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BioAssay: AID 504930

Late 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) proliferation

Name: Late 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) proliferation. ..more
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 Tested Compounds
 Tested Compounds
All(2)
 
 
Probe(1)
 
 
Active(2)
 
 
 Tested Substances
 Tested Substances
All(2)
 
 
Probe(1)
 
 
Active(2)
 
 
 Related BioAssays
 Related BioAssays
AID: 504930
Data Source: The Scripps Research Institute Molecular Screening Center (GCP_INH_RAD_0096_IC50 MDCSRUN ROUND 1 WEE1-CKD1)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
Deposit Date: 2011-07-07
Hold-until Date: 2012-07-07
Modify Date: 2012-07-11

Data Table ( Complete ):           Active    All
BioActive Compounds: Chemical Probe: 1    Active: 2
Depositor Specified Assays
Show more
AIDNameTypeProbeComment
1321Primary Cell-based High Throughput Screening Assay for Inhibitors of Wee1 Degradationscreening Primary screen (WEE1 degradation inhibitors in singlicate)
1410Confirmation cell-based high throughput screening assay for inhibitors of Wee1 degradationscreening Confirmation screen (WEE1 degradation inhibitors in triplicate)
1412Dose Response Cell-based Assay for Inhibitors of Wee1 Degradationconfirmatory Dose response (WEE1 degradation inhibitors in singlicate)
1413Cytotoxicity counterscreen assay for inhibitors of Wee1 degradationconfirmatory Cytotoxicity counterscreen (WEE1 degradation inhibitors in triplicate)
1414Counterscreen assay for inhibitors of Wee1 degradation: dose response cell-based assay to identify inhibitors of cyclin B degradationconfirmatory Dose response counterscreen (cyclin B degradation inhibitors in triplicate)
1807Summary of probe development efforts to identify inhibitors of Wee1 degradationsummary1 Summary (WEE1 degradation inhibitors)
2088Late stage results from the probe development effort to identify inhibitors of Wee1 degradation.screening Late stage results (WEE1 degradation inhibitors in triplicate)
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 Late stage screen (WEE1 degradation inhibitors in quadruplicate)
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 Late stage counterscreen (FLT3 inhibitors)
463077Late 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 inhibitorsconfirmatory Late stage dose response counterscreen (CK1d inhibitors)
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 Late stage dose response (Wee1 stabilizers 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 Late stage assay provider results (WEE1 degradation inhibitors in quadruplicate)
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 Late stage counterscreen (p21 degradation inhibitors in quadruplicate)
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 Late stage counterscreen (p27 degradation inhibitors in quadruplicate)
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 Late stage counterscreen (Hela cell apoptosis inducers in quadruplicate)
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 Late stage counterscreen (cyclin B degradation inhibitors in quadruplicate)
Description:
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRISMC)
Center Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Nagi Ayad, TSRI
Network: Molecular Library Probe Production Center Network (MLPCN)
Grant Proposal Number: 1R21NS056991-01
Grant Proposal PI: Nagi Ayad, TSRI
External Assay ID: GCP_INH_RAD_0096_IC50 MDCSRUN ROUND 1 WEE1-CKD1

Name: Late 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) proliferation.

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.
9. Hatten, M.E., Neuronal regulation of astroglial morphology and proliferation in vitro. J Cell Biol, 1985. 100(2): p. 384-96.

Keywords:

Late stage, late stage AID, powders, SAR, purchased, synthesized, Wee1, WEE1hu, FLJ16446, DKFZp686I18166, radioactivity, rad, radiation, radioactive, counts, CPM, 3H, incorporation, GCP, tritium, tritiated thymidine, cell proliferation, granule cell progenitor, percoll gradient, C57Bl6J mice, mice, cell cycle, cancer, biochemical, degradation, inhibitor, inhibition, 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 GCP (Granule cell progenitor) cell proliferation. Well-established methods for purifying GCPs were developed by the Hatten laboratory (9). Compounds that inhibit GCP proliferation will result in lower of 3H-thymidine incorporation.

Protocol Summary:

To purify immature granule cells, cerebella from postnatal mice are isolated and triturated, and a mixed cell suspension containing GCPs is applied to a Percoll gradient (35%/65%), followed by three sequential panning steps to remove glia and fibroblasts from meninges and enrich GCPs. By electron microscopy (EM) and immunocytochemistry, the final suspension is 99% pure GCPs. 40 million GCPs or more are purified from a single litter of C57Bl6J mice. 3H-thymidine incorporation of GCPs treated with DMSO, FKL-135 (analog), or probe RJR-159 was measured after 24 hours.

The extent of 3H-thymidine incorporation was normalized to DMSO control. A ratio of radioactivity signals was calculated and plotted against the concentration to generate the IC50s.

%_Inhibition = ( 1 - ( Test_Compound - Low_Control ) ) * 100

Where:

Test_Compound is defined as wells containing test compound.
Low_Control is defined as wells containing DMSO.

For each test compound, percent inhibition was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay software (GraphPad Prism software Inc). The reported IC50 values were generated from fitted curves by solving for the X-intercept value at the 50% inhibition level of the Y-intercept value.

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 is assigned a value of 100 for probe compounds, 50 for actives and 0 for inactives.

The PubChem Activity Score range for active compounds is 100-50. There are no inactive compounds.

List of Reagents:

Percoll (Sigma)
C57Bl6J mice (Jackson Laboratoies)
3H-thymidine (NEN Dupont)
Comment
This assay was performed by the assay provider. 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 kinase activity, radioactivity, or cell proliferation. 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.
Result Definitions
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; CC50) shown in micromolar.FloatμM

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

Data Table (Concise)
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