Multidrug-resistant (MDR) Acinetobacter baumannii is a major global health concern, responsible for severe hospital-acquired infections such as pneumonia, septicemia, and sepsis. Its ability to persist under adverse conditions and acquire antibiotic resistance mechanisms has led the World Health Organization to classify it as a critical priority pathogen, highlighting the urgent need for new antibacterial approaches that can overcome its impermeable outer membrane and active efflux systems.
One promising approach exploits bacterial iron uptake systems through siderophore-mediated transport, enabling antibiotic delivery directly into bacteria — the “Trojan horse” strategy. Antibiotic- siderophore conjugates derived from this concept are collectively known as sideromycins, which act as molecular vectors that utilize bacterial iron uptake mechanisms to introduce antibiotics across the cell envelope.
In this context, we have developed a protease-activated siderophore–antibiotic conjugate designed to selectively target A. baumannii. The conjugate combines a simplified amonabactin analogue (AMB), with the protease-cleavable peptide linker WSPKYM, and norfloxacin (NFX) as antibiotic. Amonabactin
was selected due to its catecholate-based structure and molecular simplicity, which allow recognition by outer membrane receptors shared among several Gram-negative bacteria, including A. baumannii.
A model conjugate (WSPKYM – NFX) was first synthesized and incubated with a cell free extract of periplasmic proteases from Aeromonas salmonicida to validate the cleavage efficiency of the linker. HPLC/MS analysis confirmed successful cleavage of the WSPKYM linker, indicating an efficient protease recognition and antibiotic release mechanism.
The conjugate (AMB – WSPKYM – NFX) was obtained through a hybrid synthetic route, combining solid-phase peptide synthesis for the preparation of the linker and the siderophore fragments with solution-phase reaction steps for the final coupling with norfloxacin.
This work represents the first report of the preparation of a protease-activated amonabactin-based conjugate designed for A. baumannii, to demonstrate the versatility of this chemical platform that combines siderophore-mediated internalization with enzyme activation. Such conjugates could serve as next-generation antibacterial agents capable of bypassing permeability barriers and targeting resistant Gram-negative pathogens.
