Deciphering Therapeutic Targeting of Cathepsin B using Repurposed Drug Darifenacin.

Cathepsins are lysosomal proteases with well-documented roles in the progression of various cancers. Among them, cathepsin B (CTSB), a cysteine protease, is notably involved in the development of breast cancer and neuroblastoma. In this study, we explored the potential of darifenacin as a repurposed therapeutic targeting CTSB, using molecular docking and simulation studies which demonstrated a significantly lower binding energy against CTSB (-456.268 kJ/mol) compared to its known inhibitor, aloxistatin (-36.601 kJ/mol). The cytotoxic efficacy of darifenacin was evaluated on IMR-32 (neuroblastoma) and MCF-7 (breast cancer) cells, yielding half-maximal inhibitory concentrations (IC50) of 38.14 and 39.96 µM, respectively. Darifenacin effectively inhibited CTSB enzymatic activity by ~1.82 and ~1.75-fold in IMR-32 and MCF-7 cells, respectively, triggering intracellular ROS generation, mitochondrial membrane potential depolarization, and cell cycle arrest. These events culminated in apoptosis-mediated cell death, with apoptotic populations reaching 51.39% in IMR-32 and 40.6% in MCF-7 cells, respectively. Additionally, darifenacin disrupted lipid droplet accumulation, cellular migration, and colony and sphere-forming abilities in both cell lines. Overall, this study identifies darifenacin as a promising therapeutic agent against CTSB-driven cancer progression.