Dose Response Cell-based Assay for 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 ..
BioActive Compounds: 8
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: Wee1Degradation_ACT_LUMI_1536_EC50
Name: Dose Response Cell-based Assay for 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.
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.
Wee1, WEE1hu, FLJ16446, DKFZp686I18166, cell cycle, cancer, HeLa, degradation, inhibitor, dose response, luminescence, luciferase, 1536, HTS, High Throughput Screening, Scripps, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
The purpose of this assay is to determine dose response curves for compounds identified as active in a previous set of experiments entitled, "Primary cell-based high throughput screening assay for inhibitors of Wee1 degradation" (PubChem AID 1321), and that confirmed activity in a set of experiments entitled, "Confirmation cell-based high throughput screening assay for inhibitors of Wee1 degradation" (PubChem AID 1410). The assay uses HeLa cells transfected with a plasmid that encodes a Wee1K328M-luciferase fusion protein to monitor Wee1 levels. Wee1K328M is a kinase negative mutant used to avoid the cell growth inhibitory effects of wildtype wee1. The Wee1K328M-luciferase complex is rapidly turned over in these cells. As designed, compounds that inhibit Wee1 degradation will increase Wee1K328M-luciferase stability, leading to increased well luminescence. Compounds were tested in triplicate using a 10-point, 1:3 dilution series, starting at a nominal concentration of 50 uM.
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 degrees C in an atmosphere of 5% 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 15 to 20 million cells per mL in freezing media (growth media containing 10% DMSO), aliquoted into cryovials, and stored at -80 degrees C until needed.
Prior to the start of the assay, cells were thawed, centrifuged and resuspended in growth media at a concentration of 800,000 cells per mL. Next, 5 ul of cell suspension were dispensed into each well of 1536-well plates (4,000 cells per well). After incubation for 4 hours at 37 degrees C, 5% CO2 and 95% RH, the assay was started by dispensing 25 nL of test compound in DMSO, DMSO alone (0.5% final concentration), or the proteasome inhibitor MG132 (30 uM final nominal concentration, set as 100% activation) to the appropriate wells. The plates were next incubated for 20 hours at 37 degrees C (5% CO2, 95% RH). The assay was then stopped by dispensing 5 ul of SteadyLite HTS luciferase substrate to each well, followed by incubation at room temperature for 15 minutes. Next, well luminescence was measured on the ViewLux plate reader.
The percent activity was defined using the following mathematical formula:
% Activity = 100*((Test_Compound - Median_Low_Control) / (Median_High_Control - Median_Low_Control))
Test_Compound is defined as wells containing test compound.
High_Control is defined as wells containing MG132.
Low_Control is defined as wells containing DMSO.
For each test compound, percent activation was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (MDL Information Systems). The reported EC50 values were generated from fitted curves by solving for the X-intercept value at the 50% activation level of the Y-intercept value. In cases where the highest concentration tested (i.e. 50 uM) did not result in greater than 50% activation, the EC50 was determined manually as greater than 50 uM. Compounds with an EC50 greater than 10 uM were considered inactive. Compounds with an EC50 equal to or less than 10 uM were considered active.
Any compound with a percent activity value <50% at all test concentrations was assigned an activity score of zero. Any compound with a percent activity value >50% at any test concentration was assigned an activity score greater than zero. Activity score was then ranked by the potency, with the most potent compounds assigned the highest activity scores.
The active compounds of this assay have activity score range of 63 to 100. All inactive compounds are assigned activity score of zero.
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)
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. 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 by the MLSMR. The MLSMR was not able to provide all compounds selected for testing in this AID.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)