotein that hydrolyzes the terminal alpha-galactosyl moieties from glycolipids and glycoproteins. Deficiency of this enzyme results in Fabry Disease with progressive accumulation of globotriaosylceramide and other glycosphingolipids in vascular endothelial cells that causes renal failure, myocardial infarction and stroke, and premature death in patients. more ..
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Screening Centers Network [MLSCN]
MLSCN Grant: 1 X01MH78932-01
PI Name: Zheng, Wei [NIH]
NCGC Assay Overview:
Alpha-galactosidase is a homodimeric glycoprotein that hydrolyzes the terminal alpha-galactosyl moieties from glycolipids and glycoproteins. Deficiency of this enzyme results in Fabry Disease with progressive accumulation of globotriaosylceramide and other glycosphingolipids in vascular endothelial cells that causes renal failure, myocardial infarction and stroke, and premature death in patients. It has reported that the improper folding and trafficking of alpha-galactosidase resulted from the genetic mutations may account for a significant numbers of Fabry patients. 1-deoxygalactonojirimycin (DGJ), an inhibitor of alpha-galactosidase was reported to exhibit the pharmacological chaperone activity which significant increased the mutant enzyme activity in cells. We optimized this alpha-galactosidase 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 is a counterscreen for the glucocerebrosidase project that includes assays AID 360 and 348.
NCGC Assay Protocol Summary:
This is a fluorogenic enzyme assay with 4-Methylumbelliferyl alpha-D-galactopyranoside 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, 0.005% Tween-20, pH 4.5. (pH 4.5 is an optimal condition for this enzyme assay)
1536-well assay protocol for the human alpha-galactosidase:
(1) Add 2 ul/well of enzyme (2 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 (80 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-galactosidase, Fabry Disease, pharmacological chaperone, chaperone therapy, high throughput screening, alpha-galactosidase 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 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.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)