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: P97C522A_INH_LUMI_384_IC50 CS

Name: Luminescence counterscreen assay for p97 inhibitors: Dose response biochemical high throughput screening assay to identify inhibitors of the C522A mutant 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 (2-4). 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.

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:

p97, ATPase, valosin-containing protein, VCP, cancer, neurodegenerative disease, mutant, C522A, dose response, counterscreen, 384, inhibitor, luciferase, Scripps, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.

Assay Overview:

The purpose of this assay is to determine whether 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), and that confirmed activity in a subsequent set of experiments entitled, "Confirmation biochemical high-throughput screening assay for inhibitors of the p97 ATPase" (PubChem AID 1517) act as covalent or non-covalent p97 inhibitors. This biochemical assay employs a mutant Cys522Ala p97 protein as the target. The assay uses 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 p97Cys522Ala 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 using a 10-point, 1:3 dilution series, starting at a nominal concentration of 50 uM.

Protocol Summary:

Prior to the start of the assay 18 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, rows O and P of 384-well assay plates. The remaining wells were filled with 18 ul of Assay Buffer supplemented with 0.42 uM C522A p97 protein. Next, 50 nL of test compounds or DMSO alone (0.8% final concentration) were distributed into the appropriate wells. The plates were then incubated for 15 minutes at 25 degrees Celsius. The assay was started by the addition of 1 ul of 10 mM Tris supplemented with 100 uM ATP to all wells. The plates were then incubated for 1 hour at 25 degrees Celsius. After incubation, 20 ul of Kinase Glo reagent were added to all 24 columns and plates were incubated for another 10 minutes at 25 degrees Celsius. Plates were centrifuged and luminescence was measured by the Envision 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
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.

For each test compound, percent inhibition was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (MDL Information Systems). The reported IC50 values were generated from fitted curves by solving for the X-intercept value at the 50% inhibition level of the Y-intercept value. In cases where the highest concentration tested (i.e. 50 uM) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 50 uM. Compounds with an IC50 greater than 10 uM were considered inactive. Compounds with an IC50 equal to or less than 10 micromolar were considered active.

Any compound with a percent inhibition value <50% at all test concentrations was assigned an activity score of zero. Any compound with a percent inhibition value >50% at any test concentration was assigned an activity score greater than zero. Activity score was then ranked by the potency, with the most potent compounds assigned the highest activity scores.

Active compound of this assay has the activity score of 100 and inactive compounds have range of activity score from 0 to 78.

List of Reagents:
Recombinant C522A 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)
384-well Plates (Corning, part 3704)

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.

Grant Number: 1 R03 MH085687-01