Late stage assay provider results from the probe development effort to identify inhibitors of WEE1 degradation: luminescence-based dose response assay to identify stabilizers of WEE1
Name: Late stage assay provider results from the probe development effort to identify inhibitors of WEE1 degradation: luminescence-based dose response assay to identify stabilizers of WEE1. ..more
BioActive Compounds: 5
Depositor Specified Assays
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: WEE1-STABILIZATION_ACT_LUMI_0096_EC50 MDRUN ROUND 1
Name: Late stage assay provider results from the probe development effort to identify inhibitors of WEE1 degradation: luminescence-based dose response assay to identify stabilizers of WEE1.
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.
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The purpose of this assay is to identify compounds that stabilize WEE1. The assay uses HeLa cells transfected with a kinase negative mutant of WEE1 (WEE1K328M) fused to a luciferase reporter gene. As designed, compounds that increase WEE1K328M-luciferase stability and/or prevent its degradation will lead to increased well luminescence compared to untreated wells. Specifically, compounds that increase luminescence are considered WEE1 degradation inhibitors. Compounds were tested in quadruplicate.
HeLa cells were routinely grown in T75 tissue culture flasks in growth media composed of Dulbecco's Modified Eagle's Media (DMEM) supplemented with 10% v/v fetal bovine serum (FBS) and 1% pen-strep-neo antibiotic mixture at 37 C in an atmosphere of 10% CO2 and 95% relative humidity (RH). Cells were transiently transfected in flasks by mixing 6 million cells with 29 ug of the WEE1K328M-luciferase plasmid complexed with 87 uL of TransIT-LT1 reagent in a final volume of 24 mL of a 1:1 mix of OptiMEM and 2X supplemented DMEM, according to the manufacturer's protocol. Cells were then returned to the incubator for 48 hours. Next, the transfected cells were trypsinized and resuspended at a concentration of 850,000 cells per mL in growth media. Fifty uL of cells were added in quadruplicate to wells of a 96 well plate. Compounds were made in DMSO at 2,000 times the required concentration and diluted in media 1:1000. Fifty uL of each concentration was added to 50 uL of cells in the appropriate wells resulting in a 1:1000 final dilution. The plates were incubated for 20 hours at 37 C (10% CO2, 95% RH). The assay was stopped by adding 100 uL of Brite-lite Reagent to each well and incubating at room temperature for 1 minute. Well luminescence was measured on the Perkin Elmer Envision Plate Reader. Cells expressing either K328M-WEE1-Luciferase or Luciferase alone were incubated with increasing concentrations of compounds. A ratio of luminescence signals was then calculated and plotted against the concentration to generate the EC50s.
PubChem Activity Outcome and Score:
Compounds with an EC50 greater than 1 uM were considered inactive. Compounds with an EC50 equal to or less than 1 uM were considered active.
Activity score was ranked by the potency of the compounds, with the most potent compounds assigned the highest activity scores.
The PubChem Activity Score range for active compounds is 100-1. There are no inactive compounds.
List of Reagents:
Dulbecco's Modified Eagle's Media (Invitrogen, part 11965-092)
Fetal Bovine Serum (Hyclone, part SH 30088.03)
Penicillin-Streptomycin-Neomycin antibiotic mix (Invitrogen, part 15640-055)
SteadyLite HTS luciferase substrate (PerkinElmer, part 6016989)
Reference agonist MG132 (American Peptide, part 81-5-15)
TransIT-LT1 (Mirus, part MIR2306)
T75 flasks (Corning, part 430641)
1536-well plates (Greiner, part 789173)
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 in the test well; compounds that nonspecifically modulate luminescence. 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. All data were normalized to SID 92092801 (the probe candidate)
* Activity Concentration. ** Test Concentration.
Data Table (Concise)