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BioAssay: AID 540327

Inhibitors of USP1/UAF1: Hit Validation in Primary 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(50)
 
 
Active(25)
 
 
Inactive(4)
 
 
Inconclusive(21)
 
 
 Tested Substances
 Tested Substances
All(50)
 
 
Active(25)
 
 
Inactive(4)
 
 
Inconclusive(21)
 
 
AID: 540327
Data Source: NCGC (USP1601)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2011-08-02
Modify Date: 2011-08-19

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 25
Related Experiments
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AIDNameTypeComment
504878Inhibitors of USP1/UAF1: SummarySummarydepositor-specified cross reference: Summary AID of project
504865Inhibitors of USP1/UAF1: Pilot qHTSConfirmatorysame 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
651623Inhibitors of USP1/UAF1: SAR in USP8 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. This miniaturized assay has a fluorescent read-out and is used to screen the NIH Molecular Libraries Small Molecule Repository (MLSMR) in order to identify a small molecule that inhibits USP1. This assay was used to confirm the hits identified from the qHTS.

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

MLPCN Grant: DA030552-01
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
Protocol
3 microliters of reagents (buffer in columns 3 and 4 as negative control and 1nM USP1/UAF1 complex in columns 1, 2, and 5-48) are dispensed into Greiner black solid bottom 1536-well assay plates. Compounds are then transferred via Kalypsys pin tool equipped with 1536-pin array (10nl slotted pins, V&P Scientific, San Diego, CA). Following an incubation step of 15 min at room temperature, 1ul of Ub-Rho substrate (150nM final concentration) is added to initiate the reaction. The plates are immediately centrifuged at 1000 rpm for 15 seconds, and subsequently transferred to a ViewLux high-throughput CCD imager (PerkinElmer) wherein kinetic measurements of fluorescence are acquired using 480 nm excitation/540 nm emission filter set (6 reads every 60 seconds, see Table 1). All reagents are diluted in an assay buffer consisting of 50mM HEPES (pH 7.8), 0.5mM EDTA, 0.1 mg/ml BSA, 1mM TCEP, and 0.01% Tween-20.
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.
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Functional
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1PhenotypeIndicates type of activity observed: inhibitor, activator, fluorescent, cytotoxic, inactive, or inconclusive.String
2Potency*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.FloatμM
3EfficacyMaximal 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%
4Analysis CommentAnnotation/notes on a particular compound's data or its analysis.String
5Curve_DescriptionA 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 significant, but below 80% of control.String
6Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
7Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
8Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
9Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
10Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
11Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
12Excluded_PointsWhich 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
13Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
14Activity at 0.0003207433 uM (0.000320743μM**)% Activity at given concentration.Float%
15Activity at 0.0006451462 uM (0.000645146μM**)% Activity at given concentration.Float%
16Activity at 0.0009622300 uM (0.00096223μM**)% Activity at given concentration.Float%
17Activity at 0.00194 uM (0.00193544μM**)% Activity at given concentration.Float%
18Activity at 0.00289 uM (0.00289μM**)% Activity at given concentration.Float%
19Activity at 0.00581 uM (0.00580632μM**)% Activity at given concentration.Float%
20Activity at 0.00866 uM (0.00866μM**)% Activity at given concentration.Float%
21Activity at 0.017 uM (0.0174189μM**)% Activity at given concentration.Float%
22Activity at 0.026 uM (0.026μM**)% Activity at given concentration.Float%
23Activity at 0.065 uM (0.065μM**)% Activity at given concentration.Float%
24Activity at 0.160 uM (0.16μM**)% Activity at given concentration.Float%
25Activity at 0.479 uM (0.479μM**)% Activity at given concentration.Float%
26Activity at 1.411 uM (1.41093μM**)% Activity at given concentration.Float%
27Activity at 2.104 uM (2.104μM**)% Activity at given concentration.Float%
28Activity at 4.233 uM (4.2328μM**)% Activity at given concentration.Float%
29Activity at 6.313 uM (6.313μM**)% Activity at given concentration.Float%
30Activity at 12.70 uM (12.6984μM**)% Activity at given concentration.Float%
31Activity at 18.94 uM (18.94μM**)% Activity at given concentration.Float%
32Activity at 38.10 uM (38.0952μM**)% Activity at given concentration.Float%
33Activity at 56.82 uM (56.82μM**)% Activity at given concentration.Float%
34Activity at 114.3 uM (114.286μM**)% Activity at given concentration.Float%
35Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: DA030552

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