Serendipitous Hinge Modulation Hypothetically Reprograms Caerin 1.1-LC Antibacterial Mechanism and Gram-Negative Selectivity

<b>Background:</b> The golden era of antibiotics has long passed, and the clinical failures caused by emerging drug-resistant bacteria have intensified the demand for novel antimicrobial agents. Antimicrobial peptides have attracted significant attention as promising candidates for next-generation antibiotics. <b>Methods:</b> In this study, we identified a novel antimicrobial peptide, Caerin 1.1-LC, from the skin secretion of the Australian green tree frog, <i>Litoria caerulea</i>. Subsequent structure-activity relationship studies led us to design a series of analogues and revealed the critical role of the peptide's intrinsic hinge structure in shaping its biological activity. <b>Results:</b> Incorporation of D-isomers at the valine residues within the hinge preserved overall helical content but altered the hinge conformation, resulting in an 8-fold increase in antibacterial activity against Gram-negative bacteria. Simultaneously, haemolytic activity was markedly reduced, leading to a 56-fold improvement in therapeutic index (from 0.47 to 26.6). Structural modulation of the hinge also switched the mechanism of action from classical membrane disruption with associated permeability changes to a non-membrane-permeabilising, 'cell-penetrating-like' behaviour, inducing membrane potential depolarisation and ATP disruption to trigger bacterial death. In vivo studies using infected larval models, along with in vitro LPS neutralisation assays, further demonstrated the therapeutic potential of the D-analogue as a novel antibacterial agent. <b>Conclusions:</b> This work highlights the pivotal role of hinge structures in Caerin-family/hinge-containing AMPs, offering a strategic avenue for optimising antibacterial efficacy.