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BioAssay: AID 652277

qHTS for Inhibitors of Ubiquitin-specific Protease USP2a Using CHOP2 as the Reporter: Caspase 6 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 proteaosome. However, ubiquitination is highly reversible more ..
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 Tested Compounds
 Tested Compounds
All(28)
 
 
Inactive(26)
 
 
Inconclusive(2)
 
 
 Tested Substances
 Tested Substances
All(28)
 
 
Inactive(26)
 
 
Inconclusive(2)
 
 
 Related BioAssays
 Related BioAssays
AID: 652277
Data Source: NCGC (USP2608)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2013-04-04
Hold-until Date: 2014-01-21
Modify Date: 2014-01-21

Data Table ( Complete ):           All
Target
Tested Compounds:
Depositor Specified Assays
AIDNameTypeComment
2281qHTS Assay for Inhibitors of Ubiquitin-specific Protease USP2a: Summarysummary
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 proteaosome. However, ubiquitination is highly reversible and dynamic. Deubiquitination, the reverse process, is catalyzed through the action of enzymes referred to as isopeptidases for 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 deubiquinates fatty acid synthase (FASN) which has recently been identified as an emerging oncology target.

To ascertain whether the USP2 inhibitors also inhibit other cysteine proteases, a caspase 6 (cysteine-aspartic acid protease) assay coupled to luciferase was developed using the Caspase 6 Glo kit (Promega, Madison, WI). This assay monitors caspase 6 activity with a luminogenic caspase 6 substrate. Upon cleavage of the substrate by caspase 6, the product can be acted upon by luciferase to lead to luminescence that is proportional to the caspase 6 activity. A compound that inhibits caspase 6 would result in a decrease in luminescence. The assay is run at 5 uM substrate concentration, which is the Km reported by Promega of their substrate. Therefore, this assay is sensitive to identify non-, un- and competitive inhibitors of caspase 6. This assay is run in kinetic mode. Inactivity is the desired outcome for this assay.

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

MLPCN Grant: MH079852
PI Name: Benjamin Nicholson, Progenra Inc, Malvern, PA
Protocol
Two and a half uL of 0.5 U/mL caspase 6 in buffer was dispensed in a 1536 well plate. The assay plate was then pinned with 23 nL compound using the Kalypsis pintool. After 30 minutes at room temperature, 25 uL of Caspase 6 Glo detection reagent was added and luminescence signal was read using the PerkinElmer ViewLux plate reader with a 1 second exposure.

The data were normalized to control columns representing maximum signal (all components) and background (no enzyme). The buffer consisted of 10 mM Hepes pH 7.2, 2 mM DTT, 10% glycerol and 0.05% CHAPS, and the assay was run in white solid-bottom Greiner plates.
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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