Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of Wee1 degradation: Amplified proximity luminescence-based biochemical assay to identify inhibitors of residues 1-40 of amyloid beta
Name: Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of Wee1 degradation: Amplified proximity luminescence-based biochemical assay to identify inhibitors of residues 1-40 of amyloid beta. ..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: AMYLOID_INH_LUMI_0096_%INH MCSRUN ROUND 1 WEE1-CKD1
Name: Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of Wee1 degradation: Amplified proximity luminescence-based biochemical assay to identify inhibitors of residues 1-40 of amyloid beta.
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. Behrend, L., D.M. Milne, M. Stoter, W. Deppert, L.E. Campbell, D.W. Meek, and U. Knippschild, IC261, a specific inhibitor of the protein kinases casein kinase 1-delta and -epsilon, triggers the mitotic checkpoint and induces p53-dependent postmitotic effects. Oncogene, 2000. 19(47): p. 5303-13.
10. Rena, G., J. Bain, M. Elliott, and P. Cohen, D4476, a cell-permeant inhibitor of CK1, suppresses the site-specific phosphorylation and nuclear exclusion of FOXO1a. EMBO Rep, 2004. 5(1): p. 60-5.
11. Walton, K.M., K. Fisher, D. Rubitski, M. Marconi, Q.J. Meng, M. Sladek, J. Adams, M. Bass, R. Chandrasekaran, T. Butler, M. Griffor, F. Rajamohan, M. Serpa, Y. Chen, M. Claffey, M. Hastings, A. Loudon, E. Maywood, J. Ohren, A. Doran, and T.T. Wager, Selective inhibition of casein kinase 1 epsilon minimally alters circadian clock period. J Pharmacol Exp Ther, 2009. 330(2): p. 430-9.
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The purpose of this assay is to determine whether powder samples of probe candidates can lower amyloid beta 40 (and 42) production in an Alzheimer's model. The reference compounds in this study, D4476 and PF60462, are CK1d inhibitors already described in the literature (9-11). DAPT is a gamma-secretase inhibitor. The AlphaLISA assay (PerkinElmer, Waltham, Massachusetts, USA) was used. An Alpha Donor bead is coated with streptavidin which captures the biotinylated antibody. An Acceptor bead is coated with analyte-specific antibody. The beads are brought into proximity through binding to the analyte. When excited by laser at 680 nm, the Alpha Donor bead releases singlet oxygen which travels to the nearby Acceptor bead where it induces emission of light.
HEK-SW cells, which artificially overexpress mutated amyloid precursor protein (APP) (K670N/M671L), the so-called Swedish mutation, were cultured on D-MEM supplemented with 10% FBS. HEK-SW cells were kindly provided Dr. Dennis Selkoe. The HEK-SW cells were plated in 96 well plates (10,000 cells/well). At 24 hours post plating the media was replaced and treatments began. Individual wells (n=3) were treated with DMSO 0.1% or 1 uM of analogs FKL-135, FKL-159, D4476, PF-607462, DAPT. DAPT, N-[(3,5-difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester, is a known inhibitor of gamma-secretase purchased from TOCRIS Bioscience (Ellisville, Missouri, USA). Supernatants of cells were collected at 48 hours post plating and the concentration of Amyloid beta 1-40 (A beta 1-40) measured by AlphaLISA method (PerkinElmer, Waltham, Massachusetts, USA) following instructions from the company. Briefly, biotinylated anti-analyte antibody binds to the Streptavidin-coated Donor beads while another anti-analyte antibody is conjugated to AlphaLISA Acceptor beads. In the presence of the analyte, the beads come in to close proximity. The excitation of the Donor beads provokes the release of singlet oxygen molecules that triggers a cascade of energy transfer in the Acceptor beads resulting in a sharp peak of light emission.
PubChem Activity Outcome and Score:
Compounds that lead to a signal that is less than or equal to 80% of the DMSO wells are considered active. Compounds that lead to a signal that is greater than 80% of the DMSO wells are considered inactive.
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:
HEK-SW cells (provided by Dr. Dennis Selkoe)
AlphaLISA kit (PerkinElmer, Waltham, Massachusetts, USA)
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 filter binding, well luminescence, or kinase activity. 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
Data Table (Concise)