Caspase 3/7 Activation in response to p97 inhibition
Assay Purpose: To evaluate the ability of specific p97 inhibitors to induce apoptosis in cells by monitoring activation of caspase 3/7 with Caspase 3/7 Glo kit from Promega after a 7 hour incubation. This assay will classify p97 inhibitors into either caspase-activating compounds, which maybe useful for developing anti-cancer agents, or non-activating compounds, which may have other therapeutic potentials. For a compound to be considered as a caspase-activating agent, it should activate caspase 3/7 more than 5 fold at a concentration less than 20 microM after 7 h treatment. ..more
BioActive Compounds: 5
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: To evaluate the ability of specific p97 inhibitors to induce apoptosis in cells by monitoring activation of caspase 3/7 with Caspase 3/7 Glo kit from Promega after a 7 hour incubation. This assay will classify p97 inhibitors into either caspase-activating compounds, which maybe useful for developing anti-cancer agents, or non-activating compounds, which may have other therapeutic potentials. For a compound to be considered as a caspase-activating agent, it should activate caspase 3/7 more than 5 fold at a concentration less than 20 microM after 7 h treatment.
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. Given that the existing p97 inhibitor, DBeQ (CID676352), rapidly induced caspase 3/7 (15) in cells, specific p97 inhibitor as determined by not inhibiting ODD-Luc degradation were assayed to measure their ability to activate caspase 3/7.
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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."
15. Chou T.-F., Brown, S. J., Minond, D., Nordin, B.E., Li, K., Jones, A.C., Chase, P., Porubsky, P. R., Stoltz, B.M., Schoenen, F. J., Patricelli, M.P., Hodder, P., Rosen, H., and Deshaies, R. J. (2011) Reversible inhibitor of p97, DBeQ, impairs both ubiquitin-dependent and autophagic protein clearance pathways. Proc Natl Acad Sci USA 108:4834-4839.
SW403 cells were seeded onto a 384 well white clear bottom plate (3000 cells per well). Cells were treated with compounds for 7 h and caspase-3/7 Glo (Promega) was added into each well and the contents of the well were mixed by shaking at 500 rpm for 1 min. Luminescence signal was determined after incubation at room temperature for 1 h with 0.1 ms integration time on the Synergy HT Microplate Reader (BioTek). Normalized Caspase3/7 activity is calculated as luminescence of test compound/luminescence of DMSO (1 %).
Score = maximal fold activation of each compound / maximal fold activation observed for all compounds x 100
For a compound to be considered as caspase activating agent, its score should be more than 15. This corresponds to activity scores greater than 50. Activity scores were calculated by normalizing the highest fold activation to 100 and the lowest fold activation to 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, luciferase
Data Table (Concise)