Antagonists of Anaphase-promoting Complex (APC)-2-Cell Cycle and Apoptosis Regulatory Protein (CARP)-1 Interaction Are Novel Regulators of Cell Growth and Apoptosis

作者：Puliyappadamba Vineshkumar Thidil; Wu Wenjuan; Bevis Debra; Zhang Liyue; Polin Lisa; Kilkuskie Robert; Finley Russell L Jr; Larsen Scott D; Levi Edi; Miller Fred R; Wali Anil; Rishi Arun K^*

来源：Journal of Biological Chemistry, 2011, 286(44): 38000-38017.

DOI：10.1074/jbc.M111.222398

CARP-1/CCAR1, a perinuclear phosphoprotein, is a regulator of cell growth and apoptosis signaling. Although CARP-1 is a regulator of chemotherapy-dependent apoptosis, it is also a part of the NF-kappa B proteome and a co-activator of steroid/thyroid nuclear receptors as well as beta-catenin signaling. Our yeast two-hybrid screen revealed CARP-1 binding with the anaphase-promoting complex/cyclosome E3 ubiquitin ligase component APC-2 protein. CARP-1 also binds with anaphase-promoting complex/cyclosome co-activators Cdc20 and Cdh1. Following mapping of the minimal epitopes involved in CARP-1 binding with APC-2, a fluorescence polarization assay was established that indicated a dissociation constant (K(d)) of 480 nM for CARP1/APC-2 binding. Fluorescence polarization assay-based high throughput screening of a chemical library yielded several small molecule antagonists of CARP-1/APC-2 binding, termed CARP-1 functional mimetics. CFM-4 (1(2-chlorobenzyl)-5'-phenyl-3'H-spiro[indoline-3,2'-[1,3,4]thiadiazol]-2-one), a lead compound, binds with and stimulates CARP-1 expression. CFM-4 prevents CARP-1 binding with APC-2, causes G(2)M cell cycle arrest, and induces apoptosis with an IC(50) range of 10-15 mu M. Apoptosis signaling by CFM-4 involves activation of caspase-8 and -9 and caspase-mediated ubiquitin-proteasome pathway-independent loss of cyclin B1 and Cdc20 proteins. Depletion of CARP-1, however, interferes with CFM-4-dependent cell growth inhibition, activation of caspases, and apoptosis. Because CFM-4 also suppresses growth of drug-resistant human breast cancer cells without affecting the growth of human breast epithelial MCF-10A cells, elevating CARP-1 by CFM-4 and consequent apoptosis could in principle be exploited to further elucidate, and perhaps effectively target, often deregulated cell cycle pathways in pathological conditions, including cancer.

• 出版日期2011-11-4