In the past 20 years, over 400 cationic antimicrobial peptides have been identified from extremely diverse sources from unicellular organisms to mammals, including humans (2
). Presently, cationic antimicrobial peptides are believed to represent an important, though poorly understood, component of the innate host defenses. Antimicrobial peptides display a large heterogeneity in primary and secondary structures but share common features such as amphipathy and net positive charge. These features seem to form the basis for their cytotoxic function. The facts that some antimicrobial peptides are active against a large spectrum of microorganisms and that isomers composed of d
amino acids display potency identical to that of the l
counterparts are consistent with the hypothesis that cytotoxic activity is not mediated by interaction with a chiral center. Although their precise mechanism of action is not fully understood, a large body of data indicates that antimicrobial peptides kill target cells by interacting with and destabilizing the ordered structure of the cell membrane(s) (9
). These findings imply that peptide-based antimicrobials could escape the mechanisms involved in multidrug resistance.
In addition to their direct membrane-disrupting activity, cationic antimicrobial peptides are reportedly able to activate cells of the innate immunity, such as leukocytes and monocytes/macrophages. Thus, the frog antimicrobial peptide dermaseptin S1 was shown to stimulate microbicidal activities of rat and human leukocytes (1
), and more recently, the insect-derived peptide CEMA (a cecropin-melittin hybrid) was reported to induce the expression of 35 genes in macrophages (27
). Moreover, an increasing number of cationic peptides and proteins, including lactoferrin (3
), bactericidal/permeability increasing protein (8
), synthetic antiendotoxin peptides (7
), dermaseptins (unpublished data), and CEMA (14
), are reportedly endowed with lipopolysaccharide (LPS) binding activity. LPS is a potent activator of macrophages and is responsible for sepsis caused by gram-negative bacteria. By binding LPS, these peptides were shown to block the interaction of LPS with LPS-binding protein, suppress the ability of LPS to stimulate the production of inflammatory cytokines by macrophages, and protect animals from lethal endotoxic shock.
Thus, cationic antibacterial peptides have in recent years been attracting increasing interest of both the scientific community and the pharmaceutical industry for their potential as new therapeutic agents. However, it is widely believed that this family of agents lacks specificity and might be too toxic for systemic treatment (15
). Therefore, topical use has been chosen for various antimicrobial peptides that are currently undergoing clinical trials (4
). In this study, we report an evaluation of the in vitro antibacterial properties of three dermaseptin S4 derivatives and present data demonstrating in vivo safety and efficacy.
Dermaseptins are a large family of antimicrobial peptides (28 to 34 amino acids) expressed in amphibian skin. These linear polycationic peptides are unstructured in polar media but readily switch to an amphipathic α-helix in apolar solvents. Dermaseptins display cytolytic activity in vitro generally against a broad spectrum of host-free microorganisms, including bacteria, protozoa, yeasts, and filamentous fungi (5
), as well as against intracellular parasites (13
). Unlike most dermaseptin members, the 28-residue dermaseptin S4 is highly toxic to erythrocytes. This toxicity is probably related to its high hydrophobicity, as both nuclear magnetic resonance and fluorescence methods have indicated that the peptide is in a high aggregation state in aqueous solutions (13
), whereas recent data (10
) revealed that N-terminal domain interaction between dermaseptin S4 monomers is responsible for the peptide's oligomerization in solution. Aggregation in solution is probably further responsible for limiting its spectrum of potential target cells. Thus, bell-shaped dose-response profiles obtained with bacteria but not with protozoa or red blood cells (RBC) (which lack a cell wall) implied that peptide aggregation in solution is an important factor affecting selective activity (10
). Tampering with the composition of the hydrophobic domains by reducing hydrophobicity or by increasing the net positive charge of native dermaseptin S4 resulted in a number of analogs that displayed enhanced antibacterial activity and reduced hemolytic activity. Among these, K4
-S4 was two- to threefold more potent than native dermaseptin S4 against protozoa and RBC, yet K4
-S4 was more potent by 2 orders of magnitude against bacteria. Also, a 16-mer version, K4
-S4(1-16), displayed antibacterial activity against Escherichia coli
that was more potent by 2 orders of magnitude (50% inhibitory concentration, 0.4 μM) than that of native dermaseptin S4 (50% inhibitory concentration, 40 μM). In contrast, this analog displayed reduced hemolytic activity (50% lethal concentration, 20 μM compared to 1.4 μM). An even shorter dermaseptin S4 derivative, K4
-S4(1-13), displayed a twofold-reduced antibacterial activity and a twofold-increased relative selectivity ratio due to its low hemolytic activity (10
). In addition, when injected intravenously into rats, K4
-S4(1-13) was well tolerated at the high dose of 10 mg of peptide/kg of body weight (11
). These peptides were selected for this study to undergo a further assessment of their potential as antibacterial agents.