Novel bifunctional antibacterial peptides mediated by a covalent conjugation strategy combat priority multidrug-resistant gram-negative pathogens through dual targets.

The escalating antibiotic resistance presents formidable challenges in the treatment of Gram-negative bacterial infections. Clinically, these bacteria have also acquired resistance to polymyxin, the last resort of defense. Novel antibiotics with a single mode of action are susceptible to rapid resistance development, and sometimes asynchronous pharmacokinetics also hinders the effectiveness of combined administration strategies in vivo. Here, we developed a class of novel bifunctional antibacterial peptides by covalently conjugating a series of modified PbgA-derived peptides with colistin analog (PE-2C-C8-DH) via a small-molecule linker (KCM02). These bifunctional peptides show remarkable synergistic antibacterial efficacy, where "1 + 1 > 2", against various priority multidrug-resistant Gram-negative bacteria, involving polymyxin-resistant strains. By optimizing the structure-activity relationship, two compounds (BP-28 and BP-37) with distinct activity preferences were obtained, which possess rapid bactericidal efficacy and a significantly lower risk of resistance compared to single-mode-of-action antibacterial agents, without hemolytic toxicity and cytotoxicity. Identification of antibacterial targets revealed that they can damage Gram-negative bacterial membrane by targeting LPS and BamA. Our study offers a referable approach for the development of novel antimicrobial agents.