Rational Leucine-Centric Design Confers Enhanced Proteolytic Stability and Reduced Toxicity of Antimicrobial Peptides against Multidrug-Resistant Bacterial Infections

Antimicrobial peptides (AMPs), emerged as appealing therapeutic agents against multidrug-resistant (MDR) bacteria, face clinical obstacles due to unsatisfactory stability and mammalian toxicity. Rational design approaches capable of balancing the efficacy, stability, and safety of AMPs are greatly needed. Here, we engineered a series of derivatives of P-α-02-B─an AMP developed by our group─through site-specific modifications targeting leucine residues to improve proteolytic stability and reduce toxicity. Notably, the d-Ile substitution in the derivative H-10 synergistically enhanced proteolytic stability and attenuated toxicity. Further investigations revealed that H-10 exhibited rapid bactericidal action via membrane disruption. The bacterial selectivity of H-10 was revealed by molecular dynamics simulations. <i>In vivo</i> assessments confirmed its favorable safety profile and pharmacokinetic properties, as well as significant efficacy against <i>Staphylococcus aureus</i> (MDR) bacterial infections. Collectively, this study positions H-10 as a promising candidate against MDR infection while establishing leucine-centric design as a valuable paradigm in AMP optimization.