Rational Design of Artificial β-Strand-Forming Antimicrobial Peptides

Sep 5, 2014 - Carlo Diaferia , Teresa Sibillano , Davide Altamura , Valentina Roviello , Luigi Vitagliano , Cinzia Giannini , Giancarlo Morelli , Anto...
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Article pubs.acs.org/molecularpharmaceutics

Rational Design of Artificial β‑Strand-Forming Antimicrobial Peptides with Biocompatible Properties Karsten Rapsch,* Frank F. Bier, and Markus von Nickisch-Rosenegk Fraunhofer Institute for Biomedical Engineering IBMT, Branch Potsdam, Am Muehlenberg 13, 14476 Potsdam, Germany S Supporting Information *

ABSTRACT: Because the intensive use of antibiotics has led to a large variety of resistant bacterial strains, therapeutic measures have become increasingly challenging. In order to ensure reliable treatment of diseases, alternative antimicrobial agents need to be explored. In this context, antimicrobial peptides have been discussed as novel bioactive molecules, which, however, may be limited in their applicability due to their high manufacturing costs and poor pharmacokinetic properties. Consequently, the design of artificial antimicrobial peptides featuring two flanking cationic regions and a hydrophobic center is presented. These sequences led to distinct antimicrobial activity on the same order of magnitude as that of naturally occurring reference peptides but with less cytotoxic or cytostatic drawbacks. Furthermore, a deletion and substitution library revealed the minimal sequence requirements. By analysis of the computed 3D structures of these peptides, a single characteristic β-strand was identified. This structural motif was pivotal for antimicrobial activity. Consequently, an optimized peptide sequence with antimicrobial and biocompatible properties was derived, and its application was demonstrated in a mixed culture experiment. Thus, it was shown that the optimized artificial antimicrobial peptide is suitable as a therapeutic agent and may be used as template for the development of new antimicrobial peptides with unique secondary structures. KEYWORDS: antimicrobial peptide, secondary structure, mode of action, therapy, permeability

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INTRODUCTION Protection against bacterial infections plays a major role in today’s society. Notably, the intensive use of antibiotics has led to the development of a large variety of drug-resistant bacteria, including multiresistant Staphylococcus aureus (MRSA) or ESBL-expressing Enterobacteriaceae. This represents one of the most challenging aspects in the treatment of bacterial infections and has emerged as a significant issue in human health care.1 Therefore, research on new antimicrobial agents for the treatment of bacterial infections has been intensified. However, in recent years, only a few new types of antibiotics have been developed.2 Alternatively, antimicrobial peptides (AMP), a promising class of molecules featuring biocompatibility and antimicrobial activity, have gained significant ground in research in recent years.3 They are a diverse class of biomolecules and are common in most pro- and eukaryotic organisms as part of the innate immune system.4 These bioactive molecules differ markedly in their peptide sequence and possess either bactericidal5 or bacteriostatic6 characteristics. Due to their distinct lytic mode of action, it is assumed that the development of resistance against this class of biomolecules is less likely.7 It has been shown elsewhere that even prolonged exposure of three individual bacterial strains to sublethal doses of different naturally occurring peptides and synthetic derivatives did not trigger the development of resistance.8 Because several AMPs possess lytic activity only toward bacterial membranes, they are considered to be biocompatible. © 2014 American Chemical Society

This is caused by the different composition and charge of prokaryotic9 and eukaryotic10 membranes. Still, some AMPs display intensive cytotoxic properties nonetheless.11 In order to use such peptides in commercial or clinical applications, such restrictions need to be considered. Furthermore, naturally occurring AMPs may possess limitations due to their manufacturing cost and poor pharmacokinetic properties.12 Moreover, clinical and industrial applications of AMPs that are closely related to the host’s AMPs might cause resistance to peptides of the innate immune system. This may compromise the natural defenses against bacterial infections and represents a significant risk to human health care.13 Because these disadvantages of AMPs hamper their application in an industrial and clinical environment, a lot of research has been done to bypass these limitations. Hence, the research has focused mainly on the development of short artificial peptides.14 Additionally, abiotic compounds have been investigated that mimic AMPs.15 The identification of new artificial AMPs can be achieved by the large-scale screening of peptide libraries based on a known template. Thus, the peptide sequence is systematically modified, and the resulting antimicrobial properties are Received: Revised: Accepted: Published: 3492

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Accordingly, these results indicate the crucial parameters and advantages of rationally designed AMPs and further indicate the optimized sequence as a promising alternative to traditional antibiotics.

monitored. Such a substitution can either favor different amino acids or can be completely random.16 Another approach is the in silico modulation of secondary structure motifs, which are often fundamental for the antimicrobial mode of action. Among these secondary structure motifs, α-helical peptides represent the most abundant, widespread, and well-characterized class within the naturally occurring AMPs.17 Hence, a lot of research has been performed on the de novo generation of α-helical antimicrobial model peptides with superior antibacterial properties. Peptides consisting mainly of leucine and lysine have been thoroughly studied,18 as these amino acids are known to have strong helixforming potential in proteins. 19 Furthermore, different biochemical properties of helical peptides influencing their antimicrobial potential and cytotoxicity toward eukaryotes have been discussed.20 The cationic properties, the amphipathicity, characterized by the hydrophobic moment (μ), the overall hydrophobicity (H), and the angle subtended by the positively charged helix face (Φ) have been identified as being crucial for antimicrobial and cytotoxic activity.21 While α-helical peptides are very effective antimicrobial agents, they often exhibit cytotoxic properties toward eukaryotic cells.21 Another secondary structure motif is represented by amphipathic β-sheet-forming peptides. These peptides can be designed in silico by continuous repeats of cationic and hydrophobic amino acids.22−25 Like the α-helical motif, the β-sheet motif possesses antimicrobial properties and can be used to design artificial AMPs.26−28 It was demonstrated that β-sheet-forming peptides feature selectivity to bacterial membranes compared to their α-helical counterparts with equal biochemical properties.29 In this study, a peptide design was performed to investigate the influence of sequence arrangements on the nature of short AMPs. Additionally, artificial peptides with enhanced antimicrobial activity and biocompatible properties were selected. Therefore, peptides were designed featuring two flanking cationic regions and a hydrophobic center. Furthermore, a peptide library was prepared to determine the influence of peptide length and composition on the antimicrobial and cytotoxic properties of these peptides. For this purpose, Escherichia coli, representing Gram-negative bacteria, and the Gram-positive bacterium Staphylococcus xylosus were tested, and the underlying mode of action was characterized. Additionally, the cytotoxic properties were investigated using the human histiocytic lymphoma cell line U937. Finally, the therapeutic ratio of each individual peptide was calculated, and an optimized sequence with extensive antimicrobial as well as biocompatible properties was concluded. For comparison, the peptide library was expanded by four naturally occurring AMPs, namely, indolicidin, melittin, BMAP-27, and protamine, as well as a highly antimicrobial αhelical peptide consisting only of lysine and leucine. Because melittin is a well-characterized and commonly used AMP with antimicrobial and cytotoxic properties, it was used to highlight the differences between α-helical and the investigated artificial peptides. Given that the secondary structure is often related to the antimicrobial mode of action, the most probable conformations of the investigated peptides were calculated. Thus, a correlation among sequence, computed structural motif, and activity could be determined. Moreover, the use of the optimized AMP for the treatment of a bacterialcontaminated cell culture was demonstrated, and the selective permeabilization of the bacterial membrane was highlighted.

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MATERIALS AND METHODS Antimicrobial Peptides. Melittin and protamine were acquired from Sigma-Aldrich. Peptides indolicidin, BMAP-27, and α-helical KL-Peptide as well as peptides from the substitution and deletion library were synthesized by standard solid-phase peptide synthesis (JPT). Peptides M4, N2, N4, C3, H4, A4, and S2 had purities between 70 and 75%, peptides M2, N1, C2, C6, C7, H3, and S3 had purities between 75 and 85%, peptides N8, C5, H7, H8, and S7 had purities above 95%, and the rest had purities between 85 and 95%. All peptides were stored at −20 °C in a lyophilized state. Prior to use, they were solubilized in deionized water. Minimal Inhibitory Concentration. The minimal inhibitory concentrations (MIC) of peptides were determined against Gram-negative E. coli Dh5α and Gram-positive S. xylosus (Deutsche Sammlung von Mikroorganismen and Zellkulturen, DSMZ) by the use of a broth microdilution method. The general guidelines of the CLSI30 and EUCAST31 were followed with small adjustments according the investigation of cationic AMPs.32 Cells were cultured in Mueller− Hinton bouillon (MHB) to mid log phase, mixed with AMPs (0.25 to 64 μM, 5 × 104 bacteria/mL), and placed in 96-well polypropylene microtiter plates (Starlab). Sterile MHB was used as a negative control, whereas MHB inoculated with bacteria served as a growth control. The microtiter plates were incubated (18 h, 37 °C, 120 rpm), and afterward the absorbance was measured at 600 nm. The MIC was determined as the lowest peptide concentration at which no bacterial growth occurred. Depolarizing Properties of Soluble Peptides toward Bacteria. Three individual peptide concentrations were analyzed with S. xylosus and E. coli DH5α (DSMZ). Flow cytometry measurements in combination with live−dead staining were used in order to detect the membrane permeabilization properties of soluble AMPs. The measurements were carried out as previously described.33 Bacteria were cultured to mid log phase, their concentration was adjusted by flow cytometry measurement, and they were mixed with peptides, resulting in final concentrations of 5, 25, or 50 μM, in combination with a final bacterial concentration of 1 × 108 bacteria/mL. Afterward, the samples were incubated (2 h, 37 °C) and analyzed by flow cytometry. The lowest concentration leading to depolarized membranes in 80% of the bacterial population was determined. Outer- and Inner-Membrane Permeabilization. E. coli ML-35p (provided by Dr. R. I. Lehrer, Center for Health Sciences, Los Angeles, CA, USA) was used to evaluate the permeabilization properties of AMPs toward the outer and inner bacterial membrane. This specific bacteria was used because it expresses a periplasmic β-lactamase and a cytosolic βgalactosidase, which can be used to specifically detect inner- or outer-membrane disruption. Thus, a serial peptide dilution was prepared and incubated with E. coli ML-35p, and the timedependent release of internal cell compounds was monitored. The general procedure was performed as previously described34 with small adjustments. Briefly, the bacteria were grown in LB medium supplemented with 100 μg/mL ampicillin (Fluka) to mid log phase. Afterward, the cells were centrifuged three times 3493

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Figure 1. Three-dimensional structures of peptides. Displayed are the most likely 3D structures in an aqueous environment, as calculated using the PEP-fold37−39 web server. α-Helices are highlighted in black, β-strands are indicated by flat gray arrows, and random coils are colored in light gray. The arrowheads point into the direction of the C-terminal region. Melittin, BMAP-27, and KL-Helix exhibit a high abundance of α-helical content, while protamine displays less helical content, and indolicidin harbors no helices. M1, M2, M4, and M5 possess β-strands, while M3 and M6 feature random coils only. Peptides N1−N4 and C1−C4 (each including M1) feature a gradual truncation of cationic residues at the N- or C-terminus by one from left to right. All peptides possess β-strands; however, as the number of cationic residues decreases, so does the β-strand coverage.

to prolonged cultivation. After 4 days, lysis solution was added, and the supernatants were measured by the CytoTox assay. The lowest peptide concentration displaying complete inhibition of cell growth after 4 days was determined as the minimal toxic concentration (MTC). The therapeutic ratio (ϑ) was calculated as the quotient of the LD50 and the appropriate MIC. Indices of peptides that displayed a LD50 > 64 as well as a MTC ≤ 64 were set into brackets.

at 4000g for 1 min each, resuspended in 10 mM HEPES buffer, pH 7.2, and mixed with nitrocefin (Merck) and AMPs (nitrocefin 20 μg/mL, AMP 4 to 32 μM, bacteria OD600 = 0.1). The absorbance at 500 nm was monitored for 90 min at 37 °C with shaking using a FLUOstar omega (BMG Labtech). Permeabilization of the inner membrane was studied analogously, yet instead of nitrocefin, o-nitrophenyl-β-Dgalactoside (ONPG, Sigma-Aldrich) at a final concentration of 100 μg/mL was monitored at 420 nm. Samples without AMP were used as negative controls, whereas 5 μM polymyxin B (Sigma-Aldrich) was used as a positive control. All sample data were subtracted by the negative control and then divided by the positive control. The curve profiles were fitted by a sigmoidal model due to the minimal error coefficient using OriginPro 8.1 G (OriginLab) software. Only samples with an adjusted coefficient of determination equal to or more than 0.92 were used. For comparison, the time points at which 50% of the signal level was reached were calculated. Cytotoxicity of AMPs and Therapeutic Ratio Calculation. The human histiocytic lymphoma cell line U937 (DSMZ) was cultured (37 °C, 5% CO2) in 15 mL of RPMI 1640 medium supplemented with 2 mM glutamine and 10% FCS (Biochrom) in T-75 culture flasks (TPP). Prior to each experiment, the cells were centrifuged three times at 100g for 5 min each, resuspended, and transferred to sterile 96-well polypropylene microtiter plates. AMPs were added (0.25 to 64 μM, 5 × 104 cells/mL), and cells were cultured for 2 h. Samples without AMP were used as negative controls, while 5% Triton X-100 (Applichem) was used as a positive control. Finally, the cell suspensions were divided equally into two wells. One of the samples was analyzed to determine the direct membrane damage, while cultivation of the other sample was extended for 4 days. The direct membrane damage was assessed using CytoTox 96 non-radiactive cytotoxicity assay (Promega). The sample data were subtracted by the negative control, divided by the permeabilization control, and plotted against the peptide concentration. The curve profiles were fitted like above, and the LD50 values were calculated. Long-term inhibition properties of the AMPs were determined by analyzing the samples subjected

ϑ=

LD50 MIC

(1)

Treating Contaminated Cell Culture. Mixed culture experiments comprising U937 cells and S. xylosus were performed to determine the potential applicability of the AMPs as therapeutic agents. Cells were cultivated as described above, and both eukaryotic cells and bacteria were mixed (1 × 106/mL each) and afterward supplemented with AMPs (25 or 50 μM). Sterile medium was used as a negative control, whereas melittin served as a toxicity control. The cell mixtures were incubated (2 h, 37 °C, 5% CO2) and afterward analyzed by flow cytometry in order to determine the membrane permeabilization as previously described.33 Calculation of Peptide Properties and Three-Dimensional Structure Modeling. The hydrophobicity (H), the hydrophobic moment (μ), and the helical wheel projection were calculated for each AMP, assuming ideal α-helical conformation. The calculations were carried out by the use of the HeliQuest35 web server, which is based on the Eisenberg scale, 36 assuming a neutral environment at pH 7.4. Furthermore, the most probable 3D structures were calculated in an aqueous environment by the use of the PEP-fold37−39 web server. Furthermore, an additional algorithm was applied by using the PEPstr40 Peptide Tertiary Structure Prediction web server to confirm the postulated 3D structures. These were calculated in hydrophilic and hydrophobic environments. All parameters were calculated for peptides with a minimum length of nine residues. Shorter peptides were omitted from calculations due to the lack of mathematical accuracy. The 3494

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resulting structures were processed using the Geneious 6.1.541 software, and visualization was achieved with the UCSF Chimera package.42,43 Secondary structures were highlighted by the use of the ribbon style.

Consequently, it was not possible to calculate the values of the corresponding ϑ (Table S1). In contrast, M4−M6 did not even feature the permeabilization of the outer membrane. Altering the primary sequence of the peptides led to distinct changes in the calculated secondary structure motifs (Figure 1). While M1 possessed an extensive β-strand motif covering most of the sequence, M2, M4, and M5 displayed less β-strand content in combination with unstructured regions. In contrast, M3 and M6 featured no characteristic secondary structure motif; instead, only flexible regions were present. Deletion Library of N-Terminal Region. The influence of the positive charge at the N-terminal region was investigated. Thus, the number of cationic amino acids was reduced stepwise from five (N1) to zero residues (N5) (Table S1). The MICs toward E. coli and S. xylosus increased with a reduced number of cationic amino acids at the N-terminus (Figure 2). Therefore,

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RESULTS Reference AMPs. The results of the five reference peptides are summarized in Table S1. All peptides displayed activity toward E. coli and S. xylosus. Thus, they showed MICs for S. xylosus ranging from 0.5 to 32 μM and for E. coli from 1 to 8 μM. Furthermore, melittin, BMAP-27, and KL-Helix indicated strong cytotoxic effects toward U937 cells, with LD50 values ranging from 1.4 to 7.0 μM. Because these concentrations were within the same order of magnitude as that of the corresponding MICs, ϑ was calculated to be no more than 3.5. Protamine and indolicidin displayed no cytotoxic properties, resulting in higher ratios compared to those of the other AMPs. Protamine attained the most pronounced ratios, with >8 and >128. However, the peptide led to a complete growth inhibition of the eukaryotic cells at a concentration of 16 μM and therefore ϑ should be valid only under reservation. The membrane disruption properties of these peptides fall into two distinct groups. While melittin, BMAP-27, and KLHelix displayed membrane disruption within minutes, indolicidin and protamine exhibited hardly any disruption of bacterial membranes (Table S2). Indolicidin disrupted the membrane of S. xylosus at concentrations above 50 μM, while protamine failed to disrupt the membranes of both tested bacteria even at the highest concentration under scrutiny. Analogously, the peptides displayed a time-dependent permeabilization of the inner and outer membrane of E. coli ML-35p. Melittin, BMAP-27, and KL-Helix indicated permeabilization of both membranes in less than 20 min. In contrast, protamine and indolicidin exhibited no permeabilization of the inner membrane. While indolicidin also featured no disruption of the outer membrane, protamin readily permabilized the membrane even at a concentration of 4 μM in less than a minute. The calculated structures of melittin, BMAP-27, and KLHelix indicated a high abundance of α-helical content covering the entire sequence except for a short disruption of the motif, while protamine displayed three individual helical regions interspersed with unstructured regions (Figure 1). In contrast, indolicidin displayed no α-helical content. Artificial Peptide Design. The influence of the primary sequence on the antimicrobial properties was investigated using six different model peptides, named M1−M6. While M1 featured a central hydrophobic and two cationic flanking regions, the other peptides incorporated extensive modifications either within the hydrophobic or cationic region (Table S1). The results demonstrated antimicrobial characteristics of peptide M1, resulting in MICs of 8 and 2 μM with no cytotoxic properties. Furthermore, this peptide caused permeabilization at 25 μM (Table S2). While the outer membrane was permeabilized at any tested peptide concentration within 16− 39 min, the inner membrane was permeabilized only at concentrations as low as 16 μM. The latter was delayed by about 30 min. In contrast, peptides M2 and M3 displayed outer-membrane permeabilization only at the two highest concentrations tested, yet no disruption of the inner membrane was detected. Furthermore, these peptides resulted in MICs > 64 μM in combination with no detectable cytotoxic properties.

Figure 2. Function of MICs depending on the deletion of the N- and C-terminal regions. Displayed are the MICs of the N- (N1−N8, including M1) and C-terminal (C1−C8, including M1) deletion library. The shaded area indicates concentrations above the measurement range. Upon reduction of cationic residues from either region, the MICs increased.

N1 resulted in MICs of 8 and 0.5 μM, while N4 required 32 and 16 μM to reach inhibition. Further reduction of cationic residues at the N-terminus to zero (N5) led to MICs of 64 μM. Additional reduction of N-terminal residues, albeit hydrophobic in nature, caused a continued increase in MICs to >64 μM (N6−N8). Only N1 and N4 displayed cytotoxic properties at 36 and 58 μM, respectively, while the other peptides featured biocompatible properties (Table S1). Therefore, ϑ of the latter decreased with decreasing MICs and thereby with the deletion of the Nterminal region. Due to the cytotoxic properties of N1 and N4, they exhibited a reduced ϑ. Moreover, N1 displayed membrane disruption at 25 and 5 μM for E. coli and S. xylosus, respectively, while N4 achieved the same with concentrations of 50 and 25 μM (Table S2). Further deletion of cationic amino acids at the N-terminus led to peptides without membrane disruption properties. This was supported by the time-dependent permeabilization of the outer and inner membrane of E. coli ML-35p. All peptides containing at least one cationic residue at the N-terminus disrupted the outer membrane at any of the concentrations tested (Figure S1). At 32 μM, the time for permeabilization varied from 11 to 17 min (N1−N4, including M1). At concentrations below 32 μM, the time for permeabilization increased as the number of 3495

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Additionally, the membrane permeabilization abilities of the peptides were influenced by the gradual truncation of the hydrophobic region (Table S2). The permeabilization time increased with reduced peptide length. Upon the reduction to four hydrophobic residues (H2), no permeabilization of the E. coli membrane could be achieved, while the S. xylosus membrane could still be permeabilized at a concentration of 25 μM. Upon further truncation, both membranes displayed no membrane disruption up to a concentration of 50 μM. This effect was also reflected by the time-dependent permeabilization of the outer and inner membrane of E. coli ML-35p. Here, the outer membrane was permeabilized at any concentration investigated (Figure S4). However, the time increased due to the deletion of hydrophobic residues. Finally, at four remaining residues, the permeabilization time increased to >90 min and remained above this limit even after further reduction. Likewise, the inner membrane was disrupted by H1 and M1 only at higher concentrations (Figure S5). Despite their MICs below 64 μM, H6 and H7 displayed no membrane disruption properties. In terms of computed 3D structures, H1 featured two β-strand motifs surrounding a random coil conformation. In addition, M1 and H2 displayed β-strand motifs covering most of the sequence (Figure S6). By reducing the hydrophobic residues in the center, two random coils at the terminal regions were formed (H3). In H8, consisting completely of cationic residues at the termini and neutral amino acids in the middle, the β-strand disappeared to give rise to a random coil comprising the entire peptide. Arrangement of Charge and Hydrophobicity. After analyzing the influence of the number of cationic and hydrophobic residues in the different regions of the peptides, the composition and arrangement of those residues was examined. Therefore, hydrophobic amino acids were incrementally transferred from the center to the outside. Thus, peptides featuring equal length and hydrophobicity H, while differing in μ, were created (Table S1). Consequently, when the central hydrophobic region of A1 was separated by cationic residues into two parts (A2), the MIC increased from 8 to 16 μM for E. coli and from 0.5 to 8 μM for S. xylosus (Figure S3). Although A3 displayed the same MIC as that of A2, further rearrangement led to an increase in MIC. Thus, A4 obtained MICs of 32 and 16 μM, whereas A5 displayed no inhibition, resulting in MICs > 64 μM. Because all investigated peptides displayed no cytotoxic properties by direct membrane damage or growth inhibition, the calculated ϑ correlated with the MICs. These antimicrobial properties were emphasized by A1’s ability to disrupt both the outer and inner membrane of E. coli ML35p (Table S2). Nevertheless, outer-membrane disruption receded upon rearrangement (Figure S4), as was also observed at the inner membrane (Figure S5). In addition, altering the order of the residues resulted in distinct differences regarding the calculated 3D structures (Figure S7). While A1 featured extensive β-strand contents, A2 contained an α-helix flanked by random coils. Further transferring the hydrophobic residues from the center to the outside in A3 caused two antiparallel βsheets to be formed. These antiparallel β-sheets were even more pronounced in A4. After completely rearranging the residues (A5), only random coils remained. Influence of Amino Acid Composition. The influence of particular amino acids on the antimicrobial and cytotoxic properties was analyzed by designing different AMPs featuring two flanking regions with three cationic residues each and a five hydrophobic residue-containing core. To determine the effect

cationic residues decreased. Thus, it took N4 54 min to disrupt the outer membrane at 4 μM. N5 featured outer-membrane disruption only at 16 and 32 μM. Peptides with less than eight residues caused no disruption. While the outer membrane was readily disrupted by N1−N4 including M1, the inner membrane displayed damage only upon the interaction with the peptides N1−N3 and M1 (Figure S2). The membrane disruption ability of the peptides decreased with peptide length and was noticeable only at 16 and 32 μM. The calculated 3D structures featured predominantly β-strands (Figure 1). This motif remained unaffected by the truncation of cationic residues as low as three (M1). Reducing the number of cationic amino acids at the N-terminus to less than three led to a shorter β-strand motif flanked by two flexible areas (N3). The β-strand motif was even less pronounced in N4. Deletion Library of C-Terminal Region. Next, the influence of cationic residues at the C-terminus was analyzed. The results indicated an analogous behavior (Table S1). Reducing the number of cationic residues at the C-terminus caused a gradual increase of the corresponding MIC (Figure 2). For C1, MICs of 16 μM for E. coli and 1 μM for S. xylosus were obtained. In contrast, with only one lysine residue remaining, C4 reached MICs of 32 and 8 μM for E. coli and S. xylosus, respectively. Upon the deletion of the last positively charged residue, the MICs rose to >64 μM. Furthermore, only the peptide C1 displayed cytotoxic properties with a LD50 value of 61 μM, whereas the gradual truncation of the C-terminal region resulted in peptides with biocompatible properties. Thus, ϑ decreased with decreasing MICs. Moreover, membrane disruption potential decreased with reduced number of cationic residues at the C-terminus (Table S2). When removing the last lysine residue (C5), membrane disruption was not detected. This was supported by the membrane disruption kinetics of the outer membrane (Figure S1). Peptides carrying at least one cationic residue displayed outer-membrane disruption at any concentration investigated. However, N5, which lacks positively charged residues, displayed membrane disruption only at 16 and 32 μM. In contrast to the investigation on the N-terminal region, where the rate of the inner-membrane disruption decreased with decreasing peptide length, the truncation of the C-terminal region did not alter the disruption time. This activity could be detected only at 16 and 32 μM and down to a peptide length of nine amino acids (C4). The truncation of the C-terminal region revealed almost the same variation of the calculated 3D motifs (Figure 1) as that from the investigation on the N-terminal region. While C1, C2, and M1 were composed almost entirely of one extended β-strand, C3 harbored two flanking flexible regions encompassing a truncated β-strand. In C4, this β-strand motif was further reduced and transferred to the N-terminus. Hydrophobic Region. The gradual truncation of the hydrophobic region indicated an influence on the MICs (Table S1). H1 and M1, which featured seven and five hydrophobic residues, respectively, displayed MICs as low as 8 μM for E. coli and 1 or 2 μM for S. xylosus (Figure S3). Upon reducing the hydrophobic residues to four (H2), the MIC for E. coli climbed beyond 64 μM. For S. xylosus, the increase in MIC was 8-fold, to 16 μM. Removing an additional hydrophobic residue resulted in MICs of more than 64 μM for both bacteria. However, H6 and H7 deviated from this tendency. While H6, lacking any hydrophobic residue, displayed a MIC for S. xylosus of 16 μM, H7 carrying one neutral amino acid showed a MIC of 64 μM. 3496

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Figure 3. Three-dimensional structures of O1. Displayed are the most likely 3D structures of O1 as calculated in an (A) aqueous (PEP-fold37−39), (B) hydrophilic (PEPstr40), and (C) hydrophobic (PEPstr40) environment. β-strands are highlighted by flat gray arrows, and random coils, in light gray. The arrowhead points into the direction of the C-terminal region. O1 displays a β-strand motif encompassing almost the whole peptide chain. This calculated motif is present in hydrophilic and hydrophobic environments.

of different amino acids, lysine and arginine served as cationic residues, whereas isoleucine, leucine, phenylalanine, and valine were applied as hydrophobic amino acids. After combination, the antimicrobial properties of peptides S1−S8 were analyzed (Table S1). The results indicated activity of all AMPs toward both bacteria, albeit with a varying degree of intensity. Consequently, MICs of 4−64 μM for E. coli and 0.5−8 μM for S. xylosus were attained. Comparing the influence of arginine and lysine, the odd-numbered peptide of each pair (S1 and S2, S3 and S4, etc.) exhibited an equal or lower MIC than that of the even-numbered ones, indicating a stronger antimicrobial effect of arginine residues as compared to that of lysine. The only partial exception was represented by peptide S2, which displayed a MIC of 0.5 toward S. xylosus and thereby was 4-fold lower than S1. The increased antimicrobial properties of arginine compared to that of lysine were emphasized by the membrane disruption properties of these peptides that correlated with the MICs (Table S2). Nevertheless, S1, S3, and S7 displayed a LD50 above 64 μM; the ϑ should be handled with care, since the peptides featured a MTC of either 64 or 32 μM toward the eukaryotic cell line U937. Concerning the influence of the hydrophobic residues, the evaluation is not as obvious. While isoleucine-containing S1 featured MICs of 8 and 2 μM, substitution by leucine, as in S3, resulted in MICs of 8 and 1 μM (Table S1). However, the inner-membrane disruption decreased. Substitution by valine in S5 increased the MIC to 32 and 2 μM and decreased the membrane disruption of E. coli ML-35p (Table S2). Permeabilization of the outer membrane was detected only at the highest concentration investigated, while the inner membrane remained intact. Finally, the insertion of the hydrophobic amino acid phenylalanine (S7) resulted in MICs of 4 and 0.5 μM for E. coli and S. xylosus, respectively, in combination with outer- and inner-membrane permeabilization. The lysine containing counterparts S2, S4, S6, and S8 indicated a comparable influence of the hydrophobic amino acids on the antimicrobial properties (Table S1). While the substitution of isoleucin (S2) with leucin (S4) resulted in an increase of the MICs by 2- and 16-fold toward E. coli and S. xylosus, respectively, the membrane disruption decreased from 50 to 25 and 5 μM (Table S2). Furthermore, the outer- and inner-membrane disruption of E. coli ML-35p decreased and could be achieved only at 32 μM. Analogous to that for the arginine containing peptide (S5), the use of valine in a lysinecontaining peptide (S6) caused an increase of MICs to 64 and 8 μM in combination with decreased membrane disruption, which were present only at the outer membrane and at the highest concentration investigated. The substitution with phenylalanine (S8) led to MICs at 16 and 4 μM and resulted in a peptide with only slight membrane disruption abilities. In

summary, isoleucine revealed the most distinct antimicrobial properties within the lysine-containing peptides, and phenylalanine, among the arginine-containing peptides. Concerning the biophysical properties, neither the substitution of cationic amino acids nor the substitution of hydrophobic residues resulted in a significant variation of the hydrophobicity or the hydrophobic moment (Table S1). Only the insertion of the hydrophobic amino acid valine resulted in a distinct decrease of the hydrophobicity. Likewise to that for M1, the calculated secondary structure of S1−S8 consisted of a β-strand, which was present in all peptides and displayed only minimal changes upon the amino acid substitutions (Figure S8). Optimization of Peptide Sequence and Treatment of Cell Culture Contaminated by Bacteria. On the basis of prior experiments, an optimized peptide sequence was determined, and the corresponding antimicrobial and cytotoxic properties were analyzed. The peptide sequence of O1 comprised two flanking regions with three lysine residues at each end combined with a center of six isoleucine residues (Table S1). The resulting MICs equaled 8 and 0.5 μM. Membrane disruption was visible at 25 and 5 μM (Table S2). Outer- and inner-membrane permeabilization of E. coli ML-35p was detected at a concentration as low as 4 μM. Permeabilization of the outer membrane occurred within 17 to 23 min, while disruption of the inner membrane was delayed by about 20 min. In both cases, the time for permeabilization was constant and independent of the concentration used. Furthermore, O1 showed no cytotoxic properties toward U937 cells, resulting in ϑ of more than 8 and 128. The computed secondary structure consisted of a β-strand encompassing almost the whole peptide chain (Figure 3). This motif was invariable in a hydrophilic and hydrophobic environment. Since O1 comprised antimicrobial and biocompatible properties, the peptide’s ability to specifically disrupt the bacterial membrane was examined in a mixed cell culture experiment (Figure 4). Without addition of any peptide, S. xylosus and U937 cells displayed unaffected membranes. In addition, the bacterial concentration increased from 0.8 × 106 to 1.8 × 106 bacteria/mL. Adding melittin caused membrane damage in bacteria and U937 cells. However, supplementing the mixed culture with O1 resulted in disruption of bacterial membranes only, while the eukaryotic cells remained unaffected.

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DISCUSSION Reference AMPs. The computed structures of melittin, BMAP-27, and KL-Helix indicated α-helical structures, and the peptides displayed increased hydrophobic moments compared to those of protamine and indolicidin (Figure 1). Although the former are very effective antimicrobial agents, they also displayed intense cytotoxic properties, rendering them 3497

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Artificial Peptide Design. M1 displayed both growth inhibition and membrane disruption properties (Table S1, Table S2). The peptide consists of three cationic amino acids each at the C- and N-terminal regions of the peptide and a central region featuring five hydrophobic residues. The substitution of any region either in peptide M2, M3, or M6 with neutral amino acids led to peptides with no antimicrobial properties. This indicates the need of these three individual regions for the antimicrobial properties. Even peptides M4 and M5, displaying comparable hydrophobicities and hydrophobic moments compared to those of M1, indicated no antimicrobial properties. Because of this, it is more likely that the antimicrobial features of these peptides are linked to their calculated 3D structure, as was seen for peptides M1−M6 (Figure 1). The β-strand motif was distinct only in M1, while all other peptides displayed shortened motifs or even totally flexible unstructured regions. This relation is further confirmed by the results regarding the length of the cationic and hydrophobic regions. Shortening either region resulted in a decrease of antimicrobial activity (Figure 2). Furthermore, the decrease in activity was not related to the change of the hydrophobicity or the hydrophobic moment, since N3 and N4 as well as C3 and C4 exhibited the highest calculated values among the peptides yet displayed some of the lowest antimicrobial activities. While the antimicrobial activity of αhelical peptides increases with increasing hydrophobic moment,21 the investigated peptides were independent thereof. This further confirms the structural difference to α-helical antimicrobial peptides and indicates that the artificial peptides, consisting of a hydrophobic core region in combination with two flanking cationic regions, most likely do not adopt helical conformation. Again, it seems that the antimicrobial properties were related to the characteristic computed secondary structure of the artificial peptides. This is corroborated by rearranging the amino acids (Figure S3 and Table S1). While the peptides possessed different antimicrobial properties, they displayed consistent hydrophobicities and no meaningful change of the hydrophobic moment, which once more indicates no correlation between the antimicrobial and biophysical properties, as has been observed in helical peptides.21 Rather, the calculated 3D structure changed from a β-strand to an antiparallel β-sheet, likely due to aggregation of the two newly created hydrophobic regions at the terminal ends of the peptide chain in an aqueous environment, resulting in a looped motif (Figure S7). Although A4 forms an antiparallel β-sheet, it differs in sequence compared to that of antimicrobial amphipathic β-sheet-forming peptides, which consist of continuous repeats of cationic and hydrophobic amino acids.26−28 In a hydrophilic environment, the latter form βsheets, in which the hydrophobic side chains are within the structure. Their mode of action is explained by the “carpet model”, wherein the peptides interact with bacterial membranes via an electrostatic driven mechanism, followed by a selfassembly to peptide-rich domains.27 Afterward, the peptides insert into the bacterial membrane and form transmembrane pores, which are stabilized by the hydrophobic interactions of side chains and membrane lipids.51 Additionally, the charged amino acids line the inside, creating a hydrophilic transmembrane channel. Thus, the repetitive motif, wherein adjacent amino acids are located on opposite sides of a stretched peptide chain, is crucial for this mode of action. Because the calculated structure of A4 indicated an antiparallel β-sheet, but lacked amphipathicity, its antimicrobial activity was meager. Moreover,

Figure 4. Concentration-dependent membrane permeabilization of S. xylosus and U937 cells in a mixed culture experiment. Without addition of any peptide, neither S. xylosus nor U937 cells display membrane permeabilization (NC). In contrast to that for melittin, O1 leads to a selective permeabilization of the bacterial membrane, while the eukaryotic membrane remains unaffected.

inappropriate for clinical applications (Table S1). The calculated structure of indolicidin displayed a flexible conformation in combination with a high hydrophobicity but a rather low hydrophobic moment. This agrees with previous NMR studies revealing no α-helical or β-sheet content.44 In contrast, the calculations regarding protamine revealed a high amount of helical content combined with a low hydrophobicity and hydrophobic moment, thus indicating that the formation of an α-helical structure alone is insufficient to trigger complete membrane disruption. Rather, amphipathicity of the formed helix is pivotal. These findings are well in accordance with previous studies correlating the amphipathicity and helixforming potential to the antimicrobial properties and the mode of action17 of artificial α-helical peptides.18,21 In contrast, protamine and indolicidin hardly displayed any membrane disruption, although they featured comparable MICs. These results are consistent with previous investigations of prolineand arginine-rich peptides that indicated an inhibition of metabolism. 45 Thus, the mode of action resembles a bacteriostatic rather than a bactericidal effect. This correlates with the ability of arginine-rich peptides to translocate across bacterial membranes.46 Nevertheless, protamine displayed a rather unique antimicrobial activity, as shown by the exclusive disruption of the outer membrane of E. coli ML-35p (Table S2). This may explain previous contradictory results. It was assumed that protamine exerts its antibacterial effect toward Salmonella typhimurium without causing cell lysis or permeabilization but rather by the disruption of energy transduction.47 Otherwise, the loss of cell internal ATP indicated a permeabilization of Gram- and Gram-negative bacteria.48 These contradictory results may be explained by the demonstrated exclusive disruption of the outer membrane. However, it may reflect only the ability of antimicrobial peptides to act by multiple mechanisms that may vary depending on conformational dynamics or target organism.49 Furthermore, protamine inhibited growth of eukaryotic cells, potentially due to the high number of arginine residues, which is often related to cytotoxic properties.50 Consequently, despite protamine’s high ϑ of >8 and >128, these values have to be evaluated meticulously, as they do not account for the inhibition properties. 3498

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amphipathic interactions are crucial for α-helical antimicrobial peptides, which exhibit hydrophilic and hydrophobic helix surfaces upon folding.52 Because these basic interactions are essential for a non-receptor-mediated antimicrobial mode of action,53 the putative β-strand-forming peptides may interact in a similar manner. However, such interactions and orientations of the peptides need to be clarified and investigated further. The difference between the artificial and α-helix-forming peptides is also reflected by the disruption kinetics of the inner and outer membrane of E. coli ML-35p (Table S2). While melittin possessed (at 8 μM) the same MIC as that of model peptide M1, the kinetic of the latter was delayed by about 20− 40 min depending on the concentration and thereby in a different order of magnitude. Amino Acid Composition. S1−S8 indicated a clear trend. While arginine-containing peptides S1, S3, and S7 displayed cytostatic properties toward the eukaryotic cells over a period of 4 days, no influence of the lysine-containing peptides S2, S4, S6, and S8 was detected (Table S1). These results are consistent with previous investigations of arginine-rich peptides indicating cytotoxic properties.50 Furthermore, the different effects of arginine and lysine on the membrane curvature were previously demonstrated.54 The guanidine group of arginine facilitates multidentate hydrogen bonding, organizing bulky lipid head groups to generate positive membrane curvature in the perpendicular direction.55 In contrast, the amine group of lysine can bind only to one lipid headgroup. This results in a negative curvature of the membrane56 and may therefore cause the differences in antimicrobial activity. Additionally, the investigation of the hydrophobic region revealed isoleucine as the amino acid resulting in the most distinct antimicrobial properties compared to those of valine, phenylalanine, and leucine. This is consistent with the individual hydrophobicitiy of single amino acids according to its physicochemical properties within short peptide sequences.57 Because a general hydrophobicity is crucial for a variety of antimicrobial peptides and their related mode of action,17 it can be assumed that the hydrophobic properties of the central region of the investigated artificial peptides is just as important for the antimicrobial activity. Accordingly, this variation of amino acid hydrophobicity elucidates the different antimicrobial activities and explains the reduced antimicrobial activity of S5 and S6 (Table S1). While phenylalanine is among the most hydrophobic amino acids, its combination with lysine (S8) resulted in reduced antimicrobial activity. It has already been demonstrated that aromatic side chains of multiple phenylalanine adapt self-stabilizing interactions within naturally occurring proteins.58 Multiple stacking patterns have been suggested, including π-stacking, T-shaped stacking, and parallel displacement. Such mechanisms may influence the conformation of the parent protein, like that indicated by the calculated 3D structures. S1−S6 displayed β-strand motifs similar to that of M1, while the structures of S7 and S8 featured small flexible regions in the central region of the β-strand, resulting in a disruption of the motif (Figure S5). Because the hydrophobic region of S8 consists of five phenylalanine residues, such interactions may influence the conformation and stability of the calculated β-strand and reduce the antimicrobial activity. In contrast, S7, which is likewise composed of phenylalanine, displayed increased antimicrobial properties. Because the stretching of the peptide chain is induced by the repulsion of both positively charged regions at either peptide terminus, the higher dissociation constant of arginine compared to that of

lysine59 may compensate the stacking of aromatic ring structures. Therefore, the antimicrobial properties are likely retained. Optimized Sequence and Application. As a result of the truncation, deletion, and substitution libraries, the optimized sequence of O1 was concluded and features a central hydrophobic region of six isoleucines and two cationic regions with three lysines each. This sequence was derived on the basis of various features, such as peptide length, antimicrobial and cytotoxic properties, and permeabilization time. On one hand, the results revealed no increase in antimicrobial activity due to the increase of cationic amino acids at the terminal region of more than three, as could be seen at N1, A1, and S2. Therefore, each flanking region of O1 was limited to three cationic amino acids in order to avoid unnecessarily long peptides. On the other hand, no measurable improvements of antimicrobial activity could be detected due to the increase of hydrophobic residues above five, as could be detected at A1 and H1. However, with an increasing amount of hydrophobic amino acids, the permeabilization time of the inner membrane was reduced, as indicated by H1 and M1. Because both a short and rapid acting peptide is desirable, six hydrophobic amino acids were selected in order to meet these requirements. Furthermore, the experiments on the substitution of amino acids revealed the peptide composition to be the most decisive parameter for antimicrobial activity. Because it is beneficial to avoid cytostatic properties, the amino acids lysine and isoleucine were selected, while arginine was avoided. O1 exhibited MICs of 8 and 0.5 μM in combination with a lack of cytotoxic properties (Table S1). Thus, ϑ for O1 (more than 8 and 128) was among the highest within the investigated peptides. These values demonstrate the benefits of an optimization of an AMP sequence. In comparison to the natural occurring AMP melittin, ϑ was increased by more than 40 and 182 times. Also, the increment to the least cytotoxic helical peptide KL-Helix was more than 4 and 36 times. The calculated secondary structure motif of O1 displayed, once again, a β-strand in hydrophilic and hydrophobic environments (Figure 3) and thereby a unique structure, which does not correlate with α-helix17 or β-sheet26−28 forming peptides. As structural motifs were calculated in a hydrophobic or hydrophilic environment, the β-strand structure may change during the membrane disruption procedure since AMPs feature an induced-fit like mechanism.49 Such a conformational change could be proven for α-helix-18 and β-sheet-forming peptides.26−28 Therefore, the calculated β-strand may not directly correlate to the active peptide conformation, but nevertheless it displays a high relevance for the rational design of AMPs. Because O1 combined antimicrobial properties and biocompatibility, it was added to a mixed culture comprising S. xylosus and U937 cells. Both peptides permeabilized the bacteria within 2 h and hampered the bacterial growth compared to that of the negative control (Figure 4). While melittin also displayed cytotoxic properties, O1 selectively disrupted the bacterial membrane while leaving the eukaryotic cells intact. As AMPs are composed of proteinogenic amino acids, they are susceptible to proteolytic digestion60 by pro- or eukaryotes. Therefore, for therapeutic applications, both stability and biocompatibility are essential.61 Because O1 remains active in the presence of eukaryotic cells, it is unaffected by proteolytic digestion, neither by consumption nor adsorption to the eukaryotic cell membrane. Therefore, a selective interaction with the bacterial cell is 3499

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for technical assistance during the cytotoxicity measurements and Anne Thomas for technical assistance during the experiments concerning the antimicrobial properties.

probable. Such specific interactions have been demonstrated previously and are consistent with proposed modes of action in which the membrane composition imparts the selectivity.62 However, as selectivity is complex and may be influenced by many parameters, limitations in therapeutic applications cannot yet be excluded. Recent studies have revealed that stability may be increased by using enantiomeric63 or diasteriomeric64 AMPs without loss of activity. Therefore, it is crucial that the parent peptide is already biocompatible. Because O1 features both biocompatible and antimicrobial properties, it is a promising candidate for therapeutic applications. Moreover, it has favorable prospects for use as a template for the development of new antimicrobial peptides with unique secondary structures.

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(1) Walsh, C. Antibiotics: Actions, Origins, Resistance; ASM Press: Washington, DC, 2003. (2) Marr, A. K.; Gooderham, W. J.; Hancock, R. E. W. Antibacterial peptides for therapeutic use: obstacles and realistic outlook. Curr. Opin. Pharmacol. 2006, 6, 468−472. (3) Gordon, Y. J.; Romanowski, E. G.; McDermott, A. M. A review of antimicrobial peptides and their therapeutic potential as anti-infective drugs. Curr. Eye Res. 2005, 30, 505−515. (4) Lehrer, R. I.; Ganz, T. Defensins of vertebrate animals. Curr. Opin. Immunol. 2002, 14, 96−102. (5) Shai, Y. Mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by alpha-helical antimicrobial and cell non-selective membrane-lytic peptides. Biochim. Biophys. Acta 1999, 1462, 55−70. (6) Gennaro, R.; Zanetti, M. Structural features and biological activities of the cathelicidin-derived antimicrobial peptides. Biopolymers 2000, 55, 31−49. (7) Wiradharma, N.; Khan, M.; Yong, L. K.; Hauser, C. A.; Seow, S. V.; Zhang, S.; Yang, Y. Y. The effect of thiol functional group incorporation into cationic helical peptides on antimicrobial activities and spectra. Biomaterials 2011, 32, 9100−9108. (8) Young, A. W.; Liu, Z.; Zhou, C.; Totsingan, F.; Jiwrajka, N.; Shi, Z.; Kallenbach, N. R. Structure and antimicrobial properties of multivalent short peptides. Med. Chem. Commun. 2011, 2, 308−314. (9) Wu, M.; Maier, E.; Benz, R.; Hancock, R. E. Mechanism of interaction of different classes of cationic antimicrobial peptides with planar bilayers and with the cytoplasmic membrane of Escherichia coli. Biochemistry 1999, 38, 7235−7242. (10) Matsuzaki, K. Why and how are peptide−lipid interactions utilized for self-defense? Magainins and tachyplesins as archetypes. Biochim. Biophys. Acta 1999, 1462, 1−10. (11) Ginsburg, I.; Koren, E. Are cationic antimicrobial peptides also ‘double-edged swords’? Expert Rev. Anti-Infect. Ther. 2008, 6, 453−462. (12) Oyston, P. C.; Fox, M. A.; Richards, S. J.; Clark, G. C. Novel peptide therapeutics for treatment of infections. J. Med. Microbiol. 2009, 58, 977−987. (13) Bell, G.; Gouyon, P. H. Arming the enemy: the evolution of resistance to self-proteins. Microbiology 2003, 149, 1367−1375. (14) Hilpert, K.; Fjell, C. D.; Cherkasov, A. Short linear cationic antimicrobial peptides: screening, optimizing, and prediction. Methods Mol. Biol. 2008, 494, 127−159. (15) Nusslein, K.; Arnt, L.; Rennie, J.; Owens, C.; Tew, G. N. Broadspectrum antibacterial activity by a novel abiogenic peptide mimic. Microbiology 2006, 152, 1913−1918. (16) Hilpert, K.; Volkmer-Engert, R.; Walter, T.; Hancock, R. E. High-throughput generation of small antibacterial peptides with improved activity. Nat. Biotechnol. 2005, 23, 1008−1012. (17) Tossi, A.; Sandri, L.; Giangaspero, A. Amphipathic, alpha-helical antimicrobial peptides. Biopolymers 2000, 55, 4−30. (18) Wiradharma, N.; Sng, M. Y. S.; Khan, M.; Ong, Z. Y.; Yang, Y. Y. Rationally designed alpha-helical broad-spectrum antimicrobial peptides with idealized facial amphiphilicity. Macromol. Rapid Commun. 2013, 34, 74−80. (19) Chou, P. Y.; Fasman, G. D. Conformational parameters for amino acids in helical, beta-sheet, and random coil regions calculated from proteins. Biochemistry 1974, 13, 211−222. (20) Dathe, M.; Meyer, J.; Beyermann, M.; Maul, B.; Hoischen, C.; Bienert, M. General aspects of peptide selectivity towards lipid bilayers and cell membranes studied by variation of the structural parameters of amphipathic helical model peptides. Biochim. Biophys. Acta 2002, 1558, 171−186. (21) Dathe, M.; Wieprecht, T.; Nikolenko, H.; Handel, L.; Maloy, W. L.; MacDonald, D. L.; Beyermann, M.; Bienert, M. Hydrophobicity,

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CONCLUSIONS The research on new antimicrobial agents for the treatment of bacterial infections has been intensified since the excessive use of antibiotics led to the development of a large variety of drugresistant bacteria. In this regard, AMPs have already been investigated as promising alternatives. Because the characteristic 3D structures of AMPs are linked to their respective mode of action, α-helix- and β-sheet-forming motifs have already been used for the research and development of optimized artificial peptide sequences. As α-helical motifs have been shown to cause cytotoxicity, their possible optimizations are limited. Therefore, we constructed AMPs with alternative peptide sequences featuring a hydrophobic core with two cationic flanking regions and identified the required minimal sequence. Differences between these artificial peptides and naturally occurring α-helical peptides were highlighted and discussed with respect to their antimicrobial activity. Furthermore, PEPfold calculations revealed that the predicted β-strand of the artificial peptides is crucial for their antimicrobial activity. Consequently, we designed an optimized peptide with a putative β-strand motif and demonstrated its potential application for therapeutic applications.

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ASSOCIATED CONTENT

S Supporting Information *

Detailed physicochemical, antimicrobial, cytotoxic, and membrane disruption properties of all antimicrobial peptides; graphical presentation of the remaining data as well as HPLC-MS data of all peptides. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Tel: +49 331 58187214. Fax: +49 331 58187119. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Central Innovation Program SME (ZIM) of the Federal Ministry of Economics and Technology (BMWi) and managed by the German Federation of Industrial Research Associations (AIF) (grant no. KF22674001MD9). We would like to thank Kathi Scheinpflug (Leibniz Institute of Molecular Pharmacology, Department Peptide-Lipid Interaction, Berlin, Germany) for support with the outer- and inner-membrane permeabilization of E. coli ML35p experiments. We gratefully acknowledge Simone Aubele 3500

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