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
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.
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.
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.
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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.
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
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:
C57Bl6J mice (Jackson Laboratoies)
3H-thymidine (NEN Dupont)
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.
Categorized Comment - additional comments and annotations
* Activity Concentration.
Data Table (Concise)