The cytotoxic effect of DEBIO 1143 on triple-negative murine breast cancer cell lines: Insights from in vitro and in silico studies.

Triple-negative breast cancer (TNBC) remains a significant therapeutic challenge due to its aggressive clinical course and inherent resistance to apoptosis. The overexpression of cellular Inhibitor of Apoptosis Protein 1 (cIAP1) serves as a critical molecular barrier to conventional treatments. This study aimed to elucidate the multimodal cell-death mechanisms of DEBIO 1143, a second-generation SMAC mimetic, in murine 4T1 and 4T1-HER2 cell lines using an integrated approach of 1-µs in silico molecular dynamics simulations and in vitro validation. Cell viability was determined via MTT assays at 24 and 48 h. Cell-death modalities were quantified using Annexin V-FITC/PI flow cytometry and monodansylcadaverine staining. Key regulatory proteins (LC3II, Beclin-1, RIP3, and cIAP1) were analyzed by Western blotting. Ligand-protein interactions were probed through molecular docking and 1-µs MD simulations. DEBIO 1143 demonstrated time- and dose-dependent cytotoxicity, with 48-h IC50 values of 36.20 µM for 4T1 and 22.45 µM for 4T1-HER2 cells. Western blotting confirmed complete cIAP1 depletion at 48 h, concomitant with the upregulation of RIP3, Beclin-1, and LC3II. Flow cytometry indicated a shift toward necroptosis-like regulated cell death, supported by a significant increase in autophagic vacuole density (p < 0.001). MD simulations revealed that DEBIO 1143 lacks high-affinity binding to RIP3, Beclin-1, or LC3II, suggesting that the upregulation of these mediators is a downstream cellular response to cIAP1 depletion rather than direct engagement. DEBIO 1143 was associated with reduced apoptotic cell death and increased necroptosis-like and autophagy-associated responses in TNBC cells. These findings support its development as a multimodal therapeutic strategy for aggressive breast cancer subtypes.