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BioAssay: AID 463077

Late stage assay provider results from the probe development effort to identify inhibitors of casein kinase 1 delta (CK1d): radioactivity-based in vitro biochemical kinase assay to identify CK1d inhibitors

Name: Late stage assay provider results from the probe development effort to identify inhibitors of casein kinase 1 delta (CK1d): radioactivity-based in vitro biochemical kinase assay to identify CK1d inhibitors. ..more
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 Tested Compounds
 Tested Compounds
All(13)
 
 
Active(12)
 
 
Inactive(1)
 
 
 Tested Substances
 Tested Substances
All(13)
 
 
Active(12)
 
 
Inactive(1)
 
 
AID: 463077
Data Source: The Scripps Research Institute Molecular Screening Center (CK1D_INH_RAD_0096_IC50 MDCSRUN ROUND 1)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2010-08-03
Hold-until Date: 2011-07-28
Modify Date: 2011-07-28

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 12
Related Experiments
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AIDNameTypeProbeComment
1321Primary Cell-based High Throughput Screening Assay for Inhibitors of Wee1 DegradationScreening depositor-specified cross reference: Primary screen (WEE1 inhibitors in singlicate)
1410Confirmation cell-based high throughput screening assay for inhibitors of Wee1 degradationScreening depositor-specified cross reference: Confirmation screen (WEE1 inhibitors in triplicate)
1412Dose Response Cell-based Assay for Inhibitors of Wee1 DegradationConfirmatory depositor-specified cross reference: Dose response (WEE1 inhibitors in singlicate)
1413Cytotoxicity counterscreen assay for inhibitors of Wee1 degradationConfirmatory depositor-specified cross reference: Cytotoxicity counterscreen (WEE1 inhibitors in triplicate)
1414Counterscreen assay for inhibitors of Wee1 degradation: dose response cell-based assay to identify inhibitors of cyclin B degradationConfirmatory depositor-specified cross reference: Dose response counterscreen (cyclin B inhibitors in triplicate)
1807Summary of probe development efforts to identify inhibitors of Wee1 degradationSummary1 depositor-specified cross reference: Summary (WEE1 inhibitors)
2088Late stage results from the probe development effort to identify inhibitors of Wee1 degradation.Screening depositor-specified cross reference: Late stage results (WEE1 inhibitors)
434972Late stage results from the probe development effort to identify inhibitors of Wee1 degradation: luminescence-based cell-based assay to determine inhibition of Wee1 degradation by kinase inhibitorsOther depositor-specified cross reference: Late stage (WEE1 inhibitors in quadruplicate)
463169Late stage assay provider results from the probe development effort to identify inhibitors of Wee1 degradation: luminescence-based cell-based assay to identify inhibitors of Wee1 degradationScreening depositor-specified cross reference
463170Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of Wee1 degradation: luminescence-based cell-based assay to identify inhibitors of p21 (CDKN1A) degradationScreening depositor-specified cross reference
463171Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of Wee1 degradation: luminescence-based cell-based assay to identify inhibitors of p27 (CDKN1B) degradationOther depositor-specified cross reference
463177Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of Wee1 degradation: fluorescence activated cell sorting (FACS)-based cell-based assay to identify inducers of Hela cell apoptosisOther depositor-specified cross reference
463186Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of Wee1 degradation: luminescence-based cell-based assay to identify inhibitors of cyclin B degradationOther depositor-specified cross reference
504929Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of Wee1 degradation: Amplified proximity luminescence-based biochemical assay to identify inhibitors of residues 1-40 of amyloid betaOther1 depositor-specified cross reference
504930Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of Wee1 degradation: radioactivity-based cell-based assay to identify inhibitors of granule cell progenitor (GCP) proliferationConfirmatory1 depositor-specified cross reference
504935Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of Wee1 degradation: luminescence-based cell-based dose response assay for cytotoxic compounds using neuroblastoma cell lineOther1 depositor-specified cross reference
463076Late stage assay provider results from the probe development effort to identify inhibitors of casein kinase 1 delta (CK1d): radioactivity-based in vitro biochemical kinase assay for inhibitors of fms-related tyrosine kinase 3 (FLT3)Confirmatory same project related to Summary assay
463080Late stage assay provider results from the probe development effort to identify inhibitors of WEE1 degradation: luminescence-based dose response assay to identify stabilizers of WEE1Confirmatory same project related to Summary assay
Description:
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: Nagi Ayad, TSRI
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1R21NS056991-01
Grant Proposal PI: Nagi Ayad, TSRI
External Assay ID: CK1D_INH_RAD_0096_IC50 MDCSRUN ROUND 1

Name: Late stage assay provider results from the probe development effort to identify inhibitors of casein kinase 1 delta (CK1d): radioactivity-based in vitro biochemical kinase assay to identify CK1d inhibitors.

Description:

Cell cycle progression and entry into mitosis are regulated by a highly conserved cellular process known as checkpoint signaling (1-4). The Wee1 nuclear tyrosine kinase functions in this process by regulating the cdc2/cyclinB protein complex. Specifically, Wee1 mediates inhibitory phosphorylation of cdc2, leading to delayed mitosis and cell cycle arrest in cells with DNA damage so that DNA repair and replication can occur (1-4). Wee1 activity is inhibited during mitosis by its phosphorylation and ubiquitination by E3 ligases, and its subsequent degradation by the proteasome (5, 6). Studies showing that Wee1 expression is reduced in colon carcinoma cells (7) and that Wee1 overexpression can block cell division (8), suggest that Wee1 may act as a tumor suppressor. Thus, the identification of probes that selectively increase levels of Wee1 may provide useful insights into the roles of Wee1 in cell cycle control and tumor pathogenesis.

References:

1. Lee MH, Yang HY. Negative regulators of cyclin-dependent kinases and their roles in cancers. Cell Mol Life Sci 2001; 58: 1907-1922.
2. Heald R, McLoughlin M, McKeon F. Human Wee1 maintains mitotic timing by protecting the nucleus from cytoplasmically activated Cdc2 kinase. Cell 1993; 74: 463-474.
3. Coleman, TR & Dunphy, WG. Cdc2 regulatory factors. Curr Opin Cell Biol. 1994 Dec;6(6):877-82.
4. Kellogg, DR. Wee1-dependent mechanisms required for coordination of cell growth and cell division. J Cell Sci. 2003 Dec 15;116(Pt 24):4883-90.
5. Smith A, Simanski S, Fallahi M, Ayad NG. Redundant ubiquitin ligase activities regulate wee1 degradation and mitotic entry. Cell Cycle. 2007 Aug;6(22):2795-9.
6. Watanabe N, Arai H, Nishihara Y, Taniguchi M, Watanabe N, Hunter T, and Osada H. M-phase kinases induce phospho-dependent ubiquitination of somatic Wee1 by SCFbeta-TrCP. PNAS 2004 101: 4419-4424.
7. Backert S, Gelos M, Kobalz U, Hanski ML, Bohm C, Mann B, Lovin N, Gratchev A, Mansmann U, Moyer MP, Riecken EO, Hanski C. Differential gene expression in colon carcinoma cells and tissues detected with a cDNA array. Int J Cancer. 1999 Sep 9;82(6):868-74.
8. McGowan, C. H.; Russell, P. Human Wee1 kinase inhibits cell division by phosphorylating p34cdc2 exclusively on Tyr15. EMBO J. 1993. 12: 75-85.

Keywords:

Late stage, late stage AID, powders, SAR, purchased, synthesized, CK1d, CSNK1D , casein kinase 1, delta, kinase, HCK1D, CKI, Wee1, WEE1hu, FLJ16446, DKFZp686I18166, cell cycle, cancer, biochemical, degradation, inhibitor, inhibition, P33, radiation, radioactivity, phosphorylation, ATP, dose response, 96, assay, assay provider, Scripps, Scripps Florida, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
Protocol
Assay Overview:

The purpose of this assay is to determine whether powder samples of probe candidates can inhibit the activity of CK1d. This in vitro kinase assay was performed by Reaction Biology Corporation (RBC). For CK1, 20 uM final CK1tide was used, sequence: [KRRRAL[pS]VASLPGL] in a standard kinase assay with 33P-ATP and purified kinase. Incorporation of 33P-ATP into the peptide was measured after a filter-binding assay. Compounds were assayed in a 10-point 1:3 dilution series starting at a nominal concentration of 50 uM.

Protocol Summary:

Kinase reactions were performed using the "Hotspot" kinase profiling service of Reaction Biology Corporation.

1. Prepare indicated substrate in freshly prepared Base Reaction Buffer.
2. Deliver any required cofactors to the substrate solution above.
3. Deliver indicated kinase into the substrate solution and gently mix.
4. Deliver compounds in DMSO into the kinase reaction mixture.
5. Deliver 33P-ATP (specific activity 500 μCi/μl) into the reaction mixture to initiate the reaction.
6. Incubate kinase reaction for 120 min. at room temperature.

A ratio of radioactivity signals was then calculated and plotted against the concentration to generate the IC50s.

PubChem Activity Outcome and Score:

Compounds with an IC50 greater than 1 uM were considered inactive. Compounds with an IC50 equal to or less than 1 uM were considered active.

The PubChem Activity Score range for active compounds is 100-97, and for inactive compounds 1-1

List of Reagents:

Base Reaction buffer (20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO)
Comment
This assay was performed by Reaction Biology Corporation (RBC). This assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. Possible artifacts of this assay can include, but are not limited to: presence of lint or dust; compounds that nonspecifically modulate filter binding or kinase activity. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Biochemical
Assay Test Type: In vitro
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1IC50*The concentration at which 50 percent of the activity in the inhibition assay is observed; (IC50) shown in micromolar.FloatμM
2Inhibition at 50 uM [1] (50μM**)Value of % inhibition at 50 micromolar inhibitor concentration; replicate one.Float%
3Inhibition at 50 uM [2] (50μM**)Value of % inhibition at 50 micromolar inhibitor concentration; replicate two.Float%
4Inhibition at 50 uM [3] (50μM**)Value of % inhibition at 50 micromolar inhibitor concentration; replicate three.Float%
5Inhibition at 16.7 uM [1] (16.7μM**)Value of % inhibition at 16.7 micromolar inhibitor concentration; replicate one.Float%
6Inhibition at 16.7 uM [2] (16.7μM**)Value of % inhibition at 16.7 micromolar inhibitor concentration; replicate two.Float%
7Inhibition at 16.7 uM [3] (16.7μM**)Value of % inhibition at 16.7 micromolar inhibitor concentration; replicate three.Float%
8Inhibition at 5.56 uM [1] (5.56μM**)Value of % inhibition at 5.56 micromolar inhibitor concentration; replicate one.Float%
9Inhibition at 5.56 uM [2] (5.56μM**)Value of % inhibition at 5.56 micromolar inhibitor concentration; replicate two.Float%
10Inhibition at 5.56 uM [3] (5.56μM**)Value of % inhibition at 5.56 micromolar inhibitor concentration; replicate three.Float%
11Inhibition at 1.85 uM [1] (1.85μM**)Value of % inhibition at 1.85 micromolar inhibitor concentration; replicate one.Float%
12Inhibition at 1.85 uM [2] (1.85μM**)Value of % inhibition at 1.85 micromolar inhibitor concentration; replicate two.Float%
13Inhibition at 1.85 uM [3] (1.85μM**)Value of % inhibition at 1.85 micromolar inhibitor concentration; replicate three.Float%
14Inhibition at 0.617 uM [1] (0.617μM**)Value of % inhibition at 0.617 micromolar inhibitor concentration; replicate one.Float%
15Inhibition at 0.617 uM [2] (0.617μM**)Value of % inhibition at 0.617 micromolar inhibitor concentration; replicate two.Float%
16Inhibition at 0.617 uM [3] (0.617μM**)Value of % inhibition at 0.617 micromolar inhibitor concentration; replicate three.Float%
17Inhibition at 0.206 uM [1] (0.206μM**)Value of % inhibition at 0.206 micromolar inhibitor concentration; replicate one.Float%
18Inhibition at 0.206 uM [2] (0.206μM**)Value of % inhibition at 0.206 micromolar inhibitor concentration; replicate two.Float%
19Inhibition at 0.206 uM [3] (0.206μM**)Value of % inhibition at 0.206 micromolar inhibitor concentration; replicate three.Float%
20Inhibition at 0.0686 uM [1] (0.0686μM**)Value of % inhibition at 0.0686 micromolar inhibitor concentration; replicate one.Float%
21Inhibition at 0.0686 uM [2] (0.0686μM**)Value of % inhibition at 0.0686 micromolar inhibitor concentration; replicate two.Float%
22Inhibition at 0.0686 uM [3] (0.0686μM**)Value of % inhibition at 0.0686 micromolar inhibitor concentration; replicate three.Float%
23Inhibition at 0.0229 uM [1] (0.0229μM**)Value of % inhibition at 0.0229 micromolar inhibitor concentration; replicate one.Float%
24Inhibition at 0.0229 uM [2] (0.0229μM**)Value of % inhibition at 0.0229 micromolar inhibitor concentration; replicate two.Float%
25Inhibition at 0.0229 uM [3] (0.0229μM**)Value of % inhibition at 0.0229 micromolar inhibitor concentration; replicate three.Float%
26Inhibition at 0.00762 uM [1] (0.00762μM**)Value of % inhibition at 0.00762 micromolar inhibitor concentration; replicate one.Float%
27Inhibition at 0.00762 uM [2] (0.00762μM**)Value of % inhibition at 0.00762 micromolar inhibitor concentration; replicate two.Float%
28Inhibition at 0.00762 uM [3] (0.00762μM**)Value of % inhibition at 0.00762 micromolar inhibitor concentration; replicate three.Float%
29Inhibition at 0.00254 uM [1] (0.00254μM**)Value of % inhibition at 0.00254 micromolar inhibitor concentration; replicate one.Float%
30Inhibition at 0.00254 uM [2] (0.00254μM**)Value of % inhibition at 0.00254 micromolar inhibitor concentration; replicate two.Float%
31Inhibition at 0.00254 uM [3] (0.00254μM**)Value of % inhibition at 0.00254 micromolar inhibitor concentration; replicate three.Float%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1R21NS056991-01

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