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BioAssay: AID 651605

Inhibitors of USP1/UAF1: SAR 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(141)
 
 
Active(104)
 
 
Inactive(8)
 
 
Inconclusive(29)
 
 
 Tested Substances
 Tested Substances
All(147)
 
 
Active(109)
 
 
Inactive(8)
 
 
Inconclusive(30)
 
 
AID: 651605
Data Source: NCGC (USP1700)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2012-09-28
Hold-until Date: 2013-09-06
Modify Date: 2013-09-06

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 104
Depositor Specified Assays
AIDNameTypeComment
504878Inhibitors of USP1/UAF1: SummarysummarySummary AID
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 was used to test the analogs that were synthesized based on SAR around the original hit.

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

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
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.
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
5Activity_ScoreActivity score.Integer
6Curve_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
7Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
8Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
9Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
10Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
11Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
12Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
13Excluded_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
14Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
15Activity at 0.0003225714 uM (0.000322571μM**)% Activity at given concentration.Float%
16Activity at 0.0009677143 uM (0.000967714μM**)% Activity at given concentration.Float%
17Activity at 0.00290 uM (0.00290314μM**)% Activity at given concentration.Float%
18Activity at 0.00871 uM (0.00870949μM**)% Activity at given concentration.Float%
19Activity at 0.026 uM (0.0261284μM**)% Activity at given concentration.Float%
20Activity at 0.046 uM (0.0457μM**)% Activity at given concentration.Float%
21Activity at 0.078 uM (0.0783853μM**)% Activity at given concentration.Float%
22Activity at 0.235 uM (0.234763μM**)% Activity at given concentration.Float%
23Activity at 0.457 uM (0.457μM**)% Activity at given concentration.Float%
24Activity at 0.709 uM (0.709194μM**)% Activity at given concentration.Float%
25Activity at 2.119 uM (2.11867μM**)% Activity at given concentration.Float%
26Activity at 5.110 uM (5.11μM**)% Activity at given concentration.Float%
27Activity at 6.348 uM (6.34766μM**)% Activity at given concentration.Float%
28Activity at 11.40 uM (11.4μM**)% Activity at given concentration.Float%
29Activity at 19.05 uM (19.0476μM**)% Activity at given concentration.Float%
30Activity at 25.50 uM (25.5μM**)% Activity at given concentration.Float%
31Activity at 57.07 uM (57.0707μM**)% Activity at given concentration.Float%
32Activity at 114.0 uM (114μM**)% Activity at given concentration.Float%
33Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

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

Data Table (Concise)
Classification
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