Efficient Screening of High Bacterial Cell-Selective Antimicrobial Peptides via Proline Regulation Combined with a Universal Secondary Structure Template

Abstract To study the ″preference″ of bacterial membranes for the secondary structure of antimicrobial peptides (AMPs), we designed a universal palindromic sequence template G(LK)n(KL)nG-NH2 (designated as GnPm, where n = 2–6 and m = 0–3) that can cover various secondary structures of AMPs, with 0 to 3 proline residues uniformly and dispersedly inserted in the middle region to regulate their secondary structures. Results confirmed that both the numbers of LK/KL repeats and proline can regulate secondary structure, thereby controlling the antibacterial activity of the AMPs. The optimized peptide G5P2 exhibits potent antibacterial activity via a typical membrane-disrupting mechanism, is not prone to inducing bacterial drug resistance, can synergize with traditional antibiotics to delay the emergence of antibiotic resistance, and has excellent in vivo antibacterial activity (a 93.52% bacterial clearance) and high safety (LD50 = 89.87 mg/kg), thus providing new chemical strategies and candidate molecules for the clinical management of drug-resistant bacterial infections.