Engineered indoleamine 2,3-dioxygenase variant: Oxidative activation of prodrug indole-3-acetic acid against cancer cells for targeted therapy.

Indoleamine 2,3-dioxygenase (IDO) is a rate-limiting enzyme involved in the kynurenine pathway of tryptophan metabolism and has been implicated in immune regulation and tumor immune evasion. Beyond its role in tryptophan metabolism, we found that IDO can catalyze the oxidative activation of indole-3-acetic acid (IAA). This reaction generates reactive oxidative intermediates and triggers potent cytotoxicity in cancer cells. Leveraging human-derived enzymes as "suicide effectors" confers a critical advantage by mitigating the risk of immunogenic rejection. However, the intrinsic kynurenine-pathway activity of wild-type IDO constrains its immediate therapeutic translatability. To surmount this limitation, we further designed and characterized a truncated variant, IDO268C. This variant retains high catalytic activity toward IAA but lacks the canonical kynurenine-pathway function of IDO. Validation assays demonstrated that both breast cancer MCF-7 and lung cancer A549 cells exhibited pronounced cytotoxic responses to the IDO268C-IAA pair. Cytotoxicity was achieved through both intracellular overexpression and extracellular protein delivery. Collectively, our findings establish the IDO268C-IAA pair as an efficient enzyme-prodrug combination, representing a promising therapeutic modality for enzyme-activated prodrug therapy in oncology.