The Testing and Characterization of the Bacterial Response to Novel Lipid Ether Amine-Based Antibiotics

Bacterial biofilms exhibit enhanced resistance to antibiotics compared to planktonic bacteria, though the mechanisms underlying this tolerance remain incompletely understood. Evidence suggests biofilm-associated bacteria maintain active lipid metabolism despite reduced metabolic rates, making lipid-targeting strategies potentially effective. We investigated isopropyl lipid ether amines (LEAs), computationally designed to bind phospholipase A2, as novel antibiotics targeting biofilm bacteria through lipid metabolism disruption. LEAs consist of variable-length alkyl chain analogs of natural fatty acids connected via ether linkages to cationic head groups. Using methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa in collagen microplate assays, minimal inhibitory concentration studies, and ex vivo porcine biofilm models, we systematically compared LEAs against their fatty acid analogs to isolate the contribution of the lipid and amine moieties. Palmitic acid analog LEA-160 and oleic acid analog LEA-181 achieved MIC and reduced microplate biofilm colony forming units (CFU) against MRSA, while short lipid chain octanoic acid analog LEA-80 reduced Pseudomonas biofilms, with no effect for natural fatty acids. This demonstrates that the cationic amine group provides essential antibacterial function beyond the alkyl chain contribution. Comprehensive lipidomics analysis using LC-IMS-MS/MS revealed that LEA treatment induces significant alterations in MRSA lipid profiles, supporting a mechanism involving disruption of bacterial membrane lipid metabolism. Structure-activity relationships confirmed that both the lipid chain and cationic moieties are necessary for LEA antibacterial efficacy, with metabolic effects distinct from their natural fatty acid analogs. These findings establish LEAs as a mechanistically distinct antibiotic class targeting bacterial lipid metabolism pathways critical for biofilm survival.