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BioAssay: AID 504654

Inhibition of p97-independent reporter turnover

Assay Purpose: Inhibition of ODD-luciferase turnover is a counterscreen for AID463185, and AID488830 to eliminate non-specific p97 inhibitors in cells. The reporter is assayed by employing the same approach as for Ub-GFP (AID463185, and AID488830) We evaluate the degradation rate constant for ODD-luciferase disappearance in the absence or presence of various concentrations of test compound. From these values, we determine an IC50. For a compound to be considered a specific probe, it should inhibit with an IC50 of >/=20 microM. ..more
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 Tested Compounds
 Tested Compounds
All(21)
 
 
Active(12)
 
 
Inactive(9)
 
 
 Tested Substances
 Tested Substances
All(21)
 
 
Active(12)
 
 
Inactive(9)
 
 
AID: 504654
Data Source: Molecular Libraries Program, Specialized Chemistry Center, University of Kansas (KUA10010)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
Deposit Date: 2011-04-12
Hold-until Date: 2012-04-08
Modify Date: 2012-04-09

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 12
Depositor Specified Assays
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AIDNameTypeProbeComment
1794Summary of probe development efforts to identify inhibitors of the p97 ATPase.summary1 Screening assay
1481Primary biochemical high-throughput screening assay to measure P97 ATPase inhibitionscreening
1517Confirmation biochemical high-throughput screening assay for inhibitors of the p97 ATPasescreening
1534Dose response biochemical high throughput screening assay for inhibitors of the p97 ATPaseconfirmatory
1544Luminescence counterscreen assay for p97 inhibitors: Dose response biochemical high throughput screening assay to identify inhibitors of the C522A mutant p97 ATPase.confirmatory
1551Luminescence dose response biochemical high throughput screening assay for inhibitors of the p97 ATPase: synthesized compounds.confirmatory Confirmatory assay
1629Luminescence counterscreen assay for p97 inhibitors: dose response biochemical high throughput screening assay to identify inhibitors of the C522A mutant p97 ATPase: synthesized compounds.confirmatory
463185Inhibition of proteasomal turnover of a p97-dependent reporter substrate in human cellsconfirmatory
488830Inhibition of proteasomal turnover of a p97-dependent reporter substrate in human cells - Hit Optimization Round 2confirmatory
463184p97 ATPase dose responseconfirmatory
488828p97 ATPase dose response - Hit Optimization Round 2confirmatory
Description:
Assay Provider: Raymond Deshaies, California Institute of Technology
Assay Performer: Tsui-Fen Chou, California Institute of Technology
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R03 MH085687-01
Grant Proposal PI: Raymond Deshaies, California Institute of Technology
Assay Purpose: Inhibition of ODD-luciferase turnover is a counterscreen for AID463185, and AID488830 to eliminate non-specific p97 inhibitors in cells. The reporter is assayed by employing the same approach as for Ub-GFP (AID463185, and AID488830) We evaluate the degradation rate constant for ODD-luciferase disappearance in the absence or presence of various concentrations of test compound. From these values, we determine an IC50. For a compound to be considered a specific probe, it should inhibit with an IC50 of >/=20 microM.
Description: Misfolded proteins accumulate in the endoplasmic reticulum (ER) in response to environmental stress (1). To reduce the burden these proteins place on the secretory pathway, eukaryotic cells have evolved a process, known as ER-associated degradation (ERAD), to recognize and eliminate these proteins (1, 2). The highly conserved p97 ATPase functions in ERAD by hydrolyzing ATP needed to export ubiquitinated substrates to the cytosol for degradation by the proteasome (2, 3). The discovery of p97 missense mutations in a genetic form of human dementia (5-7), the localization of p97 in ubiquitylated inclusions in affected neurons of amyotrophic lateral sclerosis (ALS) and Parkinson's disease (8), and the overproduction of p97 in multiple cancers (10-14), suggests that p97 has diverse and essential cellular roles. Thus, the identification of probes that selectively target p97 activity may provide insights into the biological roles of p97. ODD-luciferase is derived from the oxygen-dependent degradation domain of HIF1 fused to luciferase (15) and is shown to be a p97-independent reporter that can be used as a counterscreen for developing specific p97 inhibitors (16).
1. Raasi S, Wolf DH. Ubiquitin receptors and ERAD: a network of pathways to the proteasome. Semin Cell Dev Biol. 2007 Dec;18(6):780-91. PMID: 17942349.
2. Halawani D, Latterich M. p97: The cell's molecular purgatory? Mol Cell. 2006 (22)6: 713-717. PMID: 16793541.
3. Wang Q, Li L, Ye Y. Inhibition of p97-dependent protein degradation by Eeyarestatin I. J Biol Chem. 2008 Mar 21;283(12):7445-54. PMID: 18199748.
4. Ye Y., Meyer H., Rapoport, T. A. The AAA ATPase Cdc48/p97 and its partners transport proteins from the ER to the cytosol. Nature. 2001. 414(6864): p. 52-656. PMID: 11740563.
5. Watts, G.D., J. Wymer, M.J. Kovach, S.G. Mehta, S. Mumm, D. Darvish, A. Pestronk, M.P. Whyte, and V.E. Kimonis, Inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia is caused by mutant valosin-containing protein. Nat Genet. 2004. 36(4): p. 377-81. PMID: 15034582.
6. Weihl, C.C., Dalal, S., Pestronk, A., and Hanson, P. I., Inclusion body myopathy-associated mutations in p97/VCP impair endoplasmic reticulum-associated degradation. Hum. Mol. Genet. 2006. 15(2): p. 189- 199. PMID: 16321991.
7. Mizuno, Y., Hori, S., Kakizuka, A. and Okamoto, K. (2003) Vacuole-creating protein in neurodegenerative diseases in humans. Neurosci. Lett. 343, 77-80. PMID: 12759168.
8. Ishigaki S, Hishikawa N, Niwa J, Iemura S, Natsume T, Hori S, Kakizuka A, Tanaka K, Sobue G. (2004) Physical and functional interaction between Dorfin and Valosin-containing protein that are colocalized in ubiquitylated inclusions in neurodegenerative disorders. J Biol Chem. 2004 Dec 3;279(49):51376-85. PMID: 15456787.
9. Hirabayashi, M., Inoue, K., Tanaka, K., Nakadate, K., Ohsawa, Y., Kamei, Y., Popiel, A.H., Sinohara, A., Iwamatsu, A., Kimura, Y. Uchiyama, Y.,Hori, S., Kakizuka, A. VCP/p97 in abnormal protein aggregates, cytoplasmic vacuoles, and cell death, phenotypes relevant to neurodegeneration. Cell Death
Differ. 2001, 8, 977-984. PMID: 11598795.
10. Mitas, M., Mikhitarian, K., Walters, C., Baron, P. L., Elliott, B. M., Brothers, T. E., Robison, J. G., Metcalf, J. S., Palesch, Y. Y., Zhang, Z., Gillanders, W. E., and Cole, D. J., Quantitative real-time RT-PCR detection of breast cancer micrometastasis using a multigene marker panel. Int. J. Cancer. 2001. 93(2): p. 162-171. PMID: 11410861.
11. Marchetti, A., Buttitta, F., Bertacca, G., Zavaglia, K., Bevilacqua, G., Angelucci, D., Viacava, P., Naccarato, A., Bonadio, A., Barassi, F., Felicioni, L., Salvatore, S., Mucilli, F., mRNA markers of breast cancer nodal metastases: comparison between mammaglobin and carcinoembryonic antigen in 248 patients. J. Pathol. 2001. 195(2): p. 186-190. PMID: 11592097.
12. Smith, L.M., Nesterova, A., Alley, S. C., Torgov, M. Y., Carter, P. J., Potent cytotoxicity of an auristatin-containing antibody-drug conjugate targeting melanoma cells expressing melanotransferrin/p97. Mol. Cancer Ther, 2006. 5(6): p.1474-1482. PMID: 16818506.
13. Yamamoto, S., Tomita, Y., Uruno, T., Hoshida, Y., Qiu, Y., Iizuka, N., Nakamichi, I., Miyauchi, A., and Aozasa, K., Increased expression of valosin-containing protein (p97) is correlated with disease recurrence in follicular thyroid cancer. Ann. Surg. Oncol., 2005. 12(11): p. 925-934. PMID: 16189643.
14. Yamamoto S, Tomita Y, Hoshida Y, Iizuka N, Kidogami S, Miyata H, Takiguchi S, Fujiwara Y, Yasuda T, Yano M, Nakamori S, Sakon M, Monden M, Aozasa K. Clin Cancer Res. 2004 Aug 15;10(16):5558-65. PMID: 15328197 [PubMed - indexed for MEDLINE] Free Article"
15. Kimbrel EA, Davis, T.N., Bradner, J.E., and Kung, A.L. (2009) In Vivo Pharmacodynamic Imaging of Proteasome Inhibition. Molecular Imaging 8:141-147. PMID: 19723471
16. Chou T-F, Deshaies, R. J. (2011) Quantitative cell-based proteindegradation assays to identify and classify drugs that target the ubiquitin-proteasome system. J. Biol. Chem. Feb 22. [Epub ahead of print] PMID: 21343295.
Protocol
HeLa cells stably expressing ODD-luciferase were seeded onto a 96-well white solid bottom (5000cells/well) and grow cells for 16h. Cells are treated with DMEM containing MG132 (4 microM) for 1h and washed with 100 microL PBS twice. DMEM containing 2.5% FBS, cycloheximide (50 microg/mL) and the test compound is added into the well. Four 96-well plates are prepared and one of the plates is taken out from incubator at each time point (70, 90, 120, or 150 min). Luciferin (50 microL of 1 mg/mL in PBS) was added into each well containing 50 microL of medium and incubated at room temperature with shaking at 500 rpm for 5 min. Luminescence intensity was determined with 0.1 ms integration time on the Synergy HT Microplate Reader (BioTek).
Normalized Luciferase intensity is calculated using the
following formula:
(Test compound - Background)/(Basal intensity - Background)
Where:
Test compound is defined as luciferase intensity of cells treated with the test compound. Background is defined as background intensity of HeLa cells, which do not expressed ODD-luciferase.
Basal intensity is defined as luciferase intensity of cells treated with DMSO
The degradation rate constant (k) was obtained from the slope of plotting Ln
(Normalized intensity) versus time ranging from 90 to 150 min. The percent
of remaining k for each compound is calculated using the following formula:
(Test compound/ DMSO control) * 100
Where:
Test_compound is defined as k determined from wells containing test compound, DMSO control is defined as k determined from wells containing DMSO.
IC50 values were calculated from fitting the percentage of remaining k (%k) with various concentrations of compounds to a Langmuir equation [%k =100/(1 + [Compound]/IC50)] by non-linear regression analysis using JUMP IN program.
The result was expressed a mean +/- standard error.
IC50 of >/=20 microM will be considered active in this assay.

Compounds with an IC50 equal to or greater than 20 microM were considered active. Activity scores were calculated by normalizing data with highest IC50 having the value of 100 and the lowest IC50 having 0. The "actives" have activity scores >32.
Comment
p97, AAA ATPase, valosin-containing protein, VCP, cancer, neurodegenerative disease, inclusion body myopathy associated with Paget disease of the bone and frontotemporal
dementia (IBMPFD), dose response, inhibitor, luciferase
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1IC50*The concentration at which 50 percent of the activity in the inhibitor assay is observedFloatμM
2Std. Err. (IC50)Standard error of IC50 valueFloatμM
3IC50 inequality descriptorString
4Inhibition at 0.625 microM (0.625μM**)Value of %inhibition at 0.625 micromolar inhibitor concentration; average of triplicate measurement.Float%
5Inhibition at 1.25 microM (1.25μM**)Value of %inhibition at 1.25 micromolar inhibitor concentration; average of triplicate measurement.Float%
6Inhibition at 2.5 microM (2.5μM**)Value of %inhibition at 2.5 micromolar inhibitor concentration; average of triplicate measurement.Float%
7Inhibition at 5 microM (5μM**)Value of %inhibition at 5 micromolar inhibitor concentration; average of triplicate measurement.Float%
8Inhibition at 10 microM (10μM**)Value of %inhibition at 10 micromolar inhibitor concentration; average of triplicate measurement.Float%
9Inhibition at 20 microM (20μM**)Value of %inhibition at 20 micromolar inhibitor concentration; average of triplicate measurement.Float%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 MH085687-01

Data Table (Concise)
Classification
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