| Description |
The canonical Wnt signaling pathway is fundamental in expressing genes that promote cell proliferation, differentiation, and survival. Hyperactivation of the canonical Wnt signaling pathway has been linked to the formation of various cancers and is present in cancer stem cells. Significant efforts have been made to discover inhibitors of this pathway. Inhibition of the pathway through disruption of the transcription complex is an appealing target, for which selective small-molecule inhibitors have been designed. Both the β-catenin/T-cell factor (Tcf) and β-catenin/B-cell lymphoma 9 (BCL9) protein- protein interactions (PPIs) are two key interaction interfaces that have been investigated. The key rationale for design of selective small-molecule inhibitors for each interaction interface is described. The cellular bioavailability of these inhibitors was considered during the optimization process. The determination of the cellular bioavailability of small-molecule inhibitors is a critical step to interpret cell-based data and guides further inhibitor optimization. A HPLC/MS-based protocol was developed to determine the cellular bioavailability of small-molecule PPI inhibitors. This protocol characterized various properties for the inhibitors including stability, dose- and time-dependent intracellular concentrations, cell permeability, and nonspecific binding. The protocol was successfully applied to four small-molecule PPI inhibitors. The protocol is robust, sensitive, and generalizable, and can be routinely used in chemical biology and medicinal chemistry laboratories. iv Three separate projects to design new inhibitors for β-catenin/Tcf and β- catenin/BCL9 PPIs are presented. First, the structure-based optimization of 1,4- dibenzoylpiperazines and 4-phenoxypiperidines as β-catenin/BCL9 inhibitors produced 3.20 and 3.21. The AlphaScreen assay demonstrated that 3.20 and 3.21 can disrupt the β- catenin/BCL9 PPI with Ki values of 1.6 ± 0.41 and 2.5 ± 0.31 μM, respectively. Both effectively inhibit proliferation of SW480 cells and transactivation of canonical Wnt signaling. Second, a fragmentation and reassembly approach was used to design a benzo[b]thiophene containing inhibitor for the β-catenin/Tcf PPI. The synthetic route to access the benzo[b]thiophene core was accomplished in nine steps. Two key synthetic challenges were overcome and intermediates were synthesized in 16 steps. Finally, eight peptidomimetic inhibitors for the β-catenin/Tcf PPI were designed and synthesized. The biological assays indicated that these inhibitors disputed the β-catenin/Tcf PPI with low micromolar inhibitory activity. |
