Podophyllotoxin disrupts the XIAP–caspase-9 interaction to promote apoptosis: insights from a quantitative split-luciferase complementation assay

Podophyllotoxin (PTOX), a plant-derived lignan with potent anticancer properties, is hypothesized to mimic Smac activity by interacting with Inhibitor of Apoptosis Protein (IAP) BIR domains. However, the precise molecular mechanisms governing these interactions remain poorly understood. In this study, we developed and validated a highly sensitive, cell-free split-luciferase complementation assay designed to quantify the real-time interaction dynamics between the BIR2–BIR3 domains of XIAP and its key targets, caspase-3 and caspase-9. Our results demonstrate that the BIR2–BIR3–NLuc/caspase-9-CLuc interaction is significantly more robust and stable than the BIR2–BIR3–NLuc/caspase-3-CLuc interaction, highlighting a preferential binding mechanism at the initiator caspase level. Utilizing this platform, we investigated the effects of PTOX in modulating this interaction. Consistent with in silico docking predictions, PTOX disrupted the XIAP–caspase-9 complex in a concentration-dependent manner, leading to a marked reduction in luminescence and a subsequent restoration of caspase-9 enzymatic activity. While PTOX showed a minor inhibitory effect on the XIAP–caspase-3 interaction, its impact on the caspase-9 axis was markedly more pronounced. Furthermore, Carboplatin, a DNA-crosslinking agent, exhibited no significant impact on these protein-protein interactions, confirming the specificity of the observed PTOX-mediated effects. These findings establish a sensitive, real-time platform for monitoring the XIAP–caspase axis and provide compelling evidence that PTOX acts as a targeted modulator of the apoptotic pathway, warranting its further development as a pharmacological agent.