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BioAssay: AID 651623

Inhibitors of USP1/UAF1: SAR in USP8 diubiquitin SDS-PAGE gel assay

Deubiquitinating enzymes (DUBs) are a class of enzymes that can cleave isopeptide bond formed between the C-terminal carboxylate of ubiquitin and a lysine side-chain amino group on the target protein. Among them, ubiquitin specific proteases (USPs) constitute the largest DUB family. Human USPs are emerging as promising targets for pharmacological intervention because of their connection to a more ..
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 Tested Compounds
 Tested Compounds
All(1)
 
 
Inactive(1)
 
 
 Tested Substances
 Tested Substances
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Inactive(1)
 
 
AID: 651623
Data Source: NCGC (USP704)
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2012-10-04
Hold-until Date: 2013-09-06
Modify Date: 2013-09-06

Data Table ( Complete ):           View All Data
Target
Tested Compound:
Related Experiments
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AIDNameTypeComment
504878Inhibitors of USP1/UAF1: SummarySummarydepositor-specified cross reference
504865Inhibitors of USP1/UAF1: Pilot qHTSConfirmatorysame project related to Summary assay
540327Inhibitors of USP1/UAF1: Hit Validation in Primary AssayConfirmatorysame project related to Summary assay
624341Inhibitors of USP1/UAF1: Hit Validation in USP7 Diubiquiting Gel AssayOthersame project related to Summary assay
624345Inhibitors of USP1/UAF1: Hit Validation in USP2 Diubiquiting Gel AssayOthersame project related to Summary assay
624346Inhibitors of USP1/UAF1: Hit Validation in USP1 Diubiquiting Gel AssayOthersame project related to Summary assay
651605Inhibitors of USP1/UAF1: SAR in Primary AssayConfirmatorysame project related to Summary assay
651621Inhibitors of USP1/UAF1: SAR in USP1/UAF1 diubiquitin SDS-PAGE gel assayOthersame project related to Summary assay
651622Inhibitors of USP1/UAF1: SAR in USP46/UAF1 diubiquitin SDS-PAGE gel assayOthersame project related to Summary assay
651624Inhibitors of USP1/UAF1: SAR in USP7 diubiquitin SDS-PAGE gel assayOthersame project related to Summary assay
651625Inhibitors of USP1/UAF1: SAR in USP5 diubiquitin SDS-PAGE gel assayOthersame project related to Summary assay
651626Inhibitors of USP1/UAF1: SAR in USP2 diubiquitin SDS-PAGE gel assayOthersame project related to Summary assay
743255Inhibitors of USP1/UAF1: Primary ScreenConfirmatorysame project related to Summary assay
Description:
Deubiquitinating enzymes (DUBs) are a class of enzymes that can cleave isopeptide bond formed between the C-terminal carboxylate of ubiquitin and a lysine side-chain amino group on the target protein. Among them, ubiquitin specific proteases (USPs) constitute the largest DUB family. Human USPs are emerging as promising targets for pharmacological intervention because of their connection to a number of human diseases, including prostate, colon and breast cancer (1, 2), pediatric acute lymphoblastic leukemia (3), and familial cylindromatosis (4). The advantage of inhibiting USPs lies in the potential specificity of therapeutic intervention that can lead to better efficacy and reduce nonspecific side effects.

In a collaboration between the University of Delaware and the NIH Chemical Genomics Center, a high-throughput screen assay was developed to screen for USP1/UAF1 inhibitors (5). This assay is a selectivity assay that tested for activity in a USP8 diubiquitin SDS-PAGE gel assay and inactivity is the desired outcome for this assay.

NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]

MLPCN Grant: DA030552
Assay Submitter (PI): Zhihao Zhuang, University of Delaware

1. Priolo, C., et al. (2006) The isopeptidase USP2a protects human prostate cancer from apoptosis. Cancer Res 66, 8625-8632

2. Popov, N., et al. (2007) The ubiquitin-specific protease USP28 is required for MYC stability. Nat Cell Biol 9, 765-774

3. De Pitta, C., et al. (2005) A leukemia-enriched cDNA microarray platform identifies new transcripts with relevance to the biology of pediatric acute lymphoblastic leukemia. Haematologica 90, 890-898

4. Kovalenko, A., et al. (2003) The tumour suppressor CYLD negatively regulates NF-kappaB signalling by deubiquitination. Nature 424, 801-805

5. Chen, J., et al. (2011) Selective and cell-active inhibitors of the USP1/ UAF1 deubiquitinase complex reverse cisplatin resistance in non-small cell lung cancer cells. Chem Bio 18(11):1390-400.
Protocol
The inhibition assay contains 80 nM USP8, 2 microM K63 linked diubiquitin, and inhibitors at varied concentrations (0.08 microM - 114 microM). The reaction was allowed for 1 hour at 37 degrees C, and quenched by the addition of Laemmli sample buffer. The reaction product was separated on a 20% denaturing SDS-PAGE gel and stained with Coomassie Blue. The intensity of the individual diubiquitin and monoubiquitin band was quantified using Quantity One 4.3.1 (Biorad, Hercules, CA). The percentage of the conversion was determined and used to calculate the reaction rate. The rate was plotted against inhibitor concentrations and fitted to determine the IC50 values using GraphPad Prism (GraphPad Software, Inc. La Jolla, CA).
Comment
Compounds that show percent activity >= 60% at 114 uM are considered "active" and a score of 90 is assigned; compounds that show percent activity < 60% and <= 30% at 114 uM are considered "inconclusive" are are assigned a score of 50; compounds that show percent activity < 30% at 114 uM are considered "inactive" and a score of 10 is assigned.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Percent Activity at 0.08 uMFloat%
2Percent Activity at 0.4 uMFloat%
3Percent Activity at 2 uMFloat%
4Percent Activity at 11 uMFloat%
5Percent Activity at 57 uMFloat%
6Percent Activity at 114 uMFloat%
7Compound QCString
Additional Information
Grant Number: DA030552

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