Studying Phosphoroorganic Modification of Smac‐Mimetics to Boost Selectivity for cIAP1‐BIR3 and Unravel cIAP1/XIAP Molecular Inhibition Mechanisms

ABSTRACT Homeostasis in organisms is regulated by apoptosis, whose dysregulation in neoplastic cells is often associated with overexpression of inhibitor of apoptosis proteins (IAPs). IAPs inhibit caspases, key executors of apoptosis. Smac (Second Mitochondria‐derived Activator of Caspases), counteracts this inhibition by binding to a specific groove in IAPs, enabling caspase activation. Smac‐mimetics (SMs) are small molecules antagonizing IAPs and are promising antitumor agents. As IAP homologues employ distinct mechanisms to regulate cell death, SMs binding can lead to different molecular outcomes. We report the synthesis and characterization of two novel SMs — a bivalent ( 6 ) and a monovalent ( 7 ) derivative — exhibiting one or two mimic heads and a phosphoroorganic moiety. Their effects on cIAP1 and XIAP were analyzed combining thermal stability, affinity analysis, size exclusion chromatography, and virtual docking. Both compounds selectively target cIAP1‐BIR3 over XIAP and interact with the BIR2 and BIR3 domains of XIAP via intramolecular binding, highlighting the phosphoroorganic moiety's ability to engage BIR2. In contrast, the bivalent 6 induces cIAP1 dimerization through intermolecular binding. These findings support the rational design of novel IAP antagonists with predictable effects, emphasizing selectivity and the potential to induce distinct molecular responses, including those mediated by bifunctional proteolysis‐targeting chimera (PROTAC) compounds.