Novel, tightly structurally related N-myristoyltransferase inhibitors display equally potent yet distinct inhibitory mechanisms

N-myristoyltransferases (NMTs) catalyze essential acylations of N-terminal alpha or epsilon amino groups of glycines or lysines. Here, we reveal that peptides tightly fitting the optimal glycine recognition pattern of human NMTs are potent prodrugs relying on a single-turnover mechanism. Sequence scanning of the inhibitory potency of the series closely reflects NMT glycine substrate specificity rules, with the lead inhibitor blocking myristoylation by NMTs of various species. We further redesigned the series based on the recently recognized lysine-myristoylation mechanism by taking advantage of (1) the optimal peptide chassis and (2) lysine side chain mimicry with unnatural enantiomers. Unlike the lead series, the inhibitory properties of the new compounds rely on the protonated state of the side chain amine, which stabilizes a salt bridge with the catalytic base at the active site. Our study provides the basis for designing first-in-class NMT inhibitors tailored for infectious diseases and alternative active site targeting. [Display omitted] •Optimal octapeptide substrates of Gly-myristoylation are potent prodrug NMT inhibitors•Potent Lys-myristoylation-based inhibitors were derived from the substrate series•Inhibition relies on lysine protonation and a salt bridge with the catalytic base•Potent inhibition requires a secondary salt bridge stabilizing a disordered loop of NMT Peptides fitting the optimal human NMT Gly-myristoylation recognition pattern act as potent prodrugs via a single-turnover mechanism. Rivière et al. derived Lys-myristoylation-based inhibitors from these peptides. Despite tight structural similarities, each series’ inhibitory properties are unique, relying on distinct interactions with the catalytic base at the active site.