To understand how peptide organization in aqueous solution might affect the activity of antimicrobial peptides, the potency of various dermaseptin S4 analogs was assessed against human red blood cells (RBC), protozoa, and several Gram-negative bacteria. Dermaseptin S4 had weak antibacterial activity but potent hemolytic or antiprotozoan effects. K 4 K 20 -S4 was 2-3-fold more potent against protozoa and RBC, yet K 4 K 20 -S4 was more potent by 2 orders of magnitude against bacteria. K 4 -S4 had similar behavior as K 4 K 20 -S4, but K 20 -S4 and analogous negative charge substitutions were as active as dermaseptin S4 or had reduced activity. Binding experiments suggested that potency enhancement was not the result of increased affinity to target cells. In contrast, potency correlated well with aggregation properties. Fluorescence studies indicated that K 20 -S4 and all negative charge substitutions were as aggregated as dermaseptin S4, whereas K 4 -S4 and K 4 K 20 -S4 were clearly less aggregated. Overall, the data indicated that N-terminal domain interaction between dermaseptin S4 monomers is responsible for the peptide's oligomerization in solution and, hence, for its limited spectrum of action. Moreover, bell-shaped dose-response profiles obtained with bacteria but not with protozoa or RBC implied that aggregation can have dramatic consequences on antibacterial activity. Based on these results, we tested the feasibility of selectivity reversal in the activity of dermaseptin S4. Tampering with the composition of the hydrophobic domains by reducing hydrophobicity or by increasing the net positive charge affected dramatically the peptide's activity and resulted in various analogs that displayed potent antibacterial activity but reduced hemolytic activity. Among these, maximal antibacterial activity was displayed by a 15-mer version that was more potent by 2 orders of magnitude compared with native dermaseptin S4. These results emphasize the notion that peptide-based antibiotics represent a highly modular synthetic antimicrobial system and provide indications of how the peptide's physico-chemical properties affect potency and selectivity.
Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli were, respectively, 1 to 4, 1 to 4, and 1 to 16 g/ml. MICs of the short derivatives were rather similar or two to fourfold higher. Each of the three peptides was rapidly bactericidal in vitro, reducing the number of viable CFU of either E. coli or S. aureus by 6 log units in 30 min or less. Compared with MSI-78 or PG-1, K 4 -S4(1-13) was at least as potent against bacteria (assessed at two MIC multiples) but displayed lesser toxicity against human erythrocytes. Serial passage in subinhibitory concentrations led to emergence of resistance to commercial antibiotics but not to the L-or D isomer of either of the dermaseptin derivatives. The short derivatives were further investigated for antibacterial activity in vivo, using a peritonitis model of mice infected with P. aeruginosa. Naive mice in the vehicle control group exhibited 75% mortality, compared to 18 or 36% mortality in mice that received a single intraperitoneal injection (4.5 mg/kg) of K 4 -S4(1-16) or K 4 -S4(1-13), respectively. In vivo bactericidal activity was confirmed in neutropenic mice, where intraperitoneal administration of K 4 -S4(1-16) reduced the number of viable CFU in a dose-dependent manner by >3 log units within 1 h of exposure, and this was sustained for at least 5 h. Overall, the data suggest that dermaseptin S4 derivatives could be useful in treatment of infections, including infections caused by multidrug-resistant bacteria.
The hemolytic antimicrobial peptide dermaseptin S4 was recently shown to exert antimalarial activity. In this study, we attempted to understand the underlying mechanism(s) and identify derivatives with improved antimalarial activity. A number of dermaseptin S4 derivatives inhibited parasite growth with a 50% inhibitory concentration (IC 50 ) in the micromolar range. Among these, the substituted S4 analog K 4 K 20 -S4 was the most potent (IC 50 ؍ 0.2 M), while its shorter version, K 4 -S4(1-13)a, retained a considerable potency (IC 50 ؍ 6 M). Both K 4 K 20 -S4 and K 4 -S4(1-13)a inhibited growth of the parasites more at the trophozoite stage than at the ring stage. Significant growth inhibition was observed after as little as 1 min of exposure to peptides and proceeded with nearly linear kinetics. The peptides selectively lysed infected red blood cells (RBC) while having a weaker effect on noninfected RBC. Thus, K 4 K 20 -S4 lysed trophozoites at concentrations similar to those that inhibited their proliferation, but trophozoites were >30-fold more susceptible than normal RBC to the lytic effect of K 4 K 20 -S4, the most hemolytic dermaseptin. The same trend was observed with K 4 -S4(1-13)a. The D isomers of K 4 K 20 -S4 or K 4 -S4(1-13)a were as active as the L counterparts, indicating that antimalarial activity of these peptides, like their membrane-lytic activity, is not mediated by specific interactions with a chiral center. Moreover, dissipation of transmembrane potential experiments with infected cells indicated that the peptides induce damage in the parasite's plasma membrane. Fluorescence confocal microscopy analysis of treated infected cells also indicated that the peptide is able to find its way through the complex series of membranes and interact directly with the intracellular parasite. Overall, the data showed that dermaseptins exert antimalarial activity by lysis of infected cells. Dermaseptin derivatives are also able to disrupt the parasite plasma membrane without harming that of the host RBC.
Dermaseptins are a family of antimicrobial peptides that lyse target bacterial cells by destabilization of their membranes. Here we present a novel application of a peptide derived from the dermaseptin S4, S4(13). At nontoxic concentrations, fluorescently labeled S4(13) was able to penetrate intact cultured HeLa cells but essentially failed to enter their nuclei despite its low molecular weight. Covalent attachment of nuclear localization signal (NLS) motifs of the SV40-T-antigen and of the HIV-1 Rev protein (ARM) conferred karyophilic properties upon the S4(13). The resulting peptides, which were designated as PV-S4(13) and RR-S4(13) penetrated into intact HeLa cells and were able to accumulate within the cells' nuclei. In studies with digitonin-permeabilized cells, nuclear uptake of the PV-S4(13) and the RR-S4(13) peptides showed the same features that characterize active nuclear import. Nuclear import was observed at 37 degrees C, was ATP-dependent, and was inhibited by the free peptides bearing the SV40 NLS and the Rev and Tat ARMs. Microinjected S4(13) remained in the cytoplasm while microinjected RR-S4(13) was translocated into the cells' nuclei. The new type of cell-permeable "karyophilic" peptides described here may be of potential application as a lead compound for therapeutic purposes, as a tool to study nucleocytoplasmic shuttling in intact cells, and for the delivery of peptides to the nucleus.
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