The invention of antibiotics, which ranks among the most remarkable achievements, has made extraordinary contributions to combating pathogenic infections [1]. Regrettably, the abuse of antibiotics has expedited the advancement of antimicrobial resistance (AMR), which has emerged as a prominent menace to public health. It is estimated that by 2050, there will be an alarming annual death toll of 10 million individuals attributed to AMR [2,3]. Therefore, it has become imperative to develop new antimicrobial agents.

Antimicrobial peptides (AMPs) have garnered much attention as potential candidates or adjuvants of antibiotics due to their commendable antibacterial properties and the characteristic of not easily inducing resistance [4,5]. AMPs play a vital role in the host defense system and are extensively found in plants, animals, microorganisms, and other organisms, with multiple biological functions such as inhibiting bacterial growth, preventing fungal infections, and combating viruses [6,7]. Despite the abundant chemical diversity observed in natural AMPs, they share common structural features. Most AMPs consist of 5–50 amino acid residues in their sequences and possess an amphipathic structure formed by a substantial number of hydrophobic and positively charged amino acids [8]. The specific mechanism of membrane lysis exhibited by AMPs poses a significant obstacle to the development of AMR, as mutating the bacterial cell membrane structure is highly challenging [9]. This is different from the mechanism of action exhibited by antibiotics, which mainly encompass four mechanisms: antibiotics such as penicillin and cephalosporin kill bacteria by inhibiting the synthesis of bacterial cell walls; antibiotics like ciprofloxacin and rifampicin exert antibacterial effects by impeding the replication and transcription of nucleic acids; antibiotics such as gentamicin and tetracycline kill bacteria by inhibiting protein synthesis; Peptide-based antibiotics such as polymyxin B interact with bacterial cell membranes, enhancing their permeability and exerting antibacterial effects [10]. Over the past three decades, researchers have discovered more than 3000 naturally derived AMPs, but most of them encounter several limitations that prevent them from being reliable therapeutic agents. These limitations encompass the potential for adverse effects on the host, susceptibility to breakdown by proteases, significant binding in serum, diminished antimicrobial efficacy when exposed to physiological levels of salts, and expensive production costs [11,12]. In addition, if bacteria develop resistance to naturally derived AMPs, it will pose a substantial challenge to human and animal immune systems. Therefore, the de novo design of AMPs featuring unique structures is necessary to proactively address this potential crisis [13].

Faced with these existing obstacles of natural AMPs, we attempted to propose a minimalist design strategy to overcome them. The proposed strategy for designing AMPs is that their sequences are short (typically less than twenty amino acids) and consist of fewer than four types of amino acids, as well as possessing simple molecular structures [14]. In the minimalistic design of AMPs, the pairing of the hydrophilic charged arginine (R) with the hydrophobic tryptophan (W) is a canonical and valid “golden partner” [15,16]. R and W residues are frequently observed in numerous AMPs and play indispensable roles in the insertion into membranes and permeabilization caused by AMPs, which are momentous stages in their antimicrobial activity [17]. Initially, the cationic charge of R originating from the guanidinium group serves as an efficient means of inducing peptides to target bacterial cell membranes. After that, R forms hydrogen bonding to facilitate its interaction with anionic components present on bacterial surfaces, including LPS, phospholipid headgroups, and teichoic acid [18,19]. The subsequent role of W is pivotal, as it is widely recognized for its exceptional ability to promote the connection between peptides and the membrane of bacteria and assist in efficiently embedding these peptides within the membrane due to its unique indole side chain [20,21]. Despite the combination that can confer a powerful antibacterial effect on AMPs, a large number of AMPs rich in R and W residues are susceptible to hydrolysis by peptidases, thereby limiting their practical application [22,23]. Therefore, in this research, we endeavored to introduce a new dendritic structure for AMPs that enhances their stability while distinguishing their structures from natural linear AMPs.

A dendrimer is a polymer molecule consisting of several branched monomers that extend radially from a central core, resembling a tree, thus deriving its name from the Greek word “dendra” [24]. The pioneering studies on dendrimers were first reported in the 1970s and 1980s by research groups led by Vögtle et al. [24]. Antimicrobial peptide dendrimers (AMPDs) consist of a multivalent core that tethers an array of dendritic peptides to enhance their performance against bacteria and resistance to proteolytic digestion. In recent years, AMPDs have garnered much attention as potential alternatives to antibiotics [24]. The dendrimeric structure in AMPDs was introduced as a multiple antigen peptide (MAP), which produces a synthetic peptide in the form of an oligomer or macromolecule using the solid-phase peptide synthesis method [25]. AMPDs usually utilize natural or non-natural diamino acids, disulfide bonds, or organic scaffolds as the dendritic core, such as Cys, Lys, Orn, Asp, Dab, DTM, Glu, or triazole linkage [26,27]. For example, Lys and 3,3′-diaminobenzidine (Dab) were used to synthesize the dimer of AMP Chex-Arg20, and tetramerization of Chex1-Arg20 was achieved by dithiomaleimide (DTM) conjugation of two C-terminal-cysteine bearing dimers [[28], [29], [30]]. Among these options, Lys is one of the most commonly used scaffolds. Compared to linear peptides, AMPDs generally exhibit higher activity as a result of the increased concentration at the local level of bioactive units in this multivalent assembly and greater stability resulting from their branched-core nature that creates steric hindrance and restricts peptidase cleavage in plasma [24]. However, the synthesis yield of AMPDs with multiple branches poses challenges due to their relatively large molecular size [31]. Therefore, we attempted to introduce a simplified dendritic structure using Lys as the scaffold while applying it to the minimalist design strategy described above.

To obtain ideal AMPs, we put forward a minimalist design strategy for R and W-rich dendritic AMPs, utilizing αRn(εRn)KWm-NH2 and αWn(εWn)KRm-NH2 as the general structural formulas. After careful consideration of the amphiphilicity, amino acid quantity, and charge distribution position, the new dendritic AMPs were designed and shown in Fig. 1, with their corresponding linear peptides serving as the controls. Following synthesis and purification, the newly designed AMPs were evaluated for their antimicrobial properties and biocompatibility, and the relationships between their structure and antimicrobial potency were analyzed in detail. Subsequently, four representative peptides were selected for evaluation of stability under different conditions. Then, the dendritic peptide R2(R2)KW4 was screened out, followed by an investigation into its mechanism of action. The induced resistance level of R2(R2)KW4, as well as its combined effect with the conventional antibiotics, were also evaluated. Finally, the toxicity and antibacterial activity in vivo of R2(R2)KW4 were assessed through acute toxicity test and bacterial lung infection mice model, respectively.