| Wnt signaling plays a critical role in numerous cellular processes including embryonic development, cell proliferation and tissue homeostasis. The multifunctional protein beta-catenin is the primary mediator of canonical Wnt signaling and acts as a transcriptional activator in this context. Dysregulated Wnt signaling is a hallmark of many human cancers and results in the stabilization and accumulation of beta-catenin, leading to the increased transcription and expression of Wnt target genes. The transcriptional activation function of beta-catenin requires the formation of a nuclear super-complex with protein cofactors including BCL9/B9L, TCF and CBP. It has been demonstrated that binding to these cofactors is essential for transcriptional. Of these critical cofactors, BCL9 and its homolog B9L are the most recently identified and their exact roles are not fully understood. To explore the consequences of the BCL9/B9L-beta-catenin binding interaction we developed and optimized a quantitative, reliable and high throughput fluorescence polarization (FP) binding assay along with a surface plasmon resonance (SPR)-based secondary assay. Using our FP assay, we performed extensive mutational analysis of four key hydrophobic residues in BCL9 and determined their contribution to the interaction with beta-catenin. We also mapped the precise region of BCL9 required for high affinity binding to beta-catenin. With our optimized FP assay, we performed high throughput screening (HTS) for small molecule inhibitors and identified one molecule that warrants further characterization in cell-based assays. We also synthesized BCL9 peptides tagged with cell penetrating peptides (CPPs), but found that their usefulness was limited by their poor solubility. We then explored the design and synthesis of stabilized alpha-helical BCL9 peptides by three different methods and determined that triazole-stapling, mediated by the Huisgen 1,3-dipolar cycloaddition reaction, was the most robust and highest yielding method in our hands. Our detailed study of this stapling technique defined the optimal azido and alkynyl linker combinations required for stabilizing one turn of the BCL9 alpha-helix. In summary, we confirmed the requirement for BCL9/B9L in beta-catenin transcriptional activation, identified a potentially druggable site around the BCL9 F374 binding pocket and demonstrated that triazole stapling can increase helicity and binding affinity of our BCL9 peptides. |
