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BioAssay: AID 463184

p97 ATPase dose response

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 more ..
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 Tested Compounds
 Tested Compounds
All(29)
 
 
Active(20)
 
 
Inactive(9)
 
 
 Tested Substances
 Tested Substances
All(29)
 
 
Active(20)
 
 
Inactive(9)
 
 
AID: 463184
Data Source: Molecular Libraries Program, Specialized Chemistry Center, University of Kansas (KUA10001)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
Deposit Date: 2010-08-31
Hold-until Date: 2011-08-26
Modify Date: 2011-08-31

Data Table ( Complete ):           Active    All
Target
Sequence: Valosin-containing protein [Homo sapiens]
Description ..   
Protein Family: AAA

Gene:VCP     Related Protein 3D Structures     More BioActivity Data..
BioActive Compounds: 20
Depositor Specified Assays
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AIDNameTypeProbeComment
1794Summary of probe development efforts to identify inhibitors of the p97 ATPase.summary1
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
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
504649Inhibition of proteasomal turnover of a p97-dependent reporter substrate in human cells - Hit Optimization Round 3confirmatory
504654Inhibition of p97-independent reporter turnoverconfirmatory
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: Determine inhibition of purified p97 by small molecules for SAR study.

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.
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., Uruno, T., Hoshida, Y., Qiu, Y., Iizuka, N., Nakamichi, I., Miyauchi, A., and Aozasa, K., Expression level of valosin-containing protein (p97) is associated with prognosis of esophageal carcinoma. Clin. Cancer Res., 2004. 10(16): p. 5558-5565. PMID: 15328197."
Protocol
Dispense 20 uL of 2.5x Assay Buffer (50 mM Tris pH 7.4, 20 mM MgCl2, 1 mM EDTA) into each well of 96 well plate. Dilute enzyme (100 uL of 5 mg/mL) in 900 uL 1x Assay Buffer. Dispense 10 uL to each well. Add 10 uL compounds or DMSO control and incubate at room temperature for 10 min. Add 10 uL of 500uM ATP (pH 7.5) and incubate at 37 oC for 30 min then room temperature for 10 min. Add 50 uL Kinase Glo Plus reagent to each well and incubate at room temperture for 10 min in dark. Luminescence is read on Analyst AD (Molecular
Device).

The percent of remaining activity for each compound was calculated using the
following mathematical expression : ((Test Compound-High Control)/(Low Control-High Control)) * 100

Where:
Test_Compound is defined as wells containing test compound,
Low_Control is defined as wells containing DMSO,
High_Control is defined as wells containing no p97 protein.

IC50 values were calculated from fitting the percentage of remaining activity (%RA) with various concentrations of compounds to a Langmuir equation [%RA =100/(1 + [Compound]/IC50)] by on-linear regression analysis using JUMP IN program. The result was expressed as mean standard error."

Compounds with an IC50 equal to or less than 10 microM were considered active.

Activity score was ranked by normalizing to the lowest IC50 compounds (activity score = 100). Activity score for the active compounds of this assay can range from 0 to 100 and all inactive compounds are assigned activity score of zero.
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, The University of Kansas Specialized Chemistry Center, Raymond Deshaies, California Institute of Technology
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 observed. Compounds with an IC50 equal to or less than 10 microM were considered active.FloatμM
2Std. Err. (IC50)Standard error of IC50 valueFloat
3Inhibition at 0.12 microM (0.12μM**)Value of %inhibition at 0.12 micromolar inhibitor concentration; average of triplicate measurement.Float%
4Inhibition at 0.37 microM (0.37μM**)Value of %inhibition at 0.37 micromolar inhibitor concentration; average of triplicate measurement.Float%
5Inhibition at 1.1 microM (1.1μM**)Value of %inhibition at 1.1 micromolar inhibitor concentration; average of triplicate measurement.Float%
6Inhibition at 3.3 microM (3.3μM**)Value of %inhibition at 3.3 micromolar inhibitor concentration; average of triplicate measurement.Float%
7Inhibition at 10 microM (10μM**)Value of %inhibition at 10 micromolar inhibitor concentration; average of triplicate measurement.Float%
8Inhibition at 30 microM (30μM**)Value of %inhibition at 30 micromolar inhibitor concentration; average of triplicate measurement.Float%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1RO3MH085687-01

Data Table (Concise)
Classification
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