N-myristoylation is a common posttranslational modification in which the enzyme N-myristoyltransferase, NMT, attaches a 14-carbon fatty acid to an N-terminal glycine residue. This lipid anchor governs membrane association, protein localization, and signaling cascades relevant to viral infection and cancer. While chemists have explored synthetic myristoylation to improve peptide pharmacokinetics and activity, no one had demonstrated intracellular myristoylation of a synthetic peptide: the use of cellular NMT to lipidate an exogenous substrate in situ.

A team led by Bing Xu at Brandeis University, published in the Journal of the American Chemical Society, now reports exactly that. The researchers designed Gbb-NBD, a short D-peptide featuring an N-terminal glycine, a dibiphenylalanine backbone for membrane affinity, and a nitrobenzofurazan fluorophore for imaging. The peptide self-assembled into nanospheres in aqueous solution and potently inhibited HeLa cell growth, GI50 approximately 500 nM, while showing minimal toxicity toward neuronal SH-SY5Y cells.

Live-cell imaging revealed that Gbb-NBD rapidly crossed the plasma membrane and localized to the endoplasmic reticulum, Golgi, and mitochondria within minutes. Multiple lines of evidence confirmed intracellular myristoylation: NMT inhibitors rescued cell viability and reduced intracellular fluorescent puncta; high-resolution mass spectrometry detected myristoylated peptide in cell lysates, and click chemistry with azido myristic acid showed colocalization with the peptide. N-acetylation of the glycine abolished cytotoxicity, confirming that the free amine is essential for NMT recognition.

Mechanistic studies pointed to ferroptosis as the primary death pathway. The ferroptosis inhibitor liproxstatin-1 rescued cells in a dose-dependent manner, immunoblotting showed depletion of the ferroptosis suppressor GPX4, and lipidomics revealed accumulation of oxidized phosphatidylethanolamines and phosphatidylcholines, hallmarks of lipid peroxidation. Notably, cells subjected to escalating doses showed no acquired resistance, a common limitation of conventional therapies.

A critical control underscored the importance of in situ lipidation. A premyristoylated analog exhibited poor cellular uptake and weak cytotoxicity, GI50 greater than 20 μM, likely because the preinstalled lipid tail reduced solubility and eliminated the ionizable amine needed for membrane permeation. In contrast, Gbb-NBD enters cells in its more soluble, unmyristoylated form and acquires the lipid anchor intracellularly, where it drives assembly and ferroptotic signaling.

By co-opting the cell's own lipidation machinery, this strategy offers a new route to membrane-targeting supramolecular therapeutics. Because elevated NMT activity, which can confer resistance to NMT inhibitors, would instead amplify production of the cytotoxic lipidated peptide, the approach may prove self-reinforcing against resistant phenotypes.