A Toxicity-Reduced NA-CATH-Derived Antimicrobial Peptide Active against Multidrug-Resistant Klebsiella pneumoniae and Acinetobacter baumannii

Multidrug-resistant Klebsiella pneumoniae and Acinetobacter baumannii pose major public health threats, while biofilm formation further reduces the effectiveness of existing antibiotics. Although antimicrobial peptides are considered potential alternatives, their development is frequently limited by systemic toxicity. In this study, five antimicrobial peptides were evaluated against clinical isolates of K. pneumoniae and A. baumannii for antimicrobial activity, inhibition of biofilm biomass, and hemolytic activity, and NA-CATH was selected for subsequent optimization because of its comparatively strong in vitro activity and low hemolysis. Structure-guided rational design and computational screening of nearly 50,000 variants were then applied to improve its activity–toxicity profile. Despite its favorable in vitro performance, NA-CATH caused 100% mortality in Galleria mellonella, indicating substantial in vivo toxicity. Initial rational modifications reduced toxicity but were accompanied by decreased antimicrobial activity. Subsequent computational screening identified NA-7, which retained antimicrobial activity, increased survival in K. pneumoniae-infected larvae compared with the parental peptide, and showed lower acute toxicity and fewer renal histopathological alterations in mice. Collectively, NA-7 exhibited an improved balance between antimicrobial activity and in vivo tolerability relative to NA-CATH in the evaluated models, supporting its further investigation as an optimized lead peptide and demonstrating the utility of iterative computational screening for antimicrobial peptide optimization.