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. This article, explores the potential of darifenacin as a repurposed therapeutic targeting CTSB, using molecular docking and simulation studies which demonstrate a significantly lower binding energy against CTSB (-456.268 kJ mol^-1) compared to its known inhibitor, aloxistatin (-36.601 kJ mol^-1). The cytotoxic efficacy of darifenacin is 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 inhibits CTSB enzymatic activity by ≈1.82-fold and ≈1.75-fold in IMR-32 and MCF-7 cells, respectively, triggering intracellular reactive oxygen species generation, mitochondrial membrane potential depolarization, and cell cycle arrest. These events culminates in apoptosis-mediated cell death, with apoptotic populations reaching 51.39% in IMR-32 and 40.6% in MCF-7 cells, respectively. Additionally, darifenacin disrupts lipid droplet accumulation, cellular migration, and colony- and sphere-forming abilities in both cell lines. Overall, this article identifies darifenacin as a promising therapeutic agent against CTSB-driven cancer progression.