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BioAssay: AID 927

qHTS Assay for Inhibitors of Ubiquitin-specific Protease USP2a

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(59169)
 
 
Active(62)
 
 
Inactive(58570)
 
 
Inconclusive(549)
 
 
 Tested Substances
 Tested Substances
All(59724)
 
 
Active(64)
 
 
Inactive(59109)
 
 
Inconclusive(551)
 
 
AID: 927
Data Source: NCGC (UBPA427)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2007-12-20
Modify Date: 2008-06-03

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 62
Depositor Specified Assays
AIDNameTypeComment
2281qHTS Assay for Inhibitors of Ubiquitin-specific Protease USP2a: Summarysummary
1455Confirmation Concentration-response Assay for Inhibitors of Ubiquitin-specific Protease USP2aconfirmatory
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 assay takes advantage of attaching an Ub or UBL (ubiquitin-like protein) to a reporter enzyme, rendering the reporter catalytically inactive. The reporter enzyme chosen is phospholipase A2 (PLA2), which has an absolute requirement for a free amino terminus. Thus, fusion of a UBL to the N-terminus of PLA2 inactivates PLA2. When the UBL-PLA2 reporter enzyme is cleaved by USP2, the activated reporter can subsequently act on its substrate, which is the commercially available NBD C6-HPC (Invitrogen). Thus, this coupled assay gives a fluorescent read-out, which is generated through the cleavage of the PLA2 reporter enzyme. The assay, developed by Progenra, Inc. uses Ub-PLA2 and USP2a at near equal and low (nM) amounts and has been licensed for sale by LifeSensors, Inc. as the Ub-IsoPro1 kit.
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 40nM (20nM 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&2 two-fold dilutions of 2M NEM in duplicate; column 3 DMSO; column 4 2M NEM (final concentration 38.2mM). The assay plate is incubated at RT for 30min. Subsequently, 3uL of 40nM (20nM final) Ub-PLA2 and 40uM (20uM final) NBD C6-HPC (stored on ice and protected from light) were dispensed into the plate using the Kalypsys dispenser. Plates were read immediately (0 Hour) on an Envision (Perkin Elmer) plate reader using the following wavelengths 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". 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.

Reference:

1.Amerik, A.Y. and M. Hochstrasser, Mechanism and function of deubiquitinating enzymes. Biochim Biophys Acta, 2004. 1695(1-3): p. 189-207.
2.Nijman, S.M., et al., A genomic and functional inventory of deubiquitinating enzymes. Cell, 2005. 123(5): p. 773-86.
3.Graner, E., et al., The isopeptidase USP2a regulates the stability of fatty acid synthase in prostate cancer. Cancer Cell, 2004. 5(3): p. 253-61.
4.Nicholson, B., et al., Deubiquitinating enzymes as novel anticancer targets. Future Oncol, 2007. 3(2): p. 191-199.
5.Priolo, C., et al., The Isopeptidase USP2a Protects Human Prostate Cancer from Apoptosis. Cancer Res, 2006. 66(17): p. 8625-32.
6.Stevenson, L.F., et al., The deubiquitinating enzyme USP2a regulates the p53 pathway by targeting Mdm2. Embo J, 2007. 26(4): p. 976-86.
Result Definitions
Show more
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 signficant, but below 80% of control.String
6Fit_LogAC50The logarithm of the AC50 from a fit of the ratio data to the Hill equation (calculated based on molar units).Float
7Fit_HillSlopeThe Hill slope from a fit of the ratio data to the Hill equation.Float
8Fit_R2R^2 fit value of ratio curve. Closer to 1.0 equates to better Hill equation fit.Float
9Fit_InfiniteActivityThe asymptotic efficacy from a fit of the ratio data to the Hill equation.Float%
10Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the ratio 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 0.0000054935 uM (5.49352e-06μM**)% Activity at given concentration.Float%
15Activity at 0.0000109870 uM (1.0987e-05μM**)% Activity at given concentration.Float%
16Activity at 0.0000219741 uM (2.19741e-05μM**)% Activity at given concentration.Float%
17Activity at 0.0000439481 uM (4.39481e-05μM**)% Activity at given concentration.Float%
18Activity at 0.0000878962 uM (8.78962e-05μM**)% Activity at given concentration.Float%
19Activity at 0.0001757925 uM (0.000175793μM**)% Activity at given concentration.Float%
20Activity at 0.0003515850 uM (0.000351585μM**)% Activity at given concentration.Float%
21Activity at 0.0007031700 uM (0.00070317μM**)% Activity at given concentration.Float%
22Activity at 0.00141 uM (0.00140634μM**)% Activity at given concentration.Float%
23Activity at 0.00245 uM (0.002453μM**)% Activity at given concentration.Float%
24Activity at 0.00250 uM (0.0025μM**)% Activity at given concentration.Float%
25Activity at 0.00281 uM (0.00281268μM**)% Activity at given concentration.Float%
26Activity at 0.00563 uM (0.00562536μM**)% Activity at given concentration.Float%
27Activity at 0.011 uM (0.0112507μM**)% Activity at given concentration.Float%
28Activity at 0.012 uM (0.0122643μM**)% Activity at given concentration.Float%
29Activity at 0.023 uM (0.0225014μM**)% Activity at given concentration.Float%
30Activity at 0.027 uM (0.0274229μM**)% Activity at given concentration.Float%
31Activity at 0.045 uM (0.0450029μM**)% Activity at given concentration.Float%
32Activity at 0.061 uM (0.0613176μM**)% Activity at given concentration.Float%
33Activity at 0.090 uM (0.0900058μM**)% Activity at given concentration.Float%
34Activity at 0.137 uM (0.137106μM**)% Activity at given concentration.Float%
35Activity at 0.180 uM (0.180012μM**)% Activity at given concentration.Float%
36Activity at 0.307 uM (0.306569μM**)% Activity at given concentration.Float%
37Activity at 0.360 uM (0.360023μM**)% Activity at given concentration.Float%
38Activity at 0.685 uM (0.685489μM**)% Activity at given concentration.Float%
39Activity at 0.686 uM (0.6855μM**)% Activity at given concentration.Float%
40Activity at 0.720 uM (0.720046μM**)% Activity at given concentration.Float%
41Activity at 1.440 uM (1.44009μM**)% Activity at given concentration.Float%
42Activity at 1.533 uM (1.53275μM**)% Activity at given concentration.Float%
43Activity at 2.880 uM (2.88018μM**)% Activity at given concentration.Float%
44Activity at 3.427 uM (3.42724μM**)% Activity at given concentration.Float%
45Activity at 5.760 uM (5.76037μM**)% Activity at given concentration.Float%
46Activity at 7.663 uM (7.6633μM**)% Activity at given concentration.Float%
47Activity at 11.52 uM (11.5207μM**)% Activity at given concentration.Float%
48Activity at 17.14 uM (17.1351μM**)% Activity at given concentration.Float%
49Activity at 23.04 uM (23.0415μM**)% Activity at given concentration.Float%
50Activity at 38.31 uM (38.3142μM**)% Activity at given concentration.Float%
51Activity at 46.08 uM (46.0829μM**)% Activity at given concentration.Float%
52Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.

Data Table (Concise)
Classification
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