Primary Cell-based High Throughput Screening Assay for Inhibitors of Wee1 Degradation
The Cdc2/cyclinB protein complex plays an important role in G2/M cell cycle progression. The nuclear tyrosine kinase Wee1 mediates inhibitory phosphorylation of Tyr15 on cdc2, leading to delayed mitosis and cell cycle arrest in cells with DNA damage so that DNA repair and replication can occur (1, 2). During mitosis Wee1 activity is inhibited by its phosphorylation (3, 4), accompanied by its more ..
BioActive Compounds: 2616
Depositor Specified Assays
Source (MLSCN Center Name): The Scripps Research Institute Molecular Screening Center
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: Nagi Ayad, TSRI
Network: Molecular Library Screening Center Network (MLSCN)
Grant Proposal Number: 1R21NS056991-01
Grant Proposal PI: Nagi Ayad, TSRI
External Assay ID: Wee1_INH_LUMI_1536_%ACT
Name: Primary Cell-based High Throughput Screening Assay for Inhibitors of Wee1 Degradation
The Cdc2/cyclinB protein complex plays an important role in G2/M cell cycle progression. The nuclear tyrosine kinase Wee1 mediates inhibitory phosphorylation of Tyr15 on cdc2, leading to delayed mitosis and cell cycle arrest in cells with DNA damage so that DNA repair and replication can occur (1, 2). During mitosis Wee1 activity is inhibited by its phosphorylation (3, 4), accompanied by its ubiquitination by the E3 ligases SCFbeta-TrCP and SCF-Tome-1, and its subsequent degradation by the proteasome (3). The involvement of aberrant cell cycle control and unchecked DNA damage in cancer initiation and progression, and the finding of reduced Wee1 expression in colon carcinoma cells (5), suggest that Wee1 may act as a tumor suppressor. Cancer cells often respond to DNA-damaging treatments such as chemotherapy and radiation by implementing cell cycle arrest. These cells become resistant to apoptosis, and the above conventional therapies are rendered ineffective. Thus, the identification of probes that selectively increase levels of Wee1 may provide a potent route for cell cycle-based cancer therapy.
Compounds that inhibit Wee1 degradation will provide information about the role of Wee1 in mitotic entry and cell cycle progression. The hypothesis is based on studies which show that Wee1 stabilization inhibits mitotic entry (6). A review of currently published scientific manuscripts reveals only one small molecule reported to stabilize Wee1 in HeLa cells: the dihydropteridinone BI 2536 (7, 8). The mechanism of action is unknown and the compound is not commercially available. In contrast, several reports demonstrate that Wee1 inhibition by compounds that block the G2/M checkpoint improve the cytotoxic effects of DNA damaging agents on p53-negative cells (9-12). However, these compounds are dual Src/Wee1 kinase inhibitors and cannot elucidate the specific role of Wee1 in cell cycle-related tumorigenesis. The liabilities of these compounds necessitate the discovery of high affinity, selective probes for Wee1 stabilization.
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. 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.
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. Lenart P, Petronsczki M, Steegmaier M, Di Fiore B, Lipp JJ, Hoffman M, Rettig WJ, Kraut N, Peters JM. The small molecule inhibitor BI 2536 reveals novel insights into mitotic roles of polo-like kinase 1. Curr Biol.2007 Fev 20:17(4):304-15.
8. Steegmaier M, Hoffmann M, Baum A, Lenart P, Petronczki M, Krssak M, Gurtler U, Garin-Chesa P, Lieb S, Quant J, Grauert M, Adolf GR, Kraut N, Peters JM, Rettig WJ. BI 2536, a potent and selective inhibitor of polo-like kinase 1, inhibits tumor growth in vivo. Curr Biol. 2007 Feb 20;17(4):316-22.
9. Palmer BD, Smaill JB, Rewcastle GW, Dobrusin EM, Kraker A, Moore CW, Steinkampf RW, Denny WA. Structure-activity relationships for 2-anilino-6-phenylpyrido[2,3-d]pyrimidin-7(8H)-ones as inhibitors of the cellular checkpoint kinase Wee1. Bioorg Med Chem Lett. 2005 Apr 1;15(7):1931-5.
10. Wang Y, Li J, Booher RN, Kraker A, Lawrence T, Leopold WR, Sun Y. Radiosensitization of p53 Mutant cells by PD0166285, a novel G(2) Checkpoint Abrogator. Cancer Res. 2001;61:8211-8217.
11. Hashimoto O, Shinkawa M, Torimura T, Nakamura T, Selvendiran K, Sakamoto M, Koga H, Ueno T, Sata M. Cell cycle regulation by the Wee1 inhibitor PD0166285, pyrido [2,3-d] pyimidine, in the B16 mouse melanoma cell line. BMC Cancer. 2006 Dec 19;6:292.
12. Mizenina OA, Moasser MM. S-phase inhibition of cell cycle progression by a novel class of pyridopyrimidine tyrosine kinase inhibitors. Cell Cycle. 2004 Jun; 3(6):796-803.
Wee1, WEE1hu, FLJ16446, DKFZp686I18166, cell cycle, cancer, HeLa, degradation, inhibitor, primary, primary screen, luminescence, luciferase, 1536-well, HTS, High Throughput Screening, Scripps, Scripps Research Institute Molecular Screening Center, SRIMSC.
The purpose of this assay is to identify compounds that act as inhibitors of Wee1 degradation. 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.
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-neomycin antibiotic mixture (PSN) at 37 degrees C in an atmosphere of 5% CO2 and 95% relative humidity (RH). HeLa cells were transiently transfected in T75 flasks by mixing 6 million cells with 29 micrograms of the Luc::Wee1K328M plasmid complexed with 87 microliters 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 (final nominal 5 uM concentration) to sample wells, DMSO alone (0.5% final concentration) to Low Control wells, or the proteasome inhibitor MG132 (final nominal EC100 concentration of 30 uM, set as 100% activation) to High Control wells. The plates were then incubated for 20 hours at 37 degrees C (5% CO2, 95% RH). The assay was stopped by dispensing 5 ul of SteadyLite HTS luciferase substrate to each well, followed by incubation at room temperature for 15 minutes. 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.
A mathematical algorithm was used to determine nominally activating compounds in the primary screen. Two values were calculated: (1) the average percent activation of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % activation than the cutoff parameter was declared active.
The reported Pubchem_Activity_Score has been normalized to 100% of the highest observed activation. Negative % activation values were assigned an 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 MLSCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. In this case the results of each separate campaign were assigned "Active/Inactive" status based upon that campaign's specific compound activity cutoff value. 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 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 by the MLSMR.
The inactive compounds of this assay have activity score range of 0 to 6 and active compounds range of activity score is 6 to 100.
** Test Concentration.
Data Table (Concise)