Due to the overuse of antibiotics and their limited target sites, multidrug-resistant bacteria, such as “ESKPAE” (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, and Enterobacter spp.), pose a significant threat to human lives [1,2]. Mathematical models predicted that more than 10 million people could die annually from infections caused by drug-resistant bacteria by 2050 if the current rate of growth continues, surpassing all other causes of death, including cancer [3]. The rapid spread of antibiotic resistance is outpacing the development of new antimicrobial drugs, leading to an alarming public health crisis [4].

In recent years, antimicrobial peptides (AMPs) have been considered as potential alternatives for overcoming multidrug-resistant bacterial infections [[5], [6], [7]]. These peptides are host defense peptides, with most of them being cationic (positively charged) and amphiphilic (hydrophilic and hydrophobic) α-helical peptide molecules [8]. Cationic AMPs interact with negatively charged lipopolysaccharides (LPS) or lipoteichoic acids (LTA) on bacterial membranes through electrostatic interactions, utilizing their hydrophobicity to insert into the bacterial membrane and disrupt its integrity, ultimately leading to bacterial death [9].

AMPs have been demonstrated to have their own advantages over the traditional antibiotics with a broad-spectrum of antimicrobial activities including antibacterial, antifungal, antiviral, and anticancer properties [10,11]. However, AMPs also exhibit limitations in clinical application, such as toxicity to host cells, instability to proteases and serum, sensitivity to salt and high production costs. Recently, researchers primarily focus on exploring the hydrophobicity and positive charge, which are two key features of AMPs, to study the structure-activity relationship and modify existing AMPs in order to discover AMPs with potential clinical applications [12,13]. Hydrophobic amino acids, such as Trp (W), Phe (F), and Leu (L), along with fatty acids, play a crucial role in conferring hydrophobicity to AMPs. The presence of these hydrophobic amino acids enhances the interaction between AMPs and bacterial membranes, thus improving their antimicrobial activity [14]. On the other hand, AMPs typically have 2–9 positive charges attributed to positively charged amino acids like Arg (R), Lys (K), and His (H) [15]. Through comprehensive research and meticulous design, researchers hope to discover AMPs with enhanced potential for clinical applications and provide new ideas and possibilities for the development and application of antimicrobial drugs.

Compared to naturally occurring AMPs encoded and synthesized on ribosomes, naturally occurring lipopeptides are a class of structurally diverse antibiotics synthesized through non-ribosomal mechanisms, with their N-terminus conjugated to long-chain fatty acids [16]. Among these lipopeptide antibiotics, Polymyxin B, produced by soil bacteria, has been utilized for the treatment of infections caused by Gram-negative bacteria. However, due to its pronounced nephrotoxicity and the emergence of drug resistance, Polymyxin B is now reserved as a last resort for otherwise untreatable severe infections [17]. Brevicidine is a cyclic lipopeptide (C7H13O–N(D)-Y(D)-W(D)-Orn(D)-Orn-G-Orn(D)-W-cyclo[T-I-G-S]) discovered in Brevibacillus laterosporus DSM 25 in 2018, which conjugated with (R,S)-4-methylhexanoic acid at the N-terminus and composed of a lactone ring formed by four amino acids at the C-terminus, containing three non-natural amino acid 2, 5-diaminovaleric acid (Orn) and five d-amino acids within its linear peptide portion (Fig. 1) [18]. Previous studies have demonstrated that Brevicidine exhibited significant antimicrobial activity against Gram-negative bacteria, including multidrug-resistant Pseudomonas aeruginosa and colistin-resistant Escherichia coli. The incorporation of numerous non-natural amino acids and d-amino acids has enhanced the stability of Brevicidine. Moreover, there was no resistance was observed when Escherichia coli was continuously passaged under sub-inhibitory concentrations of Brevicidine for 30 days, indicating a low risk of resistance development [18]. Unfortunately, limited efficacy against Gram-positive bacteria along with complex synthesis procedures and low yield have impeded broader applications for Brevicidine. To further explore novel AMPs with improved properties, the structure-activity relationship of Brevicidine was studied in this work.

In this study, a series of novel Brevicidine analogs were synthesized by incorporating diverse hydrophobic amino acids, N-terminal fatty acids, and varying types and quantities of positively charged amino acids. Based on the structure-activity relationship analysis of Brevicidine, it was observed that there exists a positive correlation between the hydrophobicity of the newly designed peptides and their hemolytic activity. Furthermore, modulation of peptide hydrophobicity was found to influence their antimicrobial spectrum; Specifically, increased hydrophobicity enhanced efficacy against Gram-positive bacteria while reducing effectiveness against Gram-negative bacteria. Additionally, analog 22 with an optimal therapeutic index was selected for further investigation into its stability, development of drug resistance, antimicrobial mechanisms, as well as safety and efficacy in vivo.