Intracellular Peptide N-Myristoylation for Cancer Cell Ferroptosis without Acquired Resistance.

N-Myristoylation, a well-known protein lipidation process, has yet to be explored for in situ peptide lipidation. Here, we report intracellular peptide N-myristoylation for potently inhibiting cancer cells. A self-assembling d-peptide, Gbb-NBD (1), comprising an N-terminal glycine, a d-dibiphenylalanine backbone, and a C-terminal nitrobenzofurazan, formed nanospheres in aqueous solution and exhibited strong cytotoxicity against cancer cells (GI50 = 500 nM) while sparing neuronal cells. Live-cell imaging showed that 1 traversed the plasma membrane to the ER, Golgi and mitochondria. NMT inhibition, LC-MS of cell lysates, and click chemistry confirmed the N-myristoylation of 1. Functional studies showed that blocking NMT activity or modifying the N-terminus suppressed cytotoxicity, establishing N-myristoylation as essential for activity. Mechanistically, immunoblotting, lipidomic profiling, and rescue assays demonstrated that myristoylated 1 disrupted lipid metabolism and induced ferroptotic cell death, notably without the emergence of acquired resistance. In contrast, premyristoylated 1 displayed poor uptake and weak activity, underscoring the importance of in situ lipidation for cellular entry and function. Together, these findings reveal intracellular N-myristoylation of a short peptide as a new approach to drive ferroptosis and highlight its potential for developing membrane-targeting supramolecular therapeutics.