Confirmation biochemical high-throughput screening assay for 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 ..
BioActive Compounds: 333
Depositor Specified Assays
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center
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_Lumi_1536_3X%INH
Name: Confirmation biochemical high-throughput screening assay for 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 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 (8-9), 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.
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.
p97, ATPase, valosin-containing protein, VCP, cancer, neurodegenerative disease, confirmation, screen, HTS, high throughput screen, 1536, inhibitor, luciferase, Scripps, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
The purpose of this assay is to confirm the activity of test compounds identified as active in a previous set of experiments entitled, "Primary biochemical high-throughput screening assay to measure p97 ATPase inhibition" (PubChem AID 1481). This biochemical assay employs the Kinase-Glo reagent, which contains a luciferase that emits luminescence in direct proportion to ATP levels. As designed, compounds that inhibit the ATPase activity of p97 will reduce ATP hydrolysis, thereby increasing the relative levels of ATP available for consumption by luciferase, resulting in increased well luminescence. Compounds were tested in triplicate at a nominal concentration of 8 micromolar.
Prior to the start of the assay 2.5 ul of Assay Buffer (1 mM DTT, 50 mM Tris HCl, 20 mM MgCl2, 1 mM EDTA, pH 8.0, filtered at 0.22 um) were dispensed into columns 1 and 2 of 1536-well assay plates. The remaining 46 columns were filled with 2.5 ul of Assay Buffer supplemented with 0.42 uM p97 protein. Next, 40 nL of test compounds (8 uM final nominal concentration) or DMSO alone (0.8% final concentration) were distributed into the appropriate wells. The plates were then incubated for 30 minutes at 25 degrees Celsius. The assay was started by the addition of 2.5 ul of 10 mM Tris supplemented with 10 uM ATP to all wells. The plates were then incubated for 3 hours at 25 degrees Celsius. After incubation, 5 ul of Kinase Glo reagent were added to all 48 columns and plates were incubated for another 10 minutes at 25 degrees Celsius. Plates were centrifuged and luminescence was measured by the ViewLux microplate reader.
The percent inhibition for each compound was calculated using the following mathematical expression:
% Inhibition = [1-(Test_Compound - Median_High_Control) / (Median_Low_Control - Median_High_Control)]*100
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.
A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Two values were calculated: (1) the average percent inhibition of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition than the cutoff parameter was declared active.
The inactive compounds of this assay have activity score range of 0 to 19 and active compounds range of activity score is 19 to 100.
The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.
List of reagents:
Recombinant p97 protein (produced in the Deshaies Laboratory)
Magnesium Chloride (Fisher, part M33-500)
1M Tris, pH 8.0 (Invitrogen, part T-3038)
0.5M EDTA (Invitrogen, part 15575-025)
Dithiothreitol (Fisher, part BP172-25)
3N Sodium Acetate pH 5.2 (Sigma, part S7899)
ATP (Sigma, part A7699-1G)
Kinase-Glo (Promega, part K1214)
Methanol (Fisher, part A412-4)
DMSO (Acros Organics, part 127790025)
1536-well plates (Greiner, part 789072)
Due to the increasing size of the MLPCN compound library, 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: dust or lint located in or on wells of the microtiter plate, compounds that non-specifically modulate ATP hydrolysis, and compounds that quench or emit luminescence within the well. All test compound concentrations reported are nominal; the specific concentration for a particular test compound may vary based upon the actual sample provided by the MLSMR.
** Test Concentration.
Data Table (Concise)