New antibiotics are urgently needed to address multidrug-resistant (MDR) bacteria. Herein we report that second-generation (G2) peptide dendrimers bearing a fatty acid chain at the dendrimer core efficiently kill Gram-negative bacteria including Pseudomonas aeruginosa and Acinetobacter baumannii, two of the most problematic MDR bacteria worldwide. Our most active dendrimer TNS18 is also active against Gram-positive methicillin-resistant Staphylococcus aureus. Based on circular dichroism and molecular dynamics studies, we hypothesize that TNS18 adopts a hydrophobically collapsed conformation in water with the fatty acid chain backfolded onto the peptide dendrimer branches and that the dendrimer unfolds in contact with the membrane to expose its lipid chain and hydrophobic residues, thereby facilitating membrane disruption leading to rapid bacterial cell death. Dendrimer TNS18 shows promising in vivo activity against MDR clinical isolates of A. baumannii and Escherichia coli, suggesting that lipidated peptide dendrimers might become a new class of antibacterial agents.

Multidrug-resistant opportunistic bacteria, such as Pseudomonas aeruginosa, represent a major public health threat. Antimicrobial peptides (AMPs) and related peptidomimetic systems offer an attractive opportunity to control these pathogens. AMP dendrimers (AMPDs) with high activity against multidrug-resistant clinical isolates of P. aeruginosa and Acinetobacter baumannii were now identified by a systematic survey of the peptide sequences within the branches of a distinct type of third-generation peptide dendrimers. Combined topology and peptide sequence design as illustrated here represents a new and general strategy to discover new antimicrobial agents to fight multidrug-resistant bacterial pathogens.

We used nearest-neighbor searches in chemical space to improve the activity of the antimicrobial peptide dendrimer (AMPD) G3KL and identified dendrimer T7, which has an expanded activity range against Gram-negative pathogenic bacteria including Klebsiellae pneumoniae, increased serum stability, and promising activity in an in vivo infection model against a multidrug-resistant strain of Acinetobacter baumannii. Imaging, spectroscopic studies, and a structural model from molecular dynamics simulations suggest that T7 acts through membrane disruption. These experiments provide the first example of using virtual screening in the field of dendrimers and show that dendrimer size does not limit the activity of AMPDs.

We recently discovered that peptide dendrimers such as G3KL ((KL)8(KKL)4(KKL)2 KKL, K = branching l-lysine) exert strong activity against Gram-negative bacteria including Pseudomonas aeruginosa, Acinetobacter baumannii, and Escherichia coli. Herein, we report a detailed mechanistic study using fluorescence labeled analogs bearing fluorescein (G3KL-Fluo) or dansyl (G3KL-Dansyl), which show a similar bioactivity profile as G3KL. Imaging bacterial killing by super-resolution stimulated emission depletion (STED) microscopy, time-lapse imaging, and transmission electron microscopy (TEM) reveals that the dendrimer localizes at the bacterial membrane, induces membrane depolarization and permeabilization, and destroys the outer leaflet and the inner membrane. G3KL accumulates in bacteria against which it is active; however, it only weakly penetrates into eukaryotic cells without inducing significant toxicity. G3KL furthermore binds to lipopolysaccharide (LPS) and inhibits the LPS induced release of TNF-α by macrophages, similarly to polymyxin B. Taken together, these experiments show that G3KL behaves as a potent membrane disruptive antimicrobial peptide.

eThe in vitro activity of the novel antimicrobial peptide dendrimer G3KL was evaluated against 32 Acinetobacter baumannii (including 10 OXA-23, 7 OXA-24, and 11 OXA-58 carbapenemase producers) and 35 Pseudomonas aeruginosa (including 18 VIM and 3 IMP carbapenemase producers) strains and compared to the activities of standard antibiotics. Overall, both species collections showed MIC 50/90 values of 8/8 g/ml and minimum bactericidal concentrations at which 50% or 90% of strains tested are killed (MBC 50/90 ) of 8/8 g/ml. G3KL is a promising molecule with antibacterial activity against multidrug-resistant and extensively drug-resistant A. baumannii and P. aeruginosa isolates. The spread of Acinetobacter baumannii and Pseudomonas aeruginosa isolates that are resistant to carbapenem antibiotics due to the production of carbapenemases represents a serious threat (1). These strains are usually multidrug-resistant (MDR) due to the coexpression of mechanisms involving other classes of antibiotics, thus drastically limiting our therapeutic armamentarium (2-4). In particular, extensively drug-resistant (XDR) isolates are commonly detected worldwide (5), whereas the prevalence of pandrug-resistant (PDR) isolates is increasing worryingly in several countries (6-8). Therefore, novel antimicrobial strategies need to be rapidly developed.Recently, there has been a rising interest in evaluating naturally occurring or synthetic antimicrobial peptides (AMPs) with activity against prokaryotic membranes. This attention is due to their wide spectrum of activity against both Gram-positive and Gramnegative species, potent bactericidal activity, and ability to bypass common mechanisms of resistance that affect standard antibiotics (9, 10). However, several reasons have so far limited the clinical implementation of AMPs: (i) high susceptibility to degradation by endogenous and microbial proteases; (ii) toxicity due to the high concentration necessary to inhibit bacteria; and (iii) short half-life because of high protein binding (11). Several authors have modified AMPs to obtain proteolytically resistant versions, mostly by sequence variations and the use of D-amino acids (12-15). However, redesigning the peptide chain topology, in particular by introducing multiple branching points to obtain synthetic AMP dendrimers (AMPDs), seems a promising solution to overcome all of the the aforementioned problems (16-18).G3KL is a novel AMP dendrimer (AMPD) developed at the Department of Chemistry and Biochemistry of the University of Bern (Switzerland) by sequence optimization of an initial hit compound identified by screening a combinatorial library of dendrimers using a tailored high-throughput screening assay and presumed to act as a membrane-disrupting agent (19)(20)(21)(22). Its activity requires a dendritic topology and only natural lysine and leucine residues alternating in the branches (Fig. 1). This novel AMPD has demonstrated in vitro activity against several Gram-negative strains, low toxicity to human red blood cells (minimal hemolytic ...