Confirmation of Inhibitors of Human alpha-Galactosidase Using Spleen Homogenate
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 Probe Production Centers Network [MLPCN]
MLPCN Grant: R03 MH084842-01
Assay Submitter (PI): Wei Zheng
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 been reported that the improper folding and trafficking of alpha-galactosidase resulting from genetic mutations may account for a significant number of Fabry patients. 1-deoxygalactonojirimycin (DGJ), an inhibitor of alpha-galactosidase, was reported to exhibit pharmacological chaperone activity which significantly increased the mutant enzyme activity in cells. We optimized this alpha-galactosidase assay in 1536-well plate format for identification of novel small molecule inhibitors or activators with structures other than the sugar analogs in order to develop new pharmacological chaperones. In particular, this assay is directed towards discovering compounds active in 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.
NCGC Assay Protocol Summary:
This is a fluorogenic enzyme assay with 4-methylumbelliferyl-alpha-D-galactopyranoside as the substrate and human spleen homogenate containing alpha-galactosidase as the enzyme preparation. Upon hydrolysis of this fluorogenic substrate, the resulting product, 4-methylumbelliferone (which excites at 365 nm and emits at 440 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-galactosidase 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 37C 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-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)