Development of novel broad-spectrum amphipathic antimicrobial peptides against multidrug-resistant bacteria through a rational combination strategy

In recent years, cationic amphipathic antimicrobial peptides (AMPs) have shown great promise in combating antibiotic resistance on account of their distinctive membrane-disruptive mechanism. However, the clinical application of AMPs is restricted by their unsatisfactory stability and safety. Although attempts have been made to improve the stability and safety of AMPs, many of them are accompanied by a decline in their antimicrobial activity and bacterial selectivity. To develop AMPs with excellent and balanced antimicrobial activity, stability, and safety using a combination strategy. A series of sC184b-derived peptide analogues were designed by a combination strategy of subtly adjusting the charges, hydrophobic properties, and introducing specific unnatural amino acids in a well-balanced manner. The antimicrobial activity, cytotoxicity, hemolytic activity, stability, anti-biofilm activity, mechanism of action, synergistic effects, in vivo efficacy, and pharmacokinetics of the analogues were evaluated. Among these analogues, P-α-02-B stood out for its broad-spectrum and potent antimicrobial activity, anti-biofilm activity, desirable bacterial selectivity, high plasma stability, and synergistic effect with antibiotic levofloxacin. P-α-02-B exhibited strong membrane disturbance effect, which could be explained by its rigid α-helical structure revealed by molecular dynamics simulations. More importantly, P-α-02-B showed favorable therapeutic efficacy in vivo, whether used alone or in combination with levofloxacin. P-α-02-B is a promising antimicrobial agent for MDR bacterial infections, demonstrating the effectiveness of the combination strategy for AMP development.