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BioAssay: AID 2278

Secondary Assay for Inhibitors of Ubiquitin-specific Protease USP2a: PLA2 Counterscreen

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(42)
 
 
Active(1)
 
 
Inactive(31)
 
 
Inconclusive(10)
 
 
 Tested Substances
 Tested Substances
All(42)
 
 
Active(1)
 
 
Inactive(31)
 
 
Inconclusive(10)
 
 
 Related BioAssays
 Related BioAssays
AID: 2278
Data Source: NCGC (UBPA445)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-01-21

Data Table ( Complete ):           View Active Data    View All Data
Target
Sequence: phospholipase A2, group X precursor [Mus musculus]
Description ..   
Protein Family: PLA2c

Gene:PLA2G10     Related Protein 3D Structures     More BioActivity Data..
BioActive Compound: 1
Description:
NIH Molecular Libraries Screening Centers Network [MLSCN]
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 [3-6]. To identify inhibitors of USP2a a cell-free assay was employed.

This is assay is a counterscreen used for the for the USP2a-PLA2 coupled assay. In this assay the murine PLA2 reporter enzyme is screened alone using NBD C6-HPC (Invitrogen) as a fluorogenic substrate.
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, 0.1% DMSO. 3ul of 10nM (5nM final) free PLA2, which is stored on ice, is dispensed into a medium-binding black solid Kalypsys 1,536 well plate using the Aurora BioRaptr. The assay plate is then pinned with 23nL compound with the Kalypsys pintool. The controls were pinned as follows: row 31 2M NEM and row 32 DMSO. Subsequently, 3uL 40uM (20uM final) NBD C6-HPC (stored on ice and protected from light) were dispensed into the plate using the BioRaptr dispenser. Plates were read immediately on an Envision (Perkin Elmer) plate reader using wavelengths of Ex 460nm /Em 535nm. Then, the assay plates were incubated for 2.5 hours and read again on the Envision using the same settings.

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, MLSCN, 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".
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 0.0000068511 uM (6.85111e-06μM**)% Activity at given concentration.Float%
15Activity at 0.0000137022 uM (1.37022e-05μM**)% Activity at given concentration.Float%
16Activity at 0.0000274044 uM (2.74044e-05μM**)% Activity at given concentration.Float%
17Activity at 0.0000548089 uM (5.48089e-05μM**)% Activity at given concentration.Float%
18Activity at 0.0001096177 uM (0.000109618μM**)% Activity at given concentration.Float%
19Activity at 0.0002192355 uM (0.000219235μM**)% Activity at given concentration.Float%
20Activity at 0.0004384709 uM (0.000438471μM**)% Activity at given concentration.Float%
21Activity at 0.0008769419 uM (0.000876942μM**)% Activity at given concentration.Float%
22Activity at 0.00175 uM (0.00175388μM**)% Activity at given concentration.Float%
23Activity at 0.00351 uM (0.00350777μM**)% Activity at given concentration.Float%
24Activity at 0.00702 uM (0.00701554μM**)% Activity at given concentration.Float%
25Activity at 0.014 uM (0.0140311μM**)% Activity at given concentration.Float%
26Activity at 0.028 uM (0.0280621μM**)% Activity at given concentration.Float%
27Activity at 0.056 uM (0.0561243μM**)% Activity at given concentration.Float%
28Activity at 0.112 uM (0.112249μM**)% Activity at given concentration.Float%
29Activity at 0.224 uM (0.224497μM**)% Activity at given concentration.Float%
30Activity at 0.449 uM (0.448994μM**)% Activity at given concentration.Float%
31Activity at 0.898 uM (0.897989μM**)% Activity at given concentration.Float%
32Activity at 1.796 uM (1.79598μM**)% Activity at given concentration.Float%
33Activity at 3.592 uM (3.59195μM**)% Activity at given concentration.Float%
34Activity at 7.184 uM (7.18391μM**)% Activity at given concentration.Float%
35Activity at 14.37 uM (14.3678μM**)% Activity at given concentration.Float%
36Activity at 28.74 uM (28.7356μM**)% Activity at given concentration.Float%
37Activity at 57.47 uM (57.4713μM**)% Activity at given concentration.Float%
38Compound 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)
Data Table ( Complete ):     View Active Data    View All Data
Classification
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