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 ..
Target
| Sequence: alpha-galactosidase [Homo sapiens] Protein Family: PLN02808 Gene:GLA Related Protein 3D Structures |
Tested Compounds:
Depositor Specified Assays
| AID | Name | Type | Probe | Comment |
|---|---|---|---|---|
| 1473 | Quantitative High-Throughput Screen for Inhibitors and Activators of Human alpha-Glucosidase as a Potential Chaperone Treatment of Pompe Disease: Summary | summary | 1 | Summary AID |
| 992 | Counterscreen for Glucocerebrosidase Inhibitors: qHTS Assay for Human alpha-Galactosidase at pH 4.5 | confirmatory | ||
| 2109 | Confirmation of Inhibitors and Activators of Purified Human alpha-Galactosidase | confirmatory | ||
| 2115 | Confirmation of Inhibitors and Activators of Purified Human alpha-Glucosidase | confirmatory | ||
| 1472 | Quantitative High-Throughput Screen for Inhibitors of Human alpha-Galactosidase at pH 4.5: Summary | summary | ||
| 2108 | Confirmation of Inhibitors of Human alpha-Galactosidase Using Spleen Homogenate | confirmatory | ||
| 2107 | qHTS Assay for Inhibitors and Activators of Human alpha-Galactosidase From Spleen Homogenate | confirmatory | ||
| 2242 | qHTS Assay for Activators of Human alpha-Glucosidase as a Potential Chaperone Treatment of Pompe Disease | confirmatory |
Description:
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Production centers Network [MLPCN]
MLPCN Grant: 1R03MH084842-01
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 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. Although this assay has the same protocol as PubChem AID 992, this is a separate project and, therefore, is published as a separate assay
NIH Molecular Libraries Probe Production centers Network [MLPCN]
MLPCN Grant: 1R03MH084842-01
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 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. Although this assay has the same protocol as PubChem AID 992, this is a separate project and, therefore, is published as a separate assay
Protocol
NCGC Assay Protocol Summary:
This is a fluorogenic enzyme assay with 4-Methylumbelliferyl alpha-D-galactopyranoside as the substrate and human alpha-galactolucosidase 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. This fluorescence 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 4.5), 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 (12 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: MLSMR, MLPCN, NIH Roadmap, qHTS, NCGC, : Alpha-galactosidase, Fabry Disease, pharmacological chaperone, chaperone therapy
This is a fluorogenic enzyme assay with 4-Methylumbelliferyl alpha-D-galactopyranoside as the substrate and human alpha-galactolucosidase 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. This fluorescence 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 4.5), 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 (12 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: MLSMR, MLPCN, NIH Roadmap, qHTS, NCGC, : Alpha-galactosidase, Fabry Disease, pharmacological chaperone, chaperone therapy
Comment
Compound Ranking:
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.
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.
Result Definitions
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| TID | Name | Description | Histogram | Type | Unit | |
|---|---|---|---|---|---|---|
| Outcome | The BioAssay activity outcome | Outcome | ||||
| Score | The BioAssay activity ranking score |  | Integer | |||
| 1 | Phenotype | Indicates type of activity observed: inhibitor, activator, fluorescent, cytotoxic, inactive, or inconclusive. | String | |||
| 2 | Potency* | Concentration at which compound exhibits half-maximal efficacy, AC50. Extrapolated AC50s also include the highest efficacy observed and the concentration of compound at which it was observed. | | Float | μM | |
| 3 | Efficacy | Maximal efficacy of compound, reported as a percentage of control. These values are estimated based on fits of the Hill equation to the dose-response curves. | | Float | % | |
| 4 | Analysis Comment | Annotation/notes on a particular compound's data or its analysis. | String | |||
| 5 | Curve_Description | A description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically signficant, but below 80% of control. | String | |||
| 6 | Fit_LogAC50 | The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units). | | Float | ||
| 7 | Fit_HillSlope | The Hill slope from a fit of the data to the Hill equation. | | Float | ||
| 8 | Fit_R2 | R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit. | | Float | ||
| 9 | Fit_InfiniteActivity | The asymptotic efficacy from a fit of the data to the Hill equation. | | Float | % | |
| 10 | Fit_ZeroActivity | Efficacy at zero concentration of compound from a fit of the data to the Hill equation. | | Float | % | |
| 11 | Fit_CurveClass | Numerical encoding of curve description for the fitted Hill equation. | | Float | ||
| 12 | Excluded_Points | Which dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations. | String | |||
| 13 | Max_Response | Maximum activity observed for compound (usually at highest concentration tested). | | Float | % | |
| 14 | Activity at 0.169 uM (0.168566μM**) | % Activity at given concentration. | | Float | % | |
| 15 | Activity at 0.843 uM (0.842831μM**) | % Activity at given concentration. | | Float | % | |
| 16 | Activity at 4.214 uM (4.21415μM**) | % Activity at given concentration. | | Float | % | |
| 17 | Activity at 21.07 uM (21.0708μM**) | % Activity at given concentration. | | Float | % | |
| 18 | Activity at 105.4 uM (105.354μM**) | % Activity at given concentration. | | Float | % | |
| 19 | Compound QC | NCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling' | String |
* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1R03MH084842-01
Data Table (Concise)
Classification
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