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BioAssay: AID 588478

A screen for small molecule inhibitors of the human deubiquitinating enzyme, UCH37

Deubiquitinating enzymes (DUB) represent a group of cysteine proteases that cleave the isopeptide bond between ubiquitin and its conjugated proteins. This high-throughput screen aims to identify small molecule inhibitors of a human DUB, UCH37, which is tightly associated with proteasomes. Specifically, this screen seeks to identify small molecules that inhibit the fluorescence increase that results from cleavage by UCH37 of a fluorescent substrate, ubiquitin-7-amido-4-methylcoumarin (Ub-AMC). ..more
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 Tested Compounds
 Tested Compounds
All(330703)
 
 
Active(1075)
 
 
Inactive(329630)
 
 
 Tested Substances
 Tested Substances
All(330921)
 
 
Active(1078)
 
 
Inactive(329843)
 
 
AID: 588478
Data Source: ICCB-Longwood/NSRB Screening Facility, Harvard Medical School (HMS937_MLP)
Depositor Category: Other
Deposit Date: 2011-10-11
Hold-until Date: 2012-09-19
Modify Date: 2012-09-19

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 1075
Related Experiments
AIDNameTypeComment
624100A reconfirmation screen for small molecule inhibitors of the human deubiquitinating enzyme, UCH37Otherdepositor-specified cross reference
Description:
Deubiquitinating enzymes (DUB) represent a group of cysteine proteases that cleave the isopeptide bond between ubiquitin and its conjugated proteins. This high-throughput screen aims to identify small molecule inhibitors of a human DUB, UCH37, which is tightly associated with proteasomes. Specifically, this screen seeks to identify small molecules that inhibit the fluorescence increase that results from cleavage by UCH37 of a fluorescent substrate, ubiquitin-7-amido-4-methylcoumarin (Ub-AMC).
Protocol
10 microL of proteasome-containing buffer (0.15 nM proteasome in 50 mM Tris-HCl, pH 7.4, 1 mg/ml ovalbumin, 1 mM ATP-MgCl2, 1mM DTT) were added to each well of 384-well plates (Corning 3820), except for column 1, which was used for positive controls.
100 nL of each experimental compound were transferred to the assay plates by pin transfer, and then incubated for 30 minutes at room temperature.
10 microL of 1 microM Ub-AMC in buffer (50 mM Tris-HCl, pH 7.4, 1 mg/ml ovalbumin, 1 mM ATP-MgCl2, 1mM DTT) were added to all wells of the assay plates, and then incubated for 25 minutes at room temperature. The fluorescence intensity of each well was then read using a Perkin Elmer EnVision plate reader at 355 nm/460 nm (excitation/emission).
For positive controls, 10 microL of 1 microM Ub-AMC in buffer (50 mM Tris-HCl, pH 7.4, 1 mg/ml ovalbumin, 1 mM ATP-MgCl2, 1mM DTT) were added to all wells in column 1 on each assay plate, and incubated for 25 minutes at room temperature before fluorescence reading.
For negative controls, 10 microL of proteasome-containing buffer (0.15 nM proteasome in 50 mM Tris-HCl, pH 7.4, 1 mg/ml ovalbumin, 1 mM ATP-MgCl2, 1mM DTT) were added to all wells in column 2 on each assay plate, and then 10 microL of 1 microM Ub-AMC in buffer (50 mM Tris-HCl, pH 7.4, 1 mg/ml ovalbumin, 1 mM ATP-MgCl2, 1mM DTT) was added. After incubation at room temperature for 25 minutes, fluorescence intensity was read.
Comment
Activity scores were calculated based on percent (%) inhibition, which was in turn calculated from fluorescence intensity (FI) readings as follows: % Inhibition = (1-((Vi-Vp)/(Vn-Vp)))*100, where Vi is the FI of the individual well, Vp the average FI of the positive controls on that plate, and Vn the average FI of the negative controls on that plate. A final % inhibition value was calculated as the average of replicate results for each individual compound and used to determine the Activity Score. If the % inhibition was less than zero, the Activity Score was set to Zero. Compounds showing >20% inhibition were considered active.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Intensity_AFluorescence intensity at 460 nm, replicate AInteger
2Intensity_BFluorescence intensity at 460 nm, replicate BInteger
3% Inhibition_APercent inhibition, normalized to positive and negative controls, replicate AFloat%
4% Inhibition_BPercent inhibition, normalized to positive and negative controls, replicate BFloat%
5% Inhibition_AvgAverage of replicate A and B % inhibitionFloat%

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
Classification
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