Hot spot-driven discovery of β-catenin/Tcf4 interaction inhibitors via virtual drug repurposing study and experimental validation in colorectal cancer cells.

Aberrant activation of the Wnt/β-catenin signaling pathway is a hallmark of colorectal cancer, primarily driven by the interaction between β-catenin and T-cell factor 4 (Tcf4). Due to the large and shallow interface (~ 4800 Å2) of this protein-protein interaction, it has historically been considered "undruggable." However, emerging evidence highlights the presence of druggable hot spots within this interface. In this study, we employed a structure-based virtual screening approach to repurpose FDA-approved drugs targeting the β-catenin/Tcf4 interaction. Using physics-based molecular simulations and MM/GBSA binding free energy calculations, we identified Leucovorin (Folinic acid), Carbenicillin, and Ceforanide as promising candidate hit compounds with stable binding to critical hot spot residues (Asn430, Lys435, His470, Arg474, and Lys508) of β-catenin. Experimental validation using HT-29 colorectal cancer cells revealed that Carbenicillin and Ceforanide reduced the cell viability, impaired the colony formation, and exhibited superior anti-proliferative activity compared to the other candidates. These findings provide compelling evidence for the feasibility of targeting β-catenin/Tcf4 interactions using small molecules and suggest that Carbenicillin and Ceforanide may serve as promising repurposed candidates for therapeutic intervention in Wnt-driven malignancies.