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BioAssay: AID 504929

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
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 Tested Compounds
 Tested Compounds
All(2)
 
 
Probe(1)
 
 
Active(2)
 
 
 Tested Substances
 Tested Substances
All(2)
 
 
Probe(1)
 
 
Active(2)
 
 
AID: 504929
Data Source: The Scripps Research Institute Molecular Screening Center (AMYLOID_INH_LUMI_0096_%INH MCSRUN ROUND 1 WEE1-CKD1)
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
Deposit Date: 2011-07-07
Hold-until Date: 2012-07-07
Modify Date: 2012-07-11

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: Chemical Probe: 1    Active: 2
Related Experiments
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AIDNameTypeProbeComment
1321Primary Cell-based High Throughput Screening Assay for Inhibitors of Wee1 DegradationScreening depositor-specified cross reference: Primary screen (WEE1 degradation inhibitors in singlicate)
1410Confirmation cell-based high throughput screening assay for inhibitors of Wee1 degradationScreening depositor-specified cross reference: Confirmation screen (WEE1 degradation inhibitors in triplicate)
1412Dose Response Cell-based Assay for Inhibitors of Wee1 DegradationConfirmatory depositor-specified cross reference: Dose response (WEE1 degradation inhibitors in singlicate)
1413Cytotoxicity counterscreen assay for inhibitors of Wee1 degradationConfirmatory depositor-specified cross reference: Cytotoxicity counterscreen (WEE1 degradation inhibitors in triplicate)
1414Counterscreen assay for inhibitors of Wee1 degradation: dose response cell-based assay to identify inhibitors of cyclin B degradationConfirmatory depositor-specified cross reference: Dose response counterscreen (cyclin B degradation inhibitors in triplicate)
1807Summary of probe development efforts to identify inhibitors of Wee1 degradationSummary1 depositor-specified cross reference: Summary (WEE1 degradation inhibitors)
2088Late stage results from the probe development effort to identify inhibitors of Wee1 degradation.Screening depositor-specified cross reference: Late stage results (WEE1 degradation inhibitors in triplicate)
434972Late stage results from the probe development effort to identify inhibitors of Wee1 degradation: luminescence-based cell-based assay to determine inhibition of Wee1 degradation by kinase inhibitorsOther depositor-specified cross reference: Late stage screen (WEE1 degradation inhibitors in quadruplicate)
463076Late stage assay provider results from the probe development effort to identify inhibitors of casein kinase 1 delta (CK1d): radioactivity-based in vitro biochemical kinase assay for inhibitors of fms-related tyrosine kinase 3 (FLT3)Confirmatory depositor-specified cross reference: Late stage counterscreen (FLT3 inhibitors)
463077Late stage assay provider results from the probe development effort to identify inhibitors of casein kinase 1 delta (CK1d): radioactivity-based in vitro biochemical kinase assay to identify CK1d inhibitorsConfirmatory depositor-specified cross reference: Late stage dose response counterscreen (CK1d inhibitors)
463080Late stage assay provider results from the probe development effort to identify inhibitors of WEE1 degradation: luminescence-based dose response assay to identify stabilizers of WEE1Confirmatory depositor-specified cross reference: Late stage dose response (Wee1 stabilizers in quadruplicate)
463169Late stage assay provider results from the probe development effort to identify inhibitors of Wee1 degradation: luminescence-based cell-based assay to identify inhibitors of Wee1 degradationScreening depositor-specified cross reference: Late stage assay provider results (WEE1 degradation inhibitors in quadruplicate)
463170Late 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 p21 (CDKN1A) degradationScreening depositor-specified cross reference: Late stage counterscreen (p21 degradation inhibitors in quadruplicate)
463171Late 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) degradationOther depositor-specified cross reference: Late stage counterscreen (p27 degradation inhibitors in quadruplicate)
463177Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of Wee1 degradation: fluorescence activated cell sorting (FACS)-based cell-based assay to identify inducers of Hela cell apoptosisOther depositor-specified cross reference: Late stage counterscreen (Hela cell apoptosis inducers in quadruplicate)
463186Late 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 cyclin B degradationOther depositor-specified cross reference: Late stage counterscreen (cyclin B degradation inhibitors in quadruplicate)
504930Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of Wee1 degradation: radioactivity-based cell-based assay to identify inhibitors of granule cell progenitor (GCP) proliferationConfirmatory1 same project related to Summary assay
504935Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of Wee1 degradation: luminescence-based cell-based dose response assay for cytotoxic compounds using neuroblastoma cell lineOther1 same project related to Summary assay
Description:
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.

Description:

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.

References:

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.

Keywords:

Late stage, late stage AID, powders, SAR, purchased, synthesized, Wee1, WEE1hu, FLJ16446, DKFZp686I18166, luminescence, proximity, AlphaLISA, HEK-SW, amyloid precursor protein, amyloid beta protein, amyloid beta 1-40, cell cycle, cancer, biochemical, degradation, inhibitor, inhibition, dose response, 96, assay, assay provider, Scripps, Scripps Florida, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
Protocol
Assay Overview:
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.
Protocol Summary:
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)
Comment
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
From PubChem:
Assay Format: Biochemical
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1SignalThe percentage of luminescence energy signal released from the well, normalized to the signal of control wells treated with DMSO alone.Float%
Additional Information
Grant Number: 1R21NS056991-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
Classification
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