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BioAssay: AID 526

Ubiquitin-GFP Assay

The ubiquitin-proteasome pathway is present within all eukaryotic cells and plays roles in normal cellular functions and disease-related dysfunction. Proteins are tagged with a poly-ubiquitin chain that targets them for the proteasome, a multimeric protease, that degrades the protein into peptides and free ubiquitin. The proteasome has a key role in regulating cell cycle and growth and is known more ..
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 Tested Compounds
 Tested Compounds
All(66191)
 
 
Active(3)
 
 
Inactive(65881)
 
 
Inconclusive(307)
 
 
 Tested Substances
 Tested Substances
All(67063)
 
 
Active(3)
 
 
Inactive(66753)
 
 
Inconclusive(307)
 
 
 Related BioAssays
 Related BioAssays
AID: 526
Data Source: NCGC
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
Deposit Date: 2006-11-09
Modify Date: 2006-11-13

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 3
Description:
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Screening Centers Network [MLSCN]

NCGC Assay Overview:
The ubiquitin-proteasome pathway is present within all eukaryotic cells and plays roles in normal cellular functions and disease-related dysfunction. Proteins are tagged with a poly-ubiquitin chain that targets them for the proteasome, a multimeric protease, that degrades the protein into peptides and free ubiquitin. The proteasome has a key role in regulating cell cycle and growth and is known to control the levels of cyclins and CDK inhibitors as well as tumor suppressor proteins. Additionally, the activation of NF- B is controlled by ubiquitin-labeling and degradation of the inhibitory protein I B . Inhibition of NF- B in cancer cell lines has been shown to make these cell lines more prone to apoptosis. With this in mind, proteasome inhibitors such as MG-132 and PS-341 have been used to inhibit tumor cell growth.
A cellular assay for ubiquitin-proteasome function was used to find chemical probes of this pathway. The assay used an ubiquitin-GFP construct expressed in U2OS cells (BioImage). Under basal conditions little GFP can be detected as it is degraded by the proteasome. However, treatment of the cells with a proteasome inhibitor such as MG-132 results in a rise in GFP levels that can be detected in as little as four hours. We used the Acumen-Explorer laser plate cytometer having a 488 nm laser as an excitation light source to detect the GFP. Compounds were screened in 1536-well plates using qHTS with a concentration-titration series range that varied from 46 uM to 2.9 nM.
Protocol
NCGC Assay Protocol Summary:
Cells (U20S ps 2042) expressing ubiquitin (G76V-GFP) were maintained in DMEM with Glutamax-1 and high glucose, 10% FBS, 1% (v/v) penicillin-streptomycin, 0.5 mg/mL Geneticin (G418). The cells were seeded in 1536-well plates using a solenoid-based dispenser at 700 cells/5ul in DMEM medium with Glutamax-1 and high glucose containing 0.5% FBS, w/o phenol red, 25 mM HEPES. The plates were incubated at 37 #C/5% CO2 overnight. Then 23 nl of MG-132 in DMSO was added to the control wells at a final concentration of 10 uM. Next, 23 nL of library compounds in DMSO were added to the sample wells. The plates were incubated for 4 hrs at 37 #C/5% CO2. Following this incubation the plates were read on the Acumen using a cell object definition of 15 to 100 um in width and depth and a window of Total Intensity of between 77,200 and 2,720,000 FLU to enumerate GFP-expressing cells. See Table.
The %Activity was determined from the basal and MG-132 treated control wells. %Activity was determined by normalizing to the difference in signal between basal cells (0% Activity) and cells incubated with 10 uM of the proteasome inhibitor MG-132 (100% Activity). Concentration-response curves were fitted to the signals arising from the GFP fluorescence.
Assay Protocol Summary Table
Sequence Parameter Value Description
1. Reagent 5 uL 700 U2OS ps 2040 cells/well
2. Time O/N 37C incubation
3. Reagent 23 nL MG132
4. Compound 23 nL Libraries (46 uM to 2.9 nM)
5. Time 4 hr 37C incubation
6. Detector Acumen Total Intensity, GFP fluorescence (488 ex/515 em)

Keywords: NIH Roadmap, MLSCN, MLI, MLSMR, BioImage, proteasome, ubiquitin-GFP, qHTS, NCGC
Comment
Compound Ranking:

1. Compounds are first classified as being probable activators (compounds causing an increase in GFP flourescence, presumably through inhibition of proteasome activity), inactive, or inconclusive based on flourescence readout in the cells.

2. Within the activators, compounds were ranked by efficacy and potency.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Cell Type: U-2 OS
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1PhenotypeIndicates type of activity observed: signal activator, signal inhibitor, inactive, or inconclusive. Signal activators cause an increase in cell flourescence, presumably through inhibition of proteaseome activity.String
2PotencyConcentration 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.String
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.String%
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
6Proteasome-Fit_LogAC50The logarithm of the AC50 from a fit of the ratio data to the Hill equation.Float
7Proteasome-Fit_HillSlopeThe Hill slope from a fit of the ratio data to the Hill equation.Float
8Proteasome-Fit_R2R^2 fit value of ratio curve. Closer to 1.0 equates to better Hill equation fit.Float
9Proteasome-Fit_InfiniteActivityThe asymptotic efficacy from a fit of the ratio data to the Hill equation.Float
10Proteasome-Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the ratio data to the Hill equation.Float
11Proteasome-Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
12Proteasome_ExcludedPointsWhich 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
13Proteasome-Activity at 0.6 nM% Activity at given concentration.Float%
14Proteasome-Activity at 1.3 nM% Activity at given concentration.Float%
15Proteasome-Activity at 2.9 nM% Activity at given concentration.Float%
16Proteasome-Activity at 6.6 nM% Activity at given concentration.Float%
17Proteasome-Activity at 14.7 nM% Activity at given concentration.Float%
18Proteasome-Activity at 32.8 nM% Activity at given concentration.Float%
19Proteasome-Activity at 73 nM% Activity at given concentration.Float%
20Proteasome-Activity at 164 nM% Activity at given concentration.Float%
21Proteasome-Activity at 367 nM% Activity at given concentration.Float%
22Proteasome-Activity at 821 nM% Activity at given concentration.Float%
23Proteasome-Activity at 1.84 uM% Activity at given concentration.Float%
24Proteasome-Activity at 4.10 uM% Activity at given concentration.Float%
25Proteasome-Activity at 9.17 uM% Activity at given concentration.Float%
26Proteasome-Activity at 20.5 uM% Activity at given concentration.Float%
27Proteasome-Activity at 45.9 uM% Activity at given concentration.Float%
28Compound TypeNCGC designation for compound stage: 'qHTS Exploratory', 'NIHSMR', 'Compound Followup', 'Compound Verification', 'Probe Optimization'String
29Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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