N-Terminal polarity reversal of cysteine-terminated peptides: A strategy to develop potent metallo-β-lactamase inhibitors with in vivo efficacy.

The emergence of metallo-β-lactamase (MBL)-producing pathogens poses a critical threat to the efficacy of β-lactam antibiotics, with no clinically approved inhibitors currently available. Here, we report a rational design strategy for potent MBL inhibitors based on a "polarity reversal" of the N-terminus of cysteine-terminated peptides. A series of N-terminally protected di- and tripeptides were synthesized and evaluated. Two lead compounds, Fmoc-FFC and Fmoc-MC, emerged as potent, competitive inhibitors of clinically relevant MBLs, including NDM-1, VIM-2, and IMP-1, with Ki values in the low micromolar range. These compounds demonstrated remarkable synergistic activity with meropenem, restoring its potency against MBL-producing E. coli by up to 1024-fold. Both inhibitors exhibited excellent biocompatibility with minimal cytotoxicity and hemolytic activity. Crucially, in a murine systemic infection model, co-administration of Fmoc-FFC with meropenem achieved a 100% survival rate, compared to 0% for monotherapy. This work validates the N-terminal polarity reversal as a powerful strategy for developing MBL inhibitors and presents Fmoc-FFC as a promising lead candidate for combating infections caused by multi-drug resistant superbugs.