Engineering lipopeptide-gramicidin S conjugates to combat multidrug-resistant Acinetobacter baumannii and other ESKAPE pathogens.

Gramicidin S (GS) is a cyclic decapeptide that primarily targets Gram-positive bacteria and due to its hemolytic toxicity, is mainly used for the topical treatment of eye, ear, and throat infections caused by S. aureus. Due to its unique mechanism of action, no clinically significant resistance to GS has been reported to date. With the aim of expanding the therapeutic scope of GS to Gram-negative bacteria, we developed novel GS analogues by conjugating with cationic amino acid residues and lipopeptides on the ornithine (Orn) side-chain. A total of 14 peptides were synthesized and assessed for their antimicrobial activity against human bacterial pathogens. The results revealed that the newly synthesized peptides exhibited enhanced activity against Gram-negative bacteria, including A. baumannii, P. aeruginosa and E. coli, with MIC ranging from 2 to 8 μg/mL. The cytotoxicity data showed peptide 4, 6, 10 and 11 exhibiting lesser cytotoxicity (CC50/MIC of A. baumannii 5-37) and RBC hemolysis (HC50/MIC of A. baumannii 14-26) as compared with GS. The compounds with improved activity were found to retain activity against twenty-five drug-susceptible and MDR A. baumannii clinical isolates. Their bactericidal activity was further confirmed by time-kill kinetics, scanning electron microscopy and membrane permeability experiments. These GS analogues demonstrated broad-spectrum antibacterial activity against WHO priority pathogens, thus highlighting their potential as promising candidates for further therapeutic development.