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BioAssay: AID 1807

Summary of probe development efforts to identify inhibitors of Wee1 degradation

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 more ..
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AID: 1807
Data Source: The Scripps Research Institute Molecular Screening Center (WEE1DEG_INH_LEADS_SUMMARY)
BioAssay Type: Summary, Candidate Probes/Leads with Supporting Evidence
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-06-08
Modify Date: 2011-07-11

Data Table ( Complete ):           View Active Data    View All Data
Target
Sequence: WEE1 homolog (S. pombe) [Homo sapiens]
Description ..   
Protein Family: Catalytic domain of Protein Kinases

Gene:WEE1     Related Protein 3D Structures     More BioActivity Data..
BioActive Compound: Chemical Probe: 1    Active: 1
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 degradation inhibitors in singlicate)
1410Confirmation cell-based high throughput screening assay for inhibitors of Wee1 degradationScreening depositor-specified cross reference: Confirmation screen (WEE1 degradation inhibitors in triplicate)
1412Dose Response Cell-based Assay for Inhibitors of Wee1 DegradationConfirmatory depositor-specified cross reference: Dose response (WEE1 degradation inhibitors in singlicate)
1413Cytotoxicity counterscreen assay for inhibitors of Wee1 degradationConfirmatory depositor-specified cross reference: 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 depositor-specified cross reference: Dose response counterscreen (cyclin B degradation inhibitors in triplicate)
2088Late stage results from the probe development effort to identify inhibitors of Wee1 degradation.Screening depositor-specified cross reference: 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 depositor-specified cross reference: 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 depositor-specified cross reference: 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 depositor-specified cross reference: 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 depositor-specified cross reference: 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 depositor-specified cross reference: 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 depositor-specified cross reference: 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 depositor-specified cross reference: 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 depositor-specified cross reference: 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 depositor-specified cross reference: Late stage counterscreen (cyclin B degradation inhibitors in quadruplicate)
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: Late stage counterscreen (inhibitors of residues 1-40 of amyloid beta)
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: Late stage counterscreen(GCP proliferation inhibitors)
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: Late stage dose response counterscreen (cytotoxic compounds, neuroblastoma cell line)
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: WEE1DEG_INH_LEADS_SUMMARY

Name: Summary of probe development efforts to identify inhibitors of WEE1 degradation

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.

Summary of Probe Development Effort:

Following primary HTS in singlicate to identify Wee1 degradation inhibitors (AID 1321), confirmation of hit activity in triplicate (AID 1410), titration assays to determine compound potency (AID 1412), cytotoxicity (AID 1413), and selectivity against cyclin B (AID 1414), several compounds belonging to different chemical scaffolds were identified as possible candidates for probe development. These compounds and several analogs were ordered for testing in dose response assays to determine their potency in Wee1 degradation assays, and in counterscreen assays against cyclin B. The best probe candidates were subsequently tested in G2/M arrest assays in the laboratory of the Assay Provider. One probe was identified (SID 4243143 liquid/ SID 87235992 powder). This probe was found to induce a noticeable increase in the G2/M population, without increases in the sub-G1 population after treatment, suggesting that compound treatment was not toxic. While this compound acts as an inhibitor of cell cycle progression, it is not a proteasome inhibitor since it did not affect turnover of another proteasome substrate, N-cyclin B-luciferase.

The above probe development effort resulted in the identification of one probe. A probe report has been published (http://mlpcn.florida.scripps.edu/index.php/probes/probe-reports.htmlA probe report for SID 87235992 can be found in the Molecular Libraries Bookshelf (PubMed Books) (http://www.ncbi.nlm.nih.gov/books) under ML118.

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:

Summary AID, WEE1, WEE1hu, FLJ16446, DKFZp686I18166, cell cycle, cancer, HeLa, degradation, inhibitor, inhibition, luminescence, luciferase, dose response, counterscreen, 1536, HTS, assay, Scripps, Scripps Florida, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
Protocol
Please see Related Bioassays for protocols performed in this probe development effort.
Comment
A probe was identified.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Cell Type: HeLa
From MLP Probe Report:
Probe count: 2
MLP Probe ML# for probe 1: ML118
PubChem Substance ID (SID) for probe 1: 87235992
PubChem Compound ID (CID) for probe 1: 44552613
Probe type for probe 1: Inhibitor
IC50/EC50 (nM) for probe 1: 7870
Target for probe 1: Wee1 (gi: 47123300)
Disease relevance for probe 1: Mitotic Entry
Anti-target for probe 1: cyclin B
Fold selectivity for probe 1: >6.3
NCBI Book chapter link for probe 1: http://www.ncbi.nlm.nih.gov/books/NBK56226/ (ID: 2508644)
Grant number for probe 1: NS056991-01
MLP Probe ML# for probe 2: ML177
PubChem Substance ID (SID) for probe 2: 92092801
PubChem Compound ID (CID) for probe 2: 45100431
NCBI Book chapter link for probe 2: http://www.ncbi.nlm.nih.gov/books/NBK143190/ (ID: 3034134)
Grant number for probe 2: NS056991-01
PubMed Publication ID (PMID) for probe 1: 20660794
PubMed Publication ID (PMID) for probe 2: 20660794
NCBI Book chapter title for probe 1: Small Molecule Inhibitors of Wee1 Degradation and Mitotic Entry
NCBI Book chapter title for probe 2: Identification of Small Molecule Inhibitors of Wee1 Degradation and Mitotic Entry
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1ML #Unique alphanumeric identifier assigned to a chemical probe molecule within the Molecular Libraries Probe Production Centers Network (MLPCN).String
Additional Information
Grant Number: 1R21NS056991-01

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