The effect of charge increase on the specificity and activity of a short antimicrobial peptide

By using short linear antimicrobial peptides as a model system, the effect of peptide charge on the specificity between Candida albicans (fungi) and Gram-positive bacteria was investigated. In a present study, we added and/or deleted lysine residue(s) at the C-terminal and/or N-terminal end(s) of an antimicrobial peptide (KKVVFKVKFK-NH(2)) and synthesized the peptides that had similar alpha helical structures in a lipid membrane mimic condition. The increase of peptide charge improved antifungal activity without the change of antibacterial activity. Structure-activity relationship study about the peptides revealed that the net positive charge must play an important role in the specificity between C. albicans and Gram-positive bacteria and the increase of the net positive charge without the moderate change of secondary structure could improve activity for C. albicans rather than Gram-positive bacteria.     

Lactoferrampin, an antimicrobial peptide of bovine lactoferrin, exerts its candidacidal activity by a cluster of positively charged residues at the C-terminus in combination with a helix-facilitating N-terminal part

The antimicrobial activity of bovine lactoferrin (bLF) is attributed to lactoferricin, which is situated in the N1-domain of bLF. Recently, another antimicrobial domain consisting of residues 268-284, designated lactoferrampin (LFampin), has been identified in the N1-domain of bLF, which exhibited antimicrobial activity against Candida albicans and several bacteria. In the present study, the candidacidal activity of a series of peptides spanning this antimicrobial domain was investigated in relation to the charge and the capacity to form a helical conformation in hydrophobic environments. C-Terminal truncation of LFampin resulted in a drastic decrease in candidacidal activity. Positively charged residues clustered at the C-terminal side of the LFampin domain appeared to be crucial for the candidacidal activity. The ability to adopt helical conformations did not change when LFampin was truncated at the C-terminal side. N-Terminally truncated LFampin peptides, truncated up to the sequence 270-284, were more reluctant to adopt a helical conformation. Therefore, we conclude that the C-terminal part of LFampin 265-284, which is the most active peptide, is crucial for its candidacidal activity, due to the presence of clustered positive charges, and that the N-terminal part is essential for activity as it facilitates helix formation.     

Structure-function studies of chemokine-derived carboxy-terminal antimicrobial peptides

Recent reports which show that several chemokines can act as direct microbicidal agents have drawn renewed attention to these chemotactic signalling proteins. Here we present a structure-function analysis of peptides derived from the human chemokines macrophage inflammatory protein-3α (MIP-3α/CCL20), interleukin-8 (IL-8), neutrophil activating protein-2 (NAP-2) and thrombocidin-1 (TC-1). These peptides encompass the C-terminal α-helices of these chemokines, which have been suggested to be important for the direct antimicrobial activities. Far-UV CD spectroscopy showed that the peptides are unstructured in aqueous solution and that a membrane mimetic solvent is required to induce a helical secondary structure. A co-solvent mixture was used to determine solution structures of the peptides by two-dimensional ¹H-NMR spectroscopy. The highly cationic peptide, MIP-3α_51-70, had the most pronounced antimicrobial activity and displayed an amphipathic structure. A shorter version of this peptide, MIP-3α_59-70, remained antimicrobial but its structure and mechanism of action were unlike that of the former peptide. The NAP-2 and TC-1 proteins differ in their sequences only by the deletion of two C-terminal residues in TC-1, but intact TC-1 is a very potent antimicrobial while NAP-2 is inactive. The corresponding C-terminal peptides, NAP-2_50-70 and TC-1_50-68, had very limited and no bactericidal activity, respectively. This suggests that other regions of TC-1 contribute to its bactericidal activity. Altogether, this work provides a rational structural basis for the biological activities of these peptides and proteins and highlights the importance of experimental characterization of peptide fragments as distinct entities because their activities and structural properties may differ substantially from their parent proteins.     

Evolution of the primate cathelicidin. Correlation between structural variations and antimicrobial activity

Cathelicidin genes homologous to the human CAMP gene, coding for the host defense peptide LL-37, have been sequenced and analyzed in 20 primate species, including Great Apes, hylobatidae, cercopithecidae, callithricidae, and cebidae. The region corresponding to the putative mature antimicrobial peptide is subject to a strong selective pressure for variation, with evidence for positive selection throughout the phylogenetic tree relating the peptides, which favors alterations in the charge while little affecting overall hydrophobicity or amphipathicity. Selected peptides were chemically synthesized and characterized, and two distinct types of behavior were observed. Macaque and leaf-eating monkey RL-37 peptides, like other helical antimicrobial peptides found in insect, frog, and mammalian species, were unstructured in bulk solution and had a potent, salt and medium independent antimicrobial activity in vitro, which may be the principal function also in vivo. Human LL-37 and the orangutan, hylobates, and callithrix homologues instead showed a salt-dependent structuring and likely aggregation in bulk solution that affected antimicrobial activity and its medium dependence. The two types of peptides differ also in their interaction with host cells. The evolution of these peptides has thus resulted in distinct mechanisms of action that affect the direct antimicrobial activity and may also modulate accessory antimicrobial functions due to interactions with host cells.     

Design and synthesis of cyclic disulfide-bonded antibacterial peptides on the basis of the alpha helical domain of Tenecin 1, an insect defensin

We synthesized cyclic disulfide-bonded (i, i+4) peptides with various net positive charges (+2-+5) from linear peptides derived from the alpha helical domain of Tenecin 1, an insect defensin, and investigated the effect of the intradisulfide bridge (i, i+4) on hydrophobicity, secondary structure, leakage activity and binding activity for large unilamellar vesicles, antimicrobial activity, and hemolytic activity. Intradisulfide bridge formation of the peptides resulted in the increase of amphiphilicity and hydrophobicity. Cyclic forms of the peptides did not deeply penetrate into PG/PC (1:1, mole ratio) large unilamellar vesicles and had a decreased lipid membrane perturbation activity for PG/PC LUVs. When the peptides interacted with PG/CL (2:1, mole ratio) LUVs, cyclic peptides with a high net positive charge (+4-+5) showed similar binding affinities and leakage activities for vesicles to those of linear forms, whereas cyclic peptides with a low net positive charge (+2-+3) exhibited lower leakage activity than their linear forms. CD spectra indicate that the intradisulfide bridge (i, i+4) provided little conformational constraint to linear peptides in buffer solution but resulted in the decrease of alpha helicity of the peptides in lipid membrane mimic conditions. The cyclic peptide with the highest net positive charge had a similar antibacterial activity to that of the linear peptide, whereas the cyclic peptides with a low net positive charge (+3-+4) exhibited lower antibacterial activity than their linear forms. The cyclic peptides of an appropriate net charge showed more potent activities against some bacteria than those of linear forms under high salt conditions.     

Antimicrobial properties of the frog skin peptide, ranatuerin-1 and its [Lys-8]-substituted analog

The predicted conformation of ranatuerin-1 (SMLSVLKNLG(10)KVGLGFVACK(20)INK QC), an antimicrobial peptide first isolated from the skin of the bullfrog Rana catesbeiana, comprises three structural domains: alpha-helix (residues 1-8), beta-sheet (residues 11-16) and beta-turn (residues 20-25). Circular dichroism studies confirm significant alpha-helical character in 50% trifluoroethanol. Replacement of Cys-19 and Cys-25 by serine resulted only in decreased antimicrobial potency but deletion of either the cyclic heptapeptide region [residues (19-25)] or the N-terminal domain [residues (1-8)] produced inactive analogs. Substitution of the glycine residues in the central domain of the [Ser-19, Ser-25] analog by lysine produced inactive peptides despite increased alpha-helical content and cationicity. The substitution Asn-8-->Lys gave a ranatuerin-1 analog with increased alpha-helicity and cationicity and increased potency against a range of Gram-positive and Gram-negative bacteria and against C. albicans but only a small increase (21%) in hemolytic activity. In contrast, increasing alpha-helicity and hydrophobicity by the substitution Asn-22-->Ala resulted in a 3.5-fold increase in hemolytic activity. Effects on antimicrobial potencies of substitutions of neutral amino acids at positions 4, 18, 22, and 24 by lysine were less marked. Strains of pathogenic E. coli from different groups showed varying degrees of sensitivity to ranatuerin-1 (MIC between 5 and 40 microM) but [Lys-8] ranatuerin-1 showed increased potency (between 2- and 8-fold; P < 0.01) against all strains. The data demonstrate that [Lys-8] ranatuerin-1 shows potential as a candidate for drug development.     

Design of novel analogues with potent antibiotic activity based on the antimicrobial peptide, HP(2-9)-ME(1-12)

To develop novel antibiotic peptides useful as therapeutic drugs, a number of analogues were designed to increase the hydrophobic helix region either by Trp-substitution or net positive charge increase by Lys-substitution, from HP(2-9)-ME(1-12). The antibiotic activities of these peptides were evaluated using bacterial (Salmonella tryphimurium, Proteus vulgaris, Bacillus subtilis and Staphylococcus aureus), fungi (Saccharomyces cerevisiae, Trichosporon beigelii and Candida albicans), tumor and human erythrocyte cells. The substitution of Lys for Thr at position 18 and 19 of HP(2-9)-ME(1-12) (HM5) increased activity against Proteus vulgaris and fungal strains without hemolysis. In contrast, substitution of Trp for Lys and Thr at positions 2, 15 and 19 of HP(2-9)-ME(1-12), respectively (HM3 and HM4), decreased activity but increased hemolysis against human erythrocytes. This suggests that an increase in positive charge increases antimicrobial activity whereas an increase in hydrophobicity by introducing Trp residues at C-terminus of HP(2-9)-ME(1-12) causes a hemolytic effect. Circular dichroism spectra suggested that the alpha-helical structure of these peptides plays an important role in their antibiotic effect but that the alpha-helical property is not connected with the enhanced antibiotic activity.     

The effects of shortening lactoferrin derived peptides against tumour cells, bacteria and normal human cells.

A number of shortened derivatives of the lactoferrin model peptide L12, PAWRKAFRWAKRMLKKAA, were designed in order to elucidate the structural basis for antitumour activity of lactoferrin derivatives. Three tumour cell lines were included in the study and toxicity determined by measuring lysis of human red blood cells and fibroblasts. The results demonstrated a strong correlation between antitumour activity and net positive charge, in which a net charge close to +7 was essential for a high antitumour activity. In order to increase the antitumour activity of the shortest peptide with a net charge less than +7, the hydrophobicity had to be increased by adding a bulky Trp residue. None of the peptides were haemolytic, but toxicity against fibroblasts was observed. However, modifications of the peptides had a higher effect on reducing fibroblast toxicity than antitumour activity and thereby resulted in peptides displaying an almost 7-fold selectivity for tumour cells compared with fibroblasts. The antimicrobial activity against the Gram-negative bacteria Escherichia coil and the Gram-positive bacteria Staphylococcus aureus was also included in order to compare the structural requirements for antitumour activity with those required for a high antimicrobial activity. The results showed that most of the peptides were highly active against both bacterial strains. Less modification by shortening the peptide sequences was tolerated for maintaining a high antitumour activity and selectivity compared with antimicrobial activity. The order of the amino acid residues and thereby the conformation of the peptides was highly essential for antitumour activity, whereas the antimicrobial activity was hardly influenced by changes in this parameter. Thus, in addition to a certain net positive charge and hydrophobicity, the ability to adopt an amphipathic conformation was a more critical structural parameter for antitumour activity than for antimicrobial activity, and implied that a higher flexibility or number of active conformations was tolerated for the peptides to exert a high antimicrobial activity.     

Isolation of peptides of the brevinin-1 family with potent candidacidal activity from the skin secretions of the frog Rana boylii.

The emergence of strains of the human pathogen Candida albicans with resistance to commonly used antibiotics has necessitated a search for new types of antifungal agents. Six peptides with antimicrobial activity were isolated from norepinephrine-stimulated skin secretions from the foothill yellow-legged frog Rana boylii. Brevinin-1BYa (FLPILASLAA10KFGPKLF CLV20TKKC) was particularly potent against C. albicans [minimal inhibitory concentration (MIC) = 3 microm] and also active against Escherichia coli (MIC = 17 microm) and Staphylococcus aureus (MIC = 2 microm), but its therapeutic potential for systemic use is limited by its strong hemolytic activity (HC50 = 4 microm). The single amino acid substitution (Phe12 --> Leu) in brevinin-1BYb resulted in a fourfold lower potency against C. albicans and the additional amino acid substitutions (Lys11 --> Thr, Phe17 --> Leu and Val20 --> Ile) in brevinin-1BYc resulted in a ninefold decrease in activity. Two members of the ranatuerin-2 family and one member of the temporin family were also isolated from the secretions but showed relatively low potency against the three microorganisms tested.     

Novel short AMP: design and activity study

In a previous study, we reported that truncation of HP (2-20) (derived from the N-terminal region of Helicobacter pylori Ribosomal Protein L1 (RPL1)) at the N- (residues 2-3) and C-terminal (residues 17-20) truncated fragments to give HP (4-16) induces increased antibiotic activity against several bacterial strains without hemolysis. In this study, to develop novel short antibiotic peptides useful as therapeutic drugs, an analogue was designed to possess increased hydrophobicity by Trp substitution in position 2 region of HP (4-16). Synthetic HP (4-16)-W showed an enhanced antimicrobial and antitumor activity. The antimicrobial activity of this peptide and others was measured by their growth inhibitory effect upon S. aureus, B. subtilis, S. epidermidis, E. coli, S. typimurium, P. aeruginosa, C. albicans, T. beigelii and S. cerevisiae. None of the peptides exhibited hemolytic activity against human erythrocyte cells except melittin as a positive control. Its antibiotic activity suggests that HP (4-16)-W is an excellent candidate as a lead compound for the development of novel antibiotic agents.     

Biological consequences of improving the structural stability of hairpins that have antimicrobial activity

Designing new antimicrobial peptides (AMPs) focuses heavily on the activity of the peptide and less on the elements that stabilize the secondary structure of these peptides. Studies have shown that improving the structure of naturally occurring AMPs can affect activity and so here we explore the relationship between structure and activity of two non‐naturally occurring AMPs. We have used a backbone‐cyclized peptide as a template and designed an uncyclized analogue of this peptide that has antimicrobial activity. We focused on beta‐hairpin‐like structuring features. Improvements to the structure of this peptide reduced the activity of the peptide against gram‐negative, Escherichia coli but improved the activity against gram‐positive, Corynebacterium glutamicum. Distinctions in structuring effects on gram‐negative versus gram‐positive activity were also seen in a second peptide system. Structural improvements resulted in a peptide that was more active than the native against gram‐positive bacterium but less active against gram‐negative bacterium. Our results show that there is not always a correlation between improved hairpin‐structuring and activity. Other factors such as the type of bacteria being targeted as well as net positive charge can play a role in the potency of AMPs. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Structural requirements for potent versus selective cytotoxicity for antimicrobial dermaseptin S4 derivatives

To better understand the structural requirements for selective cytotoxicity of antimicrobial peptides, seven dermaseptin S4 analogs were produced and investigated with respect to molecular organization in solution, binding properties to model phospholipid membranes, and cytotoxic properties. Native dermaseptin S4 displayed high aggregation in solution and high binding affinity. These properties correlated with high cytotoxicity. Yet, potency was progressively limited when facing cells whose plasma membrane was surrounded by increasingly complex barriers. Increasing the positive charge of the native peptide led to partial depolymerization that correlated with higher binding affinity and with virtually non-discriminative high cytotoxicity against all cell types. The C-terminal hydrophobic domain was found responsible for binding to membranes but not for their disruption. Truncations of the C terminus combined with increased positive charge of the N-terminal domain resulted in short peptides having similar binding affinity as the parent compound but displaying selective activity against microbes with reduced toxicity toward human red blood cells. Nuclear magnetic resonance-derived three-dimensional structures of three active derivatives enabled the delineation of a common amphipathic structure with a clear separation of two lobes of positive and negative electrostatic potential surfaces. Whereas the spatial positive electrostatic potential extended considerably beyond the peptide dimensions and was required for potency, selectivity was affected primarily by hydrophobicity. The usefulness of this approach for the design of potent and/or selective cytolytic peptides is discussed herein.     

Parallel evaluation of antimicrobial peptides derived from the synthetic PAF26 and the human LL37

The antimicrobial hexapeptide PAF26 was de novo designed towards phytopathogenic fungi of agricultural importance. To analyze its clinical potential, the activity of PAF26 has been determined against several microorganisms of clinical relevance including Staphylococcus, Candida, and several dermatophytes. For comparison purposes, the peptides KR20 and KI26 derived from the human cathelicidin LL37 were selected and fungal pathogens of agronomic relevance were included. PAF26 has similar antimicrobial activity in vitro compared to KR20 despite their different lengths and amino acid compositions. Moreover, neither peptide is lytic to human erythrocytes or keratinocytes. The hybrid peptide PAF26:KR20 showed better antimicrobial properties than the original peptides against most of the pathogens tested. The structural properties of PAF26:KR20 compared to related 26-amino acid peptides support the idea that the increment in toxicity correlates with positive charge and hydrophobicity. However, the degree of peptide helicity was not a predictor of antimicrobial activity.     

Repurposing the scorpion venom peptide VmCT1 into an active peptide against Gram-negative ESKAPE pathogens

VmCT1 is a cationic antimicrobial peptide (AMP) from the venom of the scorpion Vaejovis mexicanus. VmCT1 and analogs were designed with single substitutions for verifying the influence of changes in physicochemical features described as important for AMPs antimicrobial and hemolytic activities, as well as their effect on VmCT1 analogs resistance against proteases action. The increase of the net positive charge by the introduction of an arginine residue in positions of the hydrophilic face of the helical structure affected directly the antimicrobial activity. Arg-substituted analogs presented activity against Gram-negative bacteria from the ESKAPE list of pathogens that were not observed for VmCT1. Additionally, peptides with higher net positive charge presented increased antimicrobial activity with values ranging from 0.39 to 12.5 μmol L⁻¹ against Gram-positive and Gram-negative bacteria and fungi. The phenylalanine substitution by glycine (position 1), and the valine substitution by a proline residue (position 8) led to analogs with lower hemolytic activity (at concentrations 50 and 100 μmol L⁻¹, respectively). These results revealed that it is possible to modulate the biological activities of VmCT1 derivatives by designing single substituted-analogs as prospective therapeutics against bacteria and fungi.     

Postsecretory processing generates multiple cathelicidins for enhanced topical antimicrobial defense

The production of antimicrobial peptides and proteins is essential for defense against infection. Many of the known human antimicrobial peptides are multifunctional, with stimulatory activities such as chemotaxis while simultaneously acting as natural antibiotics. In humans, eccrine appendages express DCD and CAMP, genes encoding proteins processed into the antimicrobial peptides dermcidin and LL-37. In this study we show that after secretion onto the skin surface, the CAMP gene product is processed by a serine protease-dependent mechanism into multiple novel antimicrobial peptides distinct from the cathelicidin LL-37. These peptides show enhanced antimicrobial action, acquiring the ability to kill skin pathogens such as Staphylococcus aureus and Candida albicans. Furthermore, although LL-37 may influence the host inflammatory response by stimulating IL-8 release from keratinocytes, this activity is lost in subsequently processed peptides. Thus, a single gene product encoding an important defense molecule alters structure and function in the topical environment to shift the balance of activity toward direct inhibition of microbial colonization.     

The antimicrobial potential of a new derivative of cathelicidin from <Emphasis Type="Italic">Bungarus fasciatus</Emphasis> against methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Cathelicidins are a family of antimicrobial peptides which exhibit broad antimicrobial activities against antibiotic-resistant bacteria. Considering the progressive antibiotic resistance, cathelicidin is a candidate for use as an alternative approach to treat and overcome the challenge of antimicrobial resistance. Cathelicidin-BF (Cath-BF) is a short antimicrobial peptide, which was originally extracted from the venom of Bungarus fasciatus. Recent studies have reported that Cath-BF and some related derivatives exert strong antimicrobial and weak hemolytic properties. This study investigates the bactericidal and cytotoxic effects of Cath-BF and its analogs (Cath-A and Cath-B). Cath-A and Cath-B were designed to increase their net positive charge, to have more activity against methicillin resistant S. aureus (MRSA). The results of this study show that Cath-A, with a +17-net charge, has the most noteworthy antimicrobial activity against MRSA strains, with minimum inhibitory concentration (MIC) ranging between 32–128 μg/ml. The bacterial kinetic analysis by 1 × MIC concentration of each peptide shows that Cath-A neutralizes the clinical MRSA isolate for 60 min. The present data support the notion that increasing the positive net charge of antimicrobial peptides can increase their potential antimicrobial activity. Cath-A also displayed the weakest cytotoxicity effect against human umbilical vein endothelial and H9c2 rat cardiomyoblast cell lines. Analysis of the hemolytic activity reveals that all three peptides exhibit minor hemolytic activity against human erythrocytes at concentrations up to 250 μg/ml. Altogether, these results suggest that Cath-A and Cath-B are competent candidates as novel antimicrobial compounds against MRSA and possibly other multidrug resistant bacteria.     

Antimicrobial activity of short arginine- and tryptophan-rich peptides.

Highly antimicrobial active arginine- and tryptophan-rich peptides were synthesized ranging in size from 11 to five amino acid residues in order to elucidate the main structural requirement for such short antimicrobial peptides. The amino acid sequences of the peptides were based on previous studies of longer bovine and murine lactoferricin derivatives. Most of the peptides showed strong inhibitory action against the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, and the Gram-positive bacterium Staphylococcus aureus. For the most active derivatives, the minimal inhibitory concentration values observed for the Gram-negative bacteria were 5 microg/ml (3.5 microM), whereas it was 2.5 microg/ml (1.5 microM) for the Gram-positive bacterium. It was essential for the antimicrobial activity that the peptides contained a minimum of three tryptophan and three arginine residues, and carried a free N-terminal amino group and an amidated C-terminal end. Furthermore, a minimum sequence size of seven amino acid residues was required for a high antimicrobial activity against Pseudomonas aeruginosa. The insertion of additional arginine and tryptophan residues into the peptides resulted only in small variations in the antimicrobial activity, whereas replacement of a tryptophan residue with tyrosine in the hepta- and hexapeptides resulted in reduced antimicrobial activity, especially against the Gram-negative bacteria. The peptides were non-haemolytic, making them highly potent as prospective antibiotic agents.     

Rational design of antimicrobial C3a analogues with enhanced effects against Staphylococci using an integrated structure and function-based approach

The anaphylatoxin C3a and its inactivated derivative C3adesArg, generated during complement activation, exert direct antimicrobial effects, mediated via its C-terminal region [Nordahl et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 16879-16884]. During evolution, this region of C3a displays subtle changes in net charge, while preserving a moderate but variable amphipathicity [Pasupuleti et al. (2007) J. Biol. Chem. 282, 2520-2528]. In this study, we mimic these evolutionary changes, employing a design approach utilizing selected amino acid substitutions at strategic and structurally relevant positions in the original human C3a peptide CNYITELRRQHARASHLGLA, followed by structure-activity studies incorporating sequence-dependent QSAR models as tools for generation of C3a peptide variants with enhanced effects. While the native peptide and related amphipathic analogues of moderate positive net charge were active against the Gram-negative Escherichia coli, activity against the Gram-positive Staphylococcus aureus was primarily observed for peptides characterized by a combination of a relatively high net charge (+6-7) and a propensity to adopt an alpha-helical conformation with amphipathic character. Such increased helicity and charge also conferred activity against the fungus Candida albicans. A central histidine residue (H11), evolutionarily conserved among vertebrates, conferred high selectivity toward microbes, while substitutions with leucine rendered the peptides hemolytic. Selected C3a analogues retained their specificity against staphylococci in the presence of human plasma, while showing low cytotoxicity. The work illustrates structure-activity relationships underlying the function and specificity of antimicrobial C3a and related analogues and provides insights into the forces that drive evolution of antimicrobial peptides.     

N-terminal amphipathic helix as a trigger of hemolytic activity in antimicrobial peptides: A case study in latarcins

In silico structural analyses of sets of α-helical antimicrobial peptides (AMPs) are performed. Differences between hemolytic and non-hemolytic AMPs are revealed in organization of their N-terminal region. A parameter related to hydrophobicity of the N-terminal part is proposed as a measure of the peptide propensity to exhibit hemolytic and other unwanted cytotoxic activities. Based on the information acquired, a rational approach for selective removal of these properties in AMPs is suggested. A proof of concept is gained through engineering specific mutations that resulted in elimination of the hemolytic activity of AMPs (latarcins) while leaving the beneficial antimicrobial effect intact.     

Structure-activity relationship study of antimicrobial dermaseptin S4 showing the consequences of peptide oligomerization on selective cytotoxicity

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.