Inhibition of proteasomal turnover of a p97-dependent reporter substrate in human cells - Hit Optimization Round 3
Assay Purpose: Determine whether inhibitors obtained from the p97 ATPase Dose Response (KUA10001) are able to block turnover of a p97-dependent proteasome reporter substrate in cells. ..more
BioActive Compounds: 39
Depositor Specified Assays
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 whether inhibitors obtained from the p97 ATPase Dose Response (KUA10001) are able to block turnover of a p97-dependent proteasome reporter substrate in cells.
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, 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
Trypsinize UbG76V-GFP/HeLa cells and aliquot 5000 cells into each well of a 96-well plate and grow cells for ~16h. Ub-G76V-GFP /HeLa cells are treated with DMEM containing MG132 (4 uM) for 1h and washed with 100uL PBS twice. DMEM containing 2.5% FBS, cycloheximide (50 ug/mL) and the test compound is added into the well. Five 96-well plates are prepared and one of the plates is imaged on ImageXpress Micro at each time point (70, 90, 110, 130, 150, or 170 min) after washing with 100uL PBS. Four GFP images with 100ms exposure time per well are acquired and average GFP intensity per area of a HeLa cell is determined by using MetaXpress software. Mean GFP intensity of ~500 cells is calculated using Excel. Normalized GFP intensity is calculated using the following formula: (Test compound - Background)/(Basal GFP intensity - Background)
Test compound is defined as Mean GFP intensity of UbG76V-GFP /HeLa cells treated with the test compound
Background is defined as background GFP intensity of HeLa cells, which do not expressed Ub-G76V-GFP
Basal GFP intensity is defined as mean GFP intensity of UbG76V-GFP /HeLa cells treated with DMSO
The degradation rate constant (k) was obtained from the slope of plotting Ln(Normalized GFP intensity) versus time ranging from 90 to 170 min. The percent of remaining k for each compound is calculated using the following formula:
(Test compound/ DMSO control) * 100
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.
The method of converting the IC50 data to PubChem Activity scores and what activity constitutes an "active" should also be included in the protocol section.
Compounds with an IC50 equal to or less than 10 microM were considered active. This corresponds to activity scores greater than 19.9.
Activity score was ranked by normalizing IC50 values so that 250 nM has an activity score of 100, 2.5 microM has an activity score of 10, and 25 microM has an activity score of 0.
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, ubiquitin-fusion degradation (UFD), green fluorescent protein, proteasome
* Activity Concentration. ** Test Concentration.
Data Table (Concise)