Multifunctional stapled peptides with varied orientations and linker lengths exhibit different pull-down activity.įigure S5. SAH-p53–8 and StAx-35 competed with MSA-2 in the MDM2 pull-down assay, indicating that the recruitment of MDM2 to β-catenin is a reversible process and both SAH-p53–8 and StAx-35 portion in the MSA peptides contribute to the binding.įigure S4. Competition fluorescence polarization assays using MSA peptides with (A) different orientations and (B) varied linker lengths showing that neither peptide orientation nor linker length appeared to affect the measured binding properties. HPLC traces of purified MSA peptides measured at 280 nm.įigure S2. List of MSA peptides used in this study with their chemical formula, molecular weight, calculated and found mass (m/z) for charged ions.įigure S1. Mutated amino acids in negative control peptides are highlighted in bold. StAx and SAH-p53–8 peptides and their respective negative controls with alkene containing amino acids highlighted in red and blue. Psc338-Supporting Information_Revised.docxWord 2007 document Therefore, these multifunctional stapled peptides provide a unique research tool for examining the Wnt signaling pathway by targeted knockdown of β-catenin at the protein level, and may serve as leads for potential drug candidates in the treatment of Wnt-dependent cancers. In addition, a luciferase reporter assay showed that the multifunctional stapled peptides can suppress β-catenin-mediated gene expression via the Wnt signaling pathway.
In cellulo, treatment of the human colorectal cancer cell line SW480 with the multifunctional stapled peptides showed dose-dependent degradation of endogenous β-catenin levels. We found that in vitro these multifunctional peptides can recruit the MDM2 protein to β-catenin and induce poly-ubiquitination on β-catenin. Specifically, we designed, synthesized, and evaluated a panel of multifunctional stapled peptides that have a β-catenin binding moiety (StAx-35) covalently linked to a second stapled peptide moiety (SAH-p53-8), which is capable to interact with the E3 ubiquitin ligase MDM2. Here, we report a strategy that employs multifunctional stapled peptides to recruit an E3 ubiquitin ligase to β-catenin, thereby rescuing β-catenin degradation by hijacking the endogenous ubiquitin-proteasome pathway. Targeting β-catenin has therefore emerged as an appealing approach for the treatment of Wnt-dependent cancers.
One common feature of oncogenic Wnt regulation involves an increase in the cellular levels of β-catenin due to interference with its constitutive ubiquitin-dependent degradation. Aberrant activation of the Wnt signaling pathway has been identified in numerous types of cancer.
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