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BioAssay: AID 449747

Small-molecule inhibitor of deubiquitinating enzyme USP14

Usp14, a major proteasome-associated deubiquitination enzyme, acts as an endogenous proteasome inhibitor. By high-throughput screening, we identified small-molecule inhibitors of the deubiquitinating activity of proteasome-bound human Usp14. Usp14 inhibitor can act as an enhancer of substrate degradation by proteasome. ..more
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 Tested Compounds
 Tested Compounds
All(1)
 
 
Active(1)
 
 
 Tested Substances
 Tested Substances
All(1)
 
 
Active(1)
 
 
 Related BioAssays
 Related BioAssays
AID: 449747
Data Source: Finley and King Labs, Harvard Medical School (1)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: Literature, Author
BioAssay Version:
Deposit Date: 2010-07-13
Hold-until Date: 2010-09-16
Modify Date: 2010-09-16

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Target
BioActive Compound: 1
Description:
Usp14, a major proteasome-associated deubiquitination enzyme, acts as an endogenous proteasome inhibitor. By high-throughput screening, we identified small-molecule inhibitors of the deubiquitinating activity of proteasome-bound human Usp14. Usp14 inhibitor can act as an enhancer of substrate degradation by proteasome.
Protocol
High-throughput screening was performed at ICCB-Longwood screening facility (Harvard Medical School, Boston, MA).
In summary, 10 ul of recombinant Usp14 protein was dispensed into each well of a 384-well low volume plate in duplicate, using a Wellmate plate dispenser. 33.3 nl of compound from the library was pin-transferred into the wells using a Seiko pin transfer robotic system, followed by preincubation for about 30 min. To initiate the enzyme reaction, 10 ul of Ub-VS-treated human proteasome plus Ub-AMC mixture was added to each well, using a Wellmate dispenser. Samples were then incubated for another 45 min. Ub-AMC hydrolysis was measured at Ex355/Em460. The final concentrations of Usp14, VS-proteasome, and Ub-AMC were 15 nM, 1 nM, and 0.8 uM, respectively. The final concentration of test compound was approximately 17 uM. Enzymes and substrates were prepared in Ub-AMC assay buffer (50 mM Tris-HCl [pH 7.5], 1 mM EDTA, 1 mM ATP, 5 mM MgCl2, 1 mM DTT, and 1 mg/mL ovalbumin)
Comment
PubChem score indicates % inhibition of Usp14 at 17 uM of the compound.
To measure IC50 values, various concentrations of Usp14 inhibitor were preincubated with corresponding enzymes for 30 min. The reaction was initiated by adding 1 uM of Ub-AMC. To obtain a dose-response curve, the percent inhibition for each reaction was first determined, and then curve fitting was performed according to guidelines from NIH Chemical Genomics Center (http://ncgc.nih.gov/guidance/manual_toc.html).
The results were published in Nature, volume 467 , issue 7312, pp. 179-184; DOI:10.1038/nature09299
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Functional
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1IC50*IntegerμM

* Activity Concentration.

Data Table (Concise)
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Classification
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