High-Throughput Selectivity Assays for Small-Molecule Inhibitors of β‑Catenin/T-Cell Factor Protein–Protein Interactions

Two homogeneous high-throughput assays, AlphaScreen and fluorescence polarization, were established to quantify inhibitor selectivity between different protein–protein complexes. As a first case study, they have been successfully applied to the key protein–protein interactions in the downstream sites of the canonical Wnt signaling pathway. The aberrant formation of the β-catenin/T-cell factor (Tcf) complex is the major driving force for many cancers and fibroses. Crystallographic and biochemical studies reveal that the binding modes of Tcf, E-cadherin, and adenomatous polyposis coli (APC) to β-catenin are identical and mutually exclusive. In the present study, two highly sensitive and robust assays were established to quantitatively evaluate inhibitor selectivity between β-catenin/Tcf, β-catenin/E-cadherin, and β-catenin/APC interactions. A pilot screen demonstrated the feasibility of the assays and yielded four hits for the disruption of β-catenin/Tcf interactions. A potent and dual-selective β-catenin/Tcf inhibitor was identified

Discovery of Selective Small-Molecule Inhibitors for the β‑Catenin/T-Cell Factor Protein–Protein Interaction through the Optimization of the Acyl Hydrazone Moiety

Acyl hydrazone is an important functional group for the discovery of bioactive small molecules. This functional group is also recognized as a pan assay interference structure. In this study, a new small-molecule inhibitor for the β-catenin/Tcf protein–protein interaction (PPI), ZINC02092166, was identified through AlphaScreen and FP assays. This compound contains an acyl hydrazone group and exhibits higher inhibitory activities in cell-based assays than biochemical assays. Inhibitor optimization resulted in chemically stable derivatives that disrupt the β-catenin/Tcf PPI. The binding mode of new inhibitors was characterized by site-directed mutagenesis and structure–activity relationship studies. This series of inhibitors with a new scaffold exhibits dual selectivity for β-catenin/Tcf over β-catenin/cadherin and β-catenin/APC PPIs. One derivative of this series suppresses canonical Wnt signaling, downregulates the expression of Wnt target genes, and inhibits the growth of cancer cells. This compound represents a solid starting point for the development of potent and selective β-catenin/Tcf inhibitors