Structural and functional insights into helical antimicrobial peptide-drug conjugates: a dual-action strategy against infection and inflammation.

Antimicrobial peptides (AMPs) are promising alternatives to conventional antibiotics due to their membrane-disruptive mechanisms and low propensity for resistance. We report the synthesis of twelve α-helical AMP conjugates, which are each composed of one of three AMPs (LL-I, LK6, ATRA-1) covalently linked to ibuprofen (IBF) or naproxen (NPX) via 8-aminooctanoic acid (C8) or 10-aminodecanoic acid (C10) spacers, selected based on prior lipidation-activity correlations. Conjugates were evaluated for antimicrobial potency, cytotoxicity, and anti-inflammatory activity; structural properties were assessed by circular dichroism (CD) in the presence of anionic membranes. Lead candidates (NPX-C8-LL-I, IBF-C8-LK6 and IBF-C10-ATRA-1) underwent serum stability, self-assembly, and membrane-interaction analyses. NPX-C8-LL-I exhibited aggregation driven by π-π stacking; IBF-C8-LK6 retained a stable α-helical conformation in aqueous buffer and suppressed early proinflammatory cytokine production; and IBF-C10-ATRA-1 adopted a disordered structure and lacked anti-inflammatory effects. All lead conjugates demonstrated potent, broad-spectrum antimicrobial activity with negligible cytotoxicity at bactericidal concentrations. These results highlight NSAID conjugation as a tunable strategy to modulate AMP structural dynamics and introduce dual antimicrobial-anti-inflammatory functionality.