Antagonists of anaphase-promoting complex (APC)-2-cell cycle and apoptosis regulatory protein (CARP)-1 interaction are novel regulators of cell growth and apoptosis.
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-κB proteome and a co-activator of steroid/thyroid nuclear receptors as well as β-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 CARP-1/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 μm. 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.
Mesh Terms:
Animals, Apoptosis, Apoptosis Regulatory Proteins, COS Cells, Cell Cycle, Cell Cycle Proteins, Cell Line, Tumor, Cell Proliferation, Cercopithecus aethiops, Cyclin B1, Gene Expression Regulation, HeLa Cells, Humans, Kinetics, Mice, Protein Interaction Mapping, Signal Transduction, Spiro Compounds, Thiadiazoles, Ubiquitin-Protein Ligase Complexes, Ubiquitin-Protein Ligases
Animals, Apoptosis, Apoptosis Regulatory Proteins, COS Cells, Cell Cycle, Cell Cycle Proteins, Cell Line, Tumor, Cell Proliferation, Cercopithecus aethiops, Cyclin B1, Gene Expression Regulation, HeLa Cells, Humans, Kinetics, Mice, Protein Interaction Mapping, Signal Transduction, Spiro Compounds, Thiadiazoles, Ubiquitin-Protein Ligase Complexes, Ubiquitin-Protein Ligases
J. Biol. Chem.
Date: Nov. 04, 2011
PubMed ID: 21903591
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