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BioAssay: AID 651999

uHTS identification of small molecule inhibitors of Csn-mediated Deneddylation of Cullin-Ring Ligases, vis a fluorescence polarization assay

Protein modification by the attachment of ubiquitin to cellular proteins is a key mechanism in regulating many cellular and organismal processes. Ubiquitin is covalently attached to target proteins via an isopeptide bond between the C-terminus of ubiquitin and a lysine residue of the acceptor substrate. Additional ubiquitins can be conjugated to any of the 7 lysine residues of ubiquitin or to its more ..
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 Tested Compounds
 Tested Compounds
All(363827)
 
 
Active(1401)
 
 
Inactive(362429)
 
 
 Tested Substances
 Tested Substances
All(364168)
 
 
Active(1402)
 
 
Inactive(362766)
 
 
AID: 651999
Data Source: Burnham Center for Chemical Genomics (SBCCG-A954-CSN5-Inh-Primary-Assay)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2013-01-30

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 1401
Related Experiments
AIDNameTypeComment
652009Summary assay for small molecule inhibitors of Csn-mediated Deneddylation of Cullin-Ring LigasesSummarydepositor-specified cross reference
720654Single Concentration Validation of Csn-Mediated Deneddylation of Cullin-Ring Ligase Using a Fluorescence Polarization AssayOthersame project related to Summary assay
720655Single concentration validation of Csn-Mediated Deneddylation of Cullin-Ring Ligase Hits Using a MMP-2 Fluorescence AssayOthersame project related to Summary assay
720656Single concentration validation of Csn-Mediated Deneddylation of Cullin-Ring Ligase Using a Thrombin Fluorescence Polarization AssayOthersame project related to Summary assay
Description:
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford Burnham Medical Research Institute, La Jolla, CA
Network: NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Number: 1 R21 NS071527-01A1
Assay Provider: Dr. Raymond Deshaies, California Institute of Technology

Protein modification by the attachment of ubiquitin to cellular proteins is a key mechanism in regulating many cellular and organismal processes. Ubiquitin is covalently attached to target proteins via an isopeptide bond between the C-terminus of ubiquitin and a lysine residue of the acceptor substrate. Additional ubiquitins can be conjugated to any of the 7 lysine residues of ubiquitin or to its N-terminus to form a polyubiquitin chain on the substrate. Assembly of a chain of >=4 ubiquitins linked together via Lys48 of ubiquitin marks cellular proteins for degradation by the 26S proteasome. Ubiquitination of proteins is achieved through an enzymatic cascade involving ubiquitin-activating (E1), ubiquitin-conjugating (E2), and ubiquitin-ligating (E3) enzymes. Ubiquitination occurs when an E3 binds to both substrate and an E2 thioesterified with ubiquitin (E2~Ub), bringing them in proximity so that the ubiquitin is transferred from E2 to substrate.

Among the most intensively studied E3s are members of the cullin-RING ligase (CRL) superfamily, which are regulated by a reversible covalent modification of the cullin with the ubiquitin-like protein, Nedd8. Deconjugation of Nedd8 from cullins is carried out by a novel metalloisopeptidase in the Csn5 subunit of the COP9/Signalosome (CSN). Csn5 appears to be the only enzyme capable of cleaving Nedd8 from Cul1 in vivo.

CSN is an excellent candidate for the development of a small molecule inhibitor because such a compound would greatly facilitate cell biological studies of this key regulatory factor.
Protocol
Protocol:
A. Brief Description of the Assay:
This assay attempts to identify inhibitors of the CSN catalyzed deconjugation of Nedd8. It is run in 1536-well format and is measured via fluorescence polarization.
B. Materials:
Item, source, catalog no.
SCFSkp2-Nedd8OG, derived from human proteins and expressed in E. coli
Human Csn5 enzyme, expressed and purified from HEK293T cells
Molecular Biology Grade Water, Cellgro, 46-000-CM
Tween 20, MP Biomedicals LLC, 194841
Tris pH 7.5, Fisher Scientific, ICN819638
Trehalose, Fluka, 90210
Ovalbumin, Sigma, A5503
Sodium Chloride, Fisher Scientific, S271-10
DTT, Roche, 03117006001
1536 Black solid bottom plates, Corning, 3724
C. Final Assay Conditions:
10 nM SCFSkp2-Nedd8OG substrate
0.24 nM Csn5 Enzyme
25 mM Tris pH7.5
50 mM Sodium Chloride
1 mM DTT
0.01% Tween 20
25 mM Trehalose
0.015 mg/mL Ovalbumin
12.5 uM test compound
0.25% DMSO (from compounds)
3 uL reaction volume
90 minutes incubation at room temp
D. Assay Procedures:
1. Prepare Reagents as described in section F. Recipe.
2. Using LabCyte Echo, transfer 7.5 nL from 5mM test compound source plate into assay plate Col. 5 - 48 (final concentration of test compounds is 12.5 uM).
3. Spin plates at 1000 rpm for 1 minute in centrifuge.
4. Using the Thermo Multidrop Combi, add 1.5 uL/well of control buffer to columns 1 and 2.
5. Using the Thermo Multidrop Combi, add 1.5 uL/well of enzyme solution to col. 3-48.
6. Using the Thermo Multidrop Combi, add 1.5 uL/well of substrate solution to col. 1-48.
7. Spin plates at 1000 rpm for 1 minute in centrifuge.
8. Incubate plates at room temperature for 90 minutes.
9. Read plates on BMG Pherastar FS using a Fluorescence Polarization protocol.
E. Plate Map:
Positive (High) control in column 1-2, buffer and substrate only.
Negative (Low) control in columns 3-4, enzyme and substrate.
Test wells in columns 5-48, 12.5 uM test compound, enzyme and substrate.
F. Recipe:
1X Assay Buffer
25 mM Tris pH 7.5
50 mM Sodium Chloride
1 mM DTT
0.01% Tween 20
25 mM Trehalose
0.015 mg/mL Ovalbumin
Control Buffer
1X Assay Buffer
Enzyme Solution
0.48 nM Csn5 Enzyme in 1X Assay Buffer (final enzyme concentration is 0.24 nM).
Substrate Solution
20 nM SCFSkp2-Nedd8OG Substrate in 1X Assay Buffer (final substrate concentration is 10 nM).
Comment
Compounds with corrected %Activity >=35% and 0.5 =< FRatio <= 1.5 are considered to be active in this assay.
The experimental values were normalized by the difference between values from neutral and stimulator control wells in each plate. Then normalized data was corrected to remove systematic plate patterns due to artifacts such as dispensing tip issues etc. Further information about data correction is available at http://www.genedata.com/products/screener.html.
To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented.
Activity Scoring
Activity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows:
1) First tier (0-40 range) is reserved for primary screening data. The score is correlated with % activity in the assay:
a. If outcome of the primary screen is inactive, then the assigned score is 0
b. If outcome of the primary screen is inconclusive, then the assigned score is 10
c. If outcome of the primary screen is active, then the assigned score is 20
Scoring for Single concentration confirmation screening is not applicable to this assay.
d. If outcome of the single-concentration confirmation screen is inactive, then the assigned score is 21
e. If outcome of the single-concentration confirmation screen is inconclusive, then the assigned score is 25
f. If outcome of the single-concentration confirmation screen is active, then the assigned score is 30
This scoring system helps track the stage of the testing of a particular SID. For the primary hits which are available for confirmation, their scores will be greater than 20. For those which are not further confirmed, their score will stay under 21.
2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable in this assay
3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable in this assay
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1%Activity_Corrected at 12.5 uM (12.5μM**)Genedata pattern corrected % inhibition in primary screeningFloat%
2FRatio (12.5μM**)FRatio of the sample using corrected resultsFloat
3Value at 12.5 uM (12.5μM**)Calculated mP of the sampleFloatmP
4Mean HighMean mP of positive controls in the corresponding plateFloatmP
5STD Deviation HighStandard deviation (n=64) of positive controls in the corresponding plateFloatmP
6Mean LowMean fluorescence of negative controls in the corresponding plateFloatmP
7STD Deviation LowStandard deviation (n=64) of negative controls in the corresponding plateFloatmP

** Test Concentration.
Additional Information
Grant Number: 1 R21 NS071527-01A1

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