title: Analysis of the interaction of BCL9 with beta-catenin and development of fluorescence polarization and surface plasmon resonance binding assays for this interaction.
abstract: The transcriptional activator beta-catenin is the primary mediator of the canonical Wnt signaling pathway and is frequently upregulated in many types of human cancer. Recent studies have suggested that the interaction of beta-catenin and its cofactor, B-cell lymphoma 9 (BCL9), is crucial for its transcriptional activity. Targeting this interaction using small molecules will improve our understanding of the beta-catenin/Wnt signaling pathway and may lead to the development of a new class of anticancer drugs. In this study, we developed a fluorescence polarization (FP)-based BCL9 binding assay. Using our initial FP assay, we performed extensive mutational analysis on four critical hydrophobic residues in the BCL9 peptide and determined the precise region in BCL9 responsible for binding to beta-catenin. These results led to further optimization of our FP assay, making it amenable for high-throughput screening (HTS). We also developed and validated a complementary surface plasmon resonance (SPR)-based binding assay and showed that our synthetic BCL9-based peptides are capable of fully inhibiting the binding of beta-catenin to wild-type BCL9 protein. Our studies provide not only further insight into the interaction between BCL9 and beta-catenin but also quantitative and reliable biochemical binding assays for the discovery of potent and specific small-molecule inhibitors of this interaction.
authors.affiliation: Department of Medicinal Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.
authors.forename: Steven A
authors.initials: SA
authors.lastname: Kawamoto
authors.forename: Andrea D
authors.initials: AD
authors.lastname: Thompson
authors.forename: Adriana
authors.initials: A
authors.lastname: Coleska
authors.forename: Zaneta
authors.initials: Z
authors.lastname: Nikolovska-Coleska
authors.forename: Han
authors.initials: H
authors.lastname: Yi
authors.forename: Shaomeng
authors.initials: S
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D001667:Binding, Competitive; D004252:DNA Mutational Analysis; D005454:Fluorescence Polarization / Q000379:methods; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D015203:Reproducibility of Results; D020349:Surface Plasmon Resonance / Q000379:methods; D014157:Transcription Factors; D051176:beta Catenin / Q000737:chemistry / Q000378:metabolism*
chemical_list: C111180:BCL9 protein, human; C495270:CTNNB1 protein, human; D009363:Neoplasm Proteins; D010446:Peptide Fragments; D014157:Transcription Factors; D051176:beta Catenin