The novel β-hairpin antimicrobial peptide D-G(RF)3 demonstrates exceptional antibacterial efficacy.

The clinical application of most natural antimicrobial peptides (AMPs) is hindered by their lack of a synergistic combination of high antibacterial efficacy, low toxicity, and stability, necessitating frequent complex modifications that incur significant labor and economic costs. Therefore, it is imperative to optimize the antibacterial properties of AMPs using some simplified approach. In this study, we designed a library of β-hairpin AMPs with identical β-turn sequences (-D-Pro-Gly-) and varying repetition units (IR, FR, and WK). Ultimately, candidate peptide G(RF)3 exhibited high antibacterial activity and low toxicity; however, its stability was compromised. Moreover, we synthesized the new analogue D-G(RF)3 by D-type amino acid substitution of G(RF)3, and D-G(RF)3 demonstrated concurrent high antibacterial activity, low toxicity, and remarkable stability. Interestingly, both G(RF)3 and D-G(RF)3 exerted bactericidal effects by disrupting the bacterial membrane. However, D-G(RF)3 displayed superior antibiofilm activity with a faster bactericidal rate compared to G(RF)3 and also showed enhanced synergy with antibiotics. Furthermore, D-G(RF)3 exhibited potent in vivo bactericidal activity without inducing drug resistance and has the potential to be a novel antibiotic alternative or adjuvant.