Abstract
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 (
), 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 (GI
= 500 nM) while sparing neuronal cells. Live-cell imaging showed that
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
. 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
disrupted lipid metabolism and induced ferroptotic cell death, notably without the emergence of acquired resistance. In contrast, premyristoylated
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.