Development of lysine-branched dendrimeric antimicrobial peptides targeting ESKAPE pathogens: broad-spectrum activity, biofilm eradication, and endotoxin neutralization

Antimicrobial resistance (AMR) represents a pressing global health challenge, driving the urgent need for novel therapeutic agents with improved stability and selectivity. In this study, we present the rational design and synthesis of lysine-branched dendrimeric antimicrobial peptides (AMPs) based on short arginine/tryptophan-rich motifs (Du-6 and Lf-6), yielding dimeric and tetrameric architectures. Physicochemical analyses revealed a systematic increase in net charge and hydrophobicity with higher degrees of branching. Comparative biological evaluations demonstrated that dimeric peptides (di-Du-6 and di-Lf-6) achieved optimal broad-spectrum antibacterial activity against both Gram-positive and Gram-negative bacteria, including multidrug-resistant ESKAPE pathogens. These dimers maintained low hemolytic activity and exhibited therapeutic indices of up to 40. In contrast, despite their elevated charge density and tryptophan content, tetrameric peptides showed increased cytotoxicity, likely due to deeper membrane penetration into eukaryotic cells, thereby compromising selectivity. To overcome proteolytic degradation, D-enantiomeric dimers [(di-Du-6) <i>_D</i> and (di-Lf-6) <i>_D</i> ] were synthesized. These retained potent antimicrobial efficacy, demonstrated complete resistance to trypsin digestion, and remained active under physiologically relevant conditions, including the presence of salts and serum. Beyond their antibacterial effects, the dimeric peptides effectively inhibited and eradicated biofilms formed by multidrug-resistant <i>Pseudomonas aeruginosa</i>, exhibited synergistic interactions with conventional antibiotics, and attenuated inflammatory responses by suppressing the production and expression of pro-inflammatory cytokines in LPS-stimulated macrophages. Furthermore, they neutralized endotoxins through direct binding and disaggregation of LPS aggregates. Collectively, these results establish dimeric peptides as multifunctional anti-infective agents, combining broad-spectrum antibacterial, antibiofilm, and anti-inflammatory activities. The enhanced proteolytic stability and selectivity of D-form dimers underscore their promise as next-generation therapeutics for combating multidrug-resistant infections and sepsis-associated inflammation.