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BioAssay: AID 493168

Confirmation Assay for Inhibitors of Ubiquitin-specific Protease USP2a Using CHOP2 as the Reporter

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(873)
 
 
Active(93)
 
 
Inactive(648)
 
 
Inconclusive(132)
 
 
 Tested Substances
 Tested Substances
All(873)
 
 
Active(93)
 
 
Inactive(648)
 
 
Inconclusive(132)
 
 
AID: 493168
Data Source: NCGC (UBCH003)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-02-09

Data Table ( Complete ):           Active    All
Target
Sequence: ubiquitin carboxyl-terminal hydrolase 2 isoform a [Homo sapiens]
Description ..   
Protein Family: Peptidase_C19R

Gene:USP2     Related Protein 3D Structures     More BioActivity Data..
BioActive Compounds: 93
Depositor Specified Assays
AIDNameTypeComment
2281qHTS Assay for Inhibitors of Ubiquitin-specific Protease USP2a: Summarysummarysummary assay
463106qHTS Validation Assay for Inhibitors of Ubiquitin-specific Protease USP2a Using CHOP2 as the ReporterconfirmatoryUb-CHOP2 lopac validation assay
463254qHTS Assay for Inhibitors of Ubiquitin-specific Protease USP2a Using CHOP2 as the ReporterconfirmatoryUb-CHOP2 qHTS assay
Description:
NIH Molecular Libraries Probe Centers Network [MLPCN]
NIH Chemical Genomics Center [NCGC]

MLSCN Grant: XO1-MH079852-01
PI Name: Nicholson, Ben. Progenra Inc, Malvern, PA

NCGC Assay Overview:

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) [1, 2]. 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 assay was employed.

This assay takes advantage of attaching an Ub to a reporter enzyme, rendering the reporter catalytically inactive. The reporter enzyme chosen is an enterokinase called CHOP2, which is activated when the Ub is cleaved by USP2. The free and now activated reporter can subsequently act on its reporter substrate. Thus, this coupled assay gives a fluorescent read-out at excitation/emission wavelengths of 485/531. The assay was developed by Progenra, Inc. and has been licensed for sale by LifeSensors, Inc. as Ub-CHOP2-Reporter-Kit (#PR1101).
Protocol
NCGC Assay Protocol Summary:

The assay buffer, prepared fresh at the day of the assay, contains 20mM Tris-HCl pH 8.0, 2mM CaCl2, 2mM beta-Mercaptoethanol, 0.05% CHAPS. 3ul of 20nM (10nM final) USP2 core, which is stored on ice, is dispensed into a medium-binding black solid Kalypsys 1,536 well plate using the Kalypsys dispenser. The assay plate is then pinned with 23nL compound with the Kalypsys pintool in columns 5-48. The controls are pinned as follows: column 1 two-fold dilutions of 2M NEM in duplicate; column 2 2M NEM (final concentration 38.2mM); column 3&4 DMSO. The assay plate is incubated at RT for 30min. Subsequently, 1.5uL of 100nM (25nM final) Ub-CHOP2 and 1.5uL of 100nM (25nM final) Reporter Substrate (both stored on ice and protected from light) were dispensed into the plate using the Kalypsys dispenser. Plates were read after 60min RT incubation on a ViewLux (Perkin Elmer) plate reader using the following wavelengths Ex 485nm /Em 531nm.

Data were normalized to the to AC100 inhibition (NEM). Concentration-response curves were fitted to the normalized data and the concentration-response curves were then classified based on curve quality (r2), response magnitude and degree of measured activity. The time zero reading was used to flag fluorescence artifacts.

Keywords: ubiquitination, deubiquitination, proteases, profluorescent, MLSMR, MLPCN, NIH Roadmap, qHTS, NCGC
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
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 0.00245 uM (0.00245μM**)% Activity at given concentration.Float%
15Activity at 0.012 uM (0.0123μM**)% Activity at given concentration.Float%
16Activity at 0.061 uM (0.0613μM**)% Activity at given concentration.Float%
17Activity at 0.307 uM (0.307μM**)% Activity at given concentration.Float%
18Activity at 1.530 uM (1.53μM**)% Activity at given concentration.Float%
19Activity at 7.660 uM (7.66μM**)% Activity at given concentration.Float%
20Activity at 38.30 uM (38.3μM**)% Activity at given concentration.Float%
21Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: XO1-MH079852-01

Data Table (Concise)
Classification
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