Late 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) degradation
Name: Late 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) degradation. ..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: P27_ACT_LUMI_0096_4X-FOLD-CHANGE MCSRUN ROUND 1 ASSAY 8
Name: Late 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) 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.
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 phosphodependent 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.
Late stage, late stage AID, powders, SAR, purchased, synthesized, p27, cyclin-dependent kinase inhibitor 1B, KIP1, MEN4, CDKN4, MEN1B, P27KIP1, CDKN1B, counterscreen, Wee1, WEE1hu, FLJ16446, DKFZp686I18166, cell cycle, cancer, HeLa, degradation, inhibitor, inhibition, luminescence, luciferase, stabilize, fold change, stabilization assay, stabilization, dose response, quadruplicate, 96, assay, assay provider, Scripps, Scripps Florida, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
The purpose of this assay is to determine whether powder samples of compounds identified as possible WEE1 degradation inhibitor probe candidates are non-selective inhibitors of protein turnover by the proteasome. Furthermore, since Wee1 and p27 are targeted by the same class of ubiquitin ligases (the SCF ubiquitin ligases), this P27 assay serves to identify non-selective inhibitors of SCF ligases. This assay uses HeLa cells transfected with a plasmid that encodes a P27-luciferase fusion protein to monitor P27 levels and its turnover. The P27-luciferase complex is rapidly turned over in these cells. As designed, compounds that inhibit P27 degradation will increase P27-luciferase stability, leading to increased well luminescence. Compounds were tested in quadruplicate at a nominal concentration of 100 nM.
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 P27-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. Then, 50 uL of cells were added in quadruplicate to wells of a 96 well plate. Compounds were prepared in DMSO at 2,000 times the required concentration and diluted in media 1:1000. 50 uL of compound 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. P27-luciferase levels (RLUs) were divided by luciferase only levels (RLU) for DMSO.
The fold-change inhibition for each compound was calculated as follows:
[Cells_treated_with_Test_Compound] / [Cells_treated_with_Vehicle(DMSO)]
The average fold-change of each compound tested was calculated.
PubChem Activity Outcome and Score:
Compounds that induced a fold change less than 10 were considered inactive. Compounds that induced a fold change greater than or equal to 10 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 inactive compounds is 100-1. There are no active 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)
Brite-lite reagent (PerkinElmer, part 6016769)
TransIT-LT1 (Mirus, part MIR2306)
T75 flasks (BD Falcon 353136)
96-well plates (Corning part 3917)
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 the proteasome or well 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.
** Test Concentration.
Data Table (Concise)