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 ..
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
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.
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.
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.
Please see Related Bioassays for protocols performed in this probe development effort.
Categorized Comment - additional comments and annotations
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
Data Table (Concise)