Development of a Peptide Inhibitor of Cdc20-Dependent APC Enzyme Activity

One of the most successful chemotherapeutic agents has been microtubule poisons. Although the exact mechanism leading to cancer cell death is unclear, microtubule poisons are thought to act by creating a spindle checkpoint dependent delay in mitosis, which in turn leads to cells undergoing apoptosis. Cancer cells may not respond to microtubule poison-based treatment, or may develop resistance to the therapy. Although the cellular mechanism is not known, the current leading hypothesis to explain this resistance is that in the presence of the microtubule poison, the cancer cells delay cell cycle progression, but then have the ability to under go ‘mitotic slippage’, and re-enter the cell cycle, rather than undergoing apoptosis. ‘Mitotic slippage’ requires the activity of the master cell cycle regulator, an E3-Ubiquitin Ligase called the Anaphase-Promoting Complex (APC), and the essential mitotic co-factor Cdc20 (Cell Division Cycle 20). This knowledge has led to a recent set of studies to investigate how to prevent ‘mitotic slippage’, and thus promote cancer cell apoptosis. Currently, the only successful approach has been to inhibit APCCdc20 directly. These studies were performed using tissue culture cancer cell lines, and inhibited Cdc20 by using RNAi. RNAi is currently not a viable therapeutic approach, and using cell lines to screen for inhibitory compounds can be complicated by off-target effects. To circumvent these challenges, we propose to use an in vitro biochemical APCCdc20 enzyme assay in the initial steps to screen and identify small molecule peptide inhibitors of APCCdc20. We have initially identified a 65 amino acid peptide that can disrupt APCCdc20 activity. To build upon this initial finding, we propose to accomplish in three years these Specific Aims. In aim 1, we propose to identify both the minimal and conserved amino acid residues from PQ65 sufficient for APCCdc20 inhibition, and measure the strength of inhibition. In aim 2, we propose to identify the target site(s) on the APC where the peptide inhibitor binds. In aim 3, we propose to validate the target site(s) using a yeast cell based assay in vivo.

Project ID:PA10501-1096
External Project ID:MOST103-2311-B182-005-MY3