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BioAssay: AID 652170

qHTS for Inhibitors of Ubiquitin-specific Protease USP2a Using CHOP2 as the Reporter: Cytotox Assay

Homeostasis of cellular proteins is maintained through a combination of synthesis and degradation. The pathway that accounts for the majority of protein degradation is the ubiquitin-proteasomal pathway. Ubiquitin (Ub) is highly conserved in all cells and the generation of a multi-Ub chain typically targets proteins for degradation by the proteasome. However, ubiquitination is highly reversible more ..
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 Tested Compounds
 Tested Compounds
All(297)
 
 
Active(48)
 
 
Inactive(198)
 
 
Inconclusive(53)
 
 
 Tested Substances
 Tested Substances
All(307)
 
 
Active(50)
 
 
Inactive(204)
 
 
Inconclusive(53)
 
 
AID: 652170
Data Source: NCGC (USP2603)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2013-03-25
Hold-until Date: 2014-03-24
Modify Date: 2014-03-25

Data Table ( Complete ):           Active    All
BioActive Compounds: 48
Depositor Specified Assays
AIDNameTypeComment
2281qHTS Assay for Inhibitors of Ubiquitin-specific Protease USP2a: SummarysummarySummary AID
Description:
Homeostasis of cellular proteins is maintained through a combination of synthesis and degradation. The pathway that accounts for the majority of protein degradation is the ubiquitin-proteasomal pathway. Ubiquitin (Ub) is highly conserved in all cells and the generation of a multi-Ub chain typically targets proteins for degradation by the proteasome. However, ubiquitination is highly reversible and dynamic. Deubiquitination, the reverse process, is catalyzed through the action of enzymes referred to as isopeptidases or deubiquitinating enzymes (DUBs). This group of enzymes is collectively responsible for maintaining adequate pools of free ubiquitin and regulating the ubiquitination status of cellular proteins. The class of DUBs referred to as the ubiquitin-specific proteases (USP) family functions endoproteolytically to cleave Ub chains from a wide range of protein substrates. USP2a deubiquitinates fatty acid synthase (FASN) which has recently been identified as an emerging oncology target. To identify inhibitors of USP2a a cell-free qHTS assay was employed.

Confirmed qHTS active compounds were tested for cytoxicity in HCT116 cells (human colon cancer cells) using the Cell Titer Glo luciferase-coupled ATP detection system from Promega. This assays monitors cell proliferation by quantitating the amount of ATP present by luciferase, which in the presence of ATP converts pro-luciferin into the luminescent luciferin product. A compound that inhibits cell proliferation would result in a decrease in luminescence. The data were normalized to control columns representing maximum signal (cells with DMSO) and background (no cells with DMSO). The cells were cultured in DMEM with 10% FBS and plated in white solid-bottom tissue-culture treated plates.

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

MLSCN Grant: MH079852
PI Name: Benjamin Nicholson, Progenra Inc, Malvern, PA
Protocol
5 uL of HCT116 (600 cells/well) were plated into a 1536 well plate and then incubated for 6 hr at 37 deg C, 5% CO2. Compounds are added through pinning before the plate is incubated for 72 hr at 37 deg C, 5% CO2. Then, 3 uL of Cell Titer Glo is added, the plate is left at RT for 10 min, before luminescence is detected using the ViewLux.
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 0.0007804147 uM (0.000780415μM**)% Activity at given concentration.Float%
16Activity at 0.00234 uM (0.00234124μM**)% Activity at given concentration.Float%
17Activity at 0.00702 uM (0.00702378μM**)% Activity at given concentration.Float%
18Activity at 0.021 uM (0.0210713μM**)% Activity at given concentration.Float%
19Activity at 0.063 uM (0.0632139μM**)% Activity at given concentration.Float%
20Activity at 0.190 uM (0.189642μM**)% Activity at given concentration.Float%
21Activity at 0.569 uM (0.568925μM**)% Activity at given concentration.Float%
22Activity at 1.707 uM (1.70678μM**)% Activity at given concentration.Float%
23Activity at 5.120 uM (5.12033μM**)% Activity at given concentration.Float%
24Activity at 15.36 uM (15.361μM**)% Activity at given concentration.Float%
25Activity at 46.08 uM (46.0829μM**)% Activity at given concentration.Float%
26Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: MH079852

Data Table (Concise)
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