Antibacterial Diamines Targeting Bacterial Membranes

Abstract

Antibiotic resistance is a growing threat to human health exacerbated by a lack of new antibiotics. We now describe a series of substituted diamines that produce rapid bactericidal activity against both Gram-positive and Gram-negative bacteria, including methicillin-resistant Staphylococcus aureus and stationary-phase bacteria. These compounds reduce biofilm formation and promote biofilm dispersal in Pseudomonas aeruginosa. The most potent analogue, 3 (1,13-bis{[(2,2-diphenyl)-1-ethyl]thioureido}-4,10-diazatridecane), primarily acts by depolarization of the cytoplasmic membrane and permeabilization of the bacterial outer membrane. Transmission electron microscopy confirmed that 3 disrupts membrane integrity rapidly. Compound 3 is also synergistic with kanamycin, demonstrated by the checkerboard method and by time-kill kinetic experiments. In human cell toxicity assays, 3 showed limited adverse effects against the HEK293T human kidney embryonic cells and A549 human adenocarcinoma cells. In addition, 3 produced no adverse effects on Caenorhabditis elegans development, survival, and reproduction. Collectively, diamines related to 3 represent a new class of broad-spectrum antibacterials against drug-resistant pathogens.

Figure 1

Structures of (bis)ureido or (bis)thioureido(N,N′-alkyl)-diamines 1–10 and (bis)ureido or (bis)thioureidoalkanes 10–16.

Figure 2

Effect of 3 on biofilm formation (A) and dispersal (B). The pegged-lid method was used to grow the P. aeruginosa biofilm, and the effect of 3 was compared with tetracycline (TET), norfloxacin (NOR), and kanamycin (KAN). EDTA was used as a positive control for biofilm dispersal. Each data point is the average of 3 determinations ± SEM.

Figure 3

Viability of exponential-phase and stationary-phase bacteria in the presence of 3 or the indicated antibiotics. (A) Exponential-phase S. aureus and (B) stationary-phase S. aureus were treated with antibiotics at the following concentrations: 0.5 μg/mL ampicillin sodium (1.35 μM), 32 μg/mL kanamycin sulfate (54.9 μM), 2 μg/mL norfloxacin (6.26 μM), and 8 μg/mL 3 (11.5 μM). (C) Exponential-phase P. aeruginosa and (D) stationaryphase P. aeruginosa were treated with 512 μg/mL ampicillin sodium (1.38 mM), 128 μg/mL kanamycin sulfate (219.6 μM), 2 μg/mL norfloxacin (6.26 μM), and 32 μg/mL 3 (41.6 μM).

Figure 4

(A) S. aureus cytoplasmic membrane depolarization promoted by 3. (B) P. aeruginosa cytoplasmic membrane depolarization promoted by 3. (C) P. aeruginosa outer membrane permeabilization promoted by 3. The arrow indicates the time point at which the inhibitor was added. Experiments were performed three times independently, and data from a representative experiment are shown.

Figure 5

(A) Bactericidal kinetics resulting from cytoplasmic membrane depolarization of S. aureus by 3. (B) Bactericidal kinetics resulting from cytoplasmic membrane depolarization of P. aeruginosa by 3. Each data point is the average of 3 determinations ± SEM.

Figure 6

Transmission electron micrographs of (A) untreated S. aureus; (B,C) S. aureus treated with 3 at 2× its MIC₉₉; (D) untreated P. aeruginosa; (E,F) P. aeruginosa treated with 3 at 2× its MIC₉₉. The arrows mark areas of membrane damage and cell content release.

Figure 7

Isobolograms showing the synergistic effects of 3 with kanamycin against S. aureus and P. aeruginosa.

Figure 8

Interaction of 3 and kanamycin (KAN) against (A) S. aureus and (B) P. aeruginosa, determined by time-kill experiments that were performed three times independently. Each data point is the average of 3 determinations ± SEM.

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