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BioAssay: AID 1794

Summary of probe development efforts to identify inhibitors of the p97 ATPase.

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 more ..
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AID: 1794
Data Source: The Scripps Research Institute Molecular Screening Center (P97_INH_LEADS_SUMMARY)
BioAssay Type: Summary, Candidate Probes/Leads with Supporting Evidence
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-06-01
Modify Date: 2011-04-15

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compound: Chemical Probe: 1    Active: 1
Related Experiments
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AIDNameTypeComment
1481Primary biochemical high-throughput screening assay to measure P97 ATPase inhibitionScreeningdepositor-specified cross reference: Primary HTS in singlicate to identify P97 inhibitors.
1517Confirmation biochemical high-throughput screening assay for inhibitors of the p97 ATPaseScreeningdepositor-specified cross reference: Confirmation of hit activity in triplicate.
1534Dose response biochemical high throughput screening assay for inhibitors of the p97 ATPaseConfirmatorydepositor-specified cross reference: Titration assays in triplicate to determine potency.
1544Luminescence counterscreen assay for p97 inhibitors: Dose response biochemical high throughput screening assay to identify inhibitors of the C522A mutant p97 ATPase.Confirmatorydepositor-specified cross reference: Titration assays in triplicate to determine selectivity.
1551Luminescence dose response biochemical high throughput screening assay for inhibitors of the p97 ATPase: synthesized compounds.Confirmatorydepositor-specified cross reference: Titration assays in triplicate to determine potency.
1629Luminescence counterscreen assay for p97 inhibitors: dose response biochemical high throughput screening assay to identify inhibitors of the C522A mutant p97 ATPase: synthesized compounds.Confirmatorydepositor-specified cross reference: Titration assays in triplicate to determine selectivity.
2131Late stage results from the probe development effort to identify inhibitors of the p97 ATPase.Screeningdepositor-specified cross reference: Late stage results (p97 ATPase inhibitors in triplicate)
463184p97 ATPase dose responseConfirmatorydepositor-specified cross reference: Dose response (p97 ATPase)
463185Inhibition of proteasomal turnover of a p97-dependent reporter substrate in human cellsConfirmatorydepositor-specified cross reference: Inhibition of proteasomal turnover of a p97-dependent reporter substrate in human cells
488828p97 ATPase dose response - Hit Optimization Round 2Confirmatorydepositor-specified cross reference: p97 ATPase dose response - Hit Optimization Round 2
488830Inhibition of proteasomal turnover of a p97-dependent reporter substrate in human cells - Hit Optimization Round 2Confirmatorydepositor-specified cross reference: Inhibition of proteasomal turnover of a p97-dependent reporter substrate in human cells - Hit Optimi
504649Inhibition of proteasomal turnover of a p97-dependent reporter substrate in human cells - Hit Optimization Round 3Confirmatorydepositor-specified cross reference: Inhibition of proteasomal turnover of a p97-dependent reporter substrate in human cells
504650p97 ATPase dose response - Hit Optimization Round 3Confirmatorydepositor-specified cross reference: p97 ATPase dose response
504653Caspase 3/7 Activation in response to p97 inhibitionOtherdepositor-specified cross reference: Caspase 3/7 Activation in response to p97 inhibition
504654Inhibition of p97-independent reporter turnoverConfirmatorydepositor-specified cross reference: Inhibition of p97-independent reporter turnover
Description:
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: Raymond Deshaies, 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
External Assay ID: P97_INH_LEADS_SUMMARY

Name: Summary of probe development efforts to identify inhibitors of the p97 ATPase.

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.

Summary of Probe Development Effort:

Following primary HTS in singlicate to identify P97 inhibitors (AID 1481), confirmation of hit activity in triplicate (AID 1517), titration assays in triplicate to determine potency (AID 1534) and titration assays in triplicate against mutant C522A P97 to determine whether compounds act as covalent or non-covalent P97 inhibitors (AID 1544), certain compounds were identified as possible candidates for probe development. Additional biochemical assays of 16 synthesized compounds to explore SAR of SID 14723044 were pursued against P97 (AID 1551) and mutant C522A P97 (AID 1629) to determine potency and selectivity, respectively. These efforts resulted in the identification of a 2-amino thiazole P97 inhibitor probe (SID 56432669) with an IC50 value of 4.5 muM. A series of biological assays and chemistry efforts revealed that this probe is a cell active, reversible inhibitor of P97.

One probe report has been submitted describing compound screening and analog syntheses. Details of protocols, compound structures, and results from the original assays can be found in PubChem at the respective AIDs listed below. The results of our probe development efforts can be found at http://mlpcn.florida.scripps.edu/index.php/probes/probe-reports.html. Probes were identified and this project is now closed. A probe report for SID 56432669 can be found in the Molecular Libraries Bookshelf (PubMed Books) (http://www.ncbi.nlm.nih.gov/books) under ML080.

References:

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.
2. Halawani D, Latterich M. p97: The cell's molecular purgatory? Mol Cell. 2006 (22)6: 713-717.
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.
4. Ye, Y., Meyer, H. H., and Rapoport, T. A., The AAA ATPase Cdc48/p97 and its partners transport proteins from the ER into the cytosol. Nature. 2001. 414(6864): p. 52-656.
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.
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.
7. Mizuno, Y., Hori, S., Kakizuka, A. and Okamoto, K. (2003) Vacuole-creating protein in neurodegenerative diseases in humans. Neurosci. Lett. 343, 77-80.
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.
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.
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.
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.
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.
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.
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.

Keywords:

Summary AID, probe, probe report, late stage, p97, ML080, ATPase, valosin-containing protein, VCP, cancer, neurodegenerative disease, confirmation, mutant C522A, counterscreen, dose response, HTS, high throughput screen, 1536, inhibitor, inhibition, luciferase, Scripps, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Bookshelf, Molecular Libraries Probe Production Centers Network, MLPCN
Comment
A probe was identified.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1ML #Unique alphanumeric identifier assigned to a chemical probe molecule within the Molecular Libraries Probe Production Centers Network (MLPCN).String
Additional Information
Grant Number: 1 R03 MH085687-01

Data Table (Concise)
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