Characterization of Antimicrobial, Cytotoxic, and Antiendotoxin

Article pubs.acs.org/jmc

Characterization of Antimicrobial, Cytotoxic, and Antiendotoxin Properties of Short Peptides with Different Hydrophobic Amino Acids at “a” and “d” Positions of a Heptad Repeat Sequence Sarfuddin Azmi,† Saurabh Srivastava,† Nripendra N. Mishra,‡ Jitendra K. Tripathi,† Praveen K. Shukla,‡ and Jimut Kanti Ghosh*,† †

Molecular and Structural Biology Division, ‡Fermentation Technology Division, CSIR-Central Drug Research Institute, Lucknow 226 001, India S Supporting Information *

ABSTRACT: To understand the influence of different hydrophobic amino acids at “a” and “d” positions of a heptad repeat sequence on antimicrobial, cytotoxic, and antiendotoxin properties, four 15-residue peptides with leucine (LRP), phenylalanine (FRP), valine (VRP), and alanine (ARP) residues at these positions were designed, synthesized, and characterized. Although valine is similarly hydrophobic to leucine and phenylalanine, VRP showed significantly lesser cytotoxicity than LRP and FRP; further, the replacement of leucines with valines at “a” and “d” positions of melittinheptads drastically reduced its cytotoxicity. However, all four peptides exhibited significant antimicrobial activities that correlate well with their interactions with mammalian and bacterial cell membranes and the corresponding lipid vesicles. LRP most efficiently neutralized the LPS-induced proinflammatory mediators like NO, TNF-α, and IL-6 in macrophages followed by FRP, VRP, and ARP. The results could be useful for designing short antimicrobial and antiendotoxin peptides with understanding the basis of their activity.

■

INTRODUCTION Antimicrobial peptides have been identified from a wide variety of living organisms and are regarded as an early defense measure1,2 in host. Usually, these peptides are cationic and amphipathic in nature with less than 10 KDa in size3 and are produced in those parts of organisms which are more likely to get exposed to the pathogens.4,5 Antimicrobial peptides exhibit antibacterial, antifungal, antiviral, anticancerous, and immunomodulatory activities.6−11 Even though the exact mechanism of action of these peptides is not deeply understood, it is considered that interaction of these peptides with the target cell membrane is a primary step.12 Many times, after bacterial infection or treatments, the remains of gram negative and gram positive bacteria, for example, lipopolysaccharides, lipoteichoic acid, and CpG initiate a complex regulated process by which activated host cells like monocyte and macrophages produce cytokines, growth factors, nitric oxide (NO), and prostaglandin.13,14 The overexpression of cytokines locally can cause serious diseases like rheumatoid arthritis, multiple sclerosis, and psoriasis, while at systemic level, it can cause septic shock.6,15,16 Current therapeutic approaches to the treatment of inflammatory diseases are centered on the suppression of the NO or TNFα production.17 Therefore, there is urgent need of such antimicrobial molecules which also possess antiendotoxin © 2013 American Chemical Society

activity. Several peptide-based molecules are in different phases of clinical trial, and fewer are ahead of this phase and have entered in clinical applications but at topical level only. Higher cytoxicity of these molecules at systemic level is a major concern of drug development. Therefore, it is important to minimize the cytotoxicity of antimicrobial peptides by gaining knowledge over the parameters that determines different antimicrobial properties. Heptad repeat sequence is the repetition of seven amino acids where each “a” and “d” position is occupied by hydrophobic residues. Leucine zipper, first identified in DNA binding proteins, is a special case of heptad repeat sequence in which every seventh residue (“a” position) is a leucine residue. Often the “d” positions of a leucine zipper sequence are also occupied by leucine/isoleucine residues. We identified and characterized a leucine zipper like sequence in the naturally occurring bee venom antimicrobial peptide, melittin, which plays a crucial role in maintaining cytotoxicity of this peptide.18 The role of this motif in maintaining cytotoxicity has also been tested in other antimicrobial peptides.19−21 Very recently, the leucine zipper sequence in melittin has been implicated in its interaction with LPS and in its antiendotoxin properties.22 The Received: September 27, 2012 Published: January 16, 2013 924

dx.doi.org/10.1021/jm301407k | J. Med. Chem. 2013, 56, 924−939

Journal of Medicinal Chemistry

Article

Figure 1. (A) Designations of the peptides along with their amino acid sequences, calculated and observed molecular masses, and their HPLC retention times. (B) Helical wheel projections of all designed peptides LRP, VRP, FRP, and ARP (box indicates the different hydrophobic amino acids of “a” and “d” positions).

Figure 2. Dose dependent cytotoxicity of LRP, VRP, FRP, and ARP against (A), hRBCs as measured by hemolytic activity assay of the peptides, and (B) murine 3T3 cells as measured by MTT assay for cell viability in the presence of these peptides. Symbols: square, LRP; circle, FRP; upright triangle, VRP and inverted triangle, ARP. Each data point is an average of three independent experiments and error bar represents the standard deviation.

substitution of leucine residue(s) at “a” and/or “d” position(s) can alter the assembly as well as stability of protein/peptide containing this motif. Thus this structural element provides a convenient opportunity to modulate the assembly of a peptide by placing different amino acids at “a” and “d” positions of its sequence and then to look into the antimicrobial, cytotoxic, and antiendotoxin activities of these peptides in order to understand the influence of amino acid sequence and their self-assembly properties on their biological activities. Leucine, phenylalanine, and valine possess comparable mean hydrophobicity, while alanine possesses lower mean hydrophobicity than the other three amino acids. However, despite having similar hydrophobicity, leucine, phenylalanine, and valine differ in participating helical assembly of the peptide molecules.23 Therefore, it was of our interest to address how amino acids with varying hydrophobicity at these “a” and “d” positions influence the bactericidal, cytotoxic, and LPS neutralization properties of the peptides. For this purpose, four short peptides of 15 residues

were designed on the basis of the same heptad repeat sequence with leucine, phenylalanine, valine, and alanine at the “a” and “d” positions of this sequence. Besides, it was also addressed whether a similar hydrophobicity at these positions confers a similar microbicidal, cytotoxicity, and inhibition of LPS-induced pro-inflammatory activity to these novel short antimicrobial peptides. The results have been discussed in terms of how different hydrophobic amino acids at “a” and “d” positions of a heptad repeat sequence contribute in determining their biological properties.

■

RESULTS Design of Short Novel Antimicrobial Peptides over Heptad Repeat Template. A leucine zipper sequence was first reported in a DNA binding protein C/EBP and later on surface protein of enveloped virus(s) which assist in cell−cell fusion24−26 and very recently in antimicrobial peptides.18 The side chains of the leucine and isoleucine residues positioned in 925

dx.doi.org/10.1021/jm301407k | J. Med. Chem. 2013, 56, 924−939

Journal of Medicinal Chemistry

Article

the “a” and “d” positions of a heptad repeat sequence interact with the similar amino acid side chains of another heptad repeat and thus a dimeric assembly of this peptide is formed. The substitution of leucine/isoleucine at “a” and/or “d” position(s) can alter the assembly of a protein/peptide containing this motif.27 Results from several research groups including ours suggest that self-assembly of an antimicrobial peptide greatly influences its cell selectivity/cytotoxicity.18,19,28−30 However, how different hydrophobic amino acids at “a” and “d” positions of a heptad repeat sequence determine the biological properties and helical assembly of the concerned peptides is not clearly known. To unravel this, four 15-residue amphipathic peptides were designed on the basis of a heptad repeat sequence. The first designed peptide was a leucine zipper peptide comprising leucine residues at all the “a” and “d” positions named as LRP, and the other three peptides VRP, FRP, and ARP, where valine, phenylalanine, and alanine were placed at the corresponding “a” and “d” positions of the peptides (Figure 1). The number and positions of the positively charged residues were kept the same for all four peptides (Figure 1). In these designed peptides, a tryptophan residue was included to study the intrinsic fluorescence of peptides. LRP and FRP Exhibited Higher Cytotoxicity against Mammalian Cells than VRP and ARP. To know the role of different hydrophobic amino acids at the heptadic positions in cytotoxic activity of the peptides, hemolytic activity of these designed short peptides were assayed. We observed that the heptads of leucine and phenylalanine, i.e., LRP and FRP were appreciably more hemolytic than alanine and valine containing heptads, i.e., ARP and VRP (Figure 2A) against human red blood cells (hRBCs). The extent of hemolytic activity exhibited by LRP, FRP, VRP, and ARP were ∼80, ∼70, ∼10, and ∼1%, respectively, at ∼60 μM concentration of each of these peptides. To further look into the toxic activity of these peptides against other mammalian cells, viability of the murine fibroblasts 3T3 cells was determined in the presence of these peptides. MTT assay was performed to determine the activity of the mitochondrial dehydrogenase, which ultimately suggests the viability of the cells. Result of MTT assay followed the same trend as the hemolytic activity assay against hRBCs (Figure 2B). The results probably indicate a role of the leucine and phenylalanine zipper sequences in controlling the cytotoxic activity of these peptides, which is consistent with the previous findings.18,20,31 However, it is very interesting to note that despite having significant hydrophobicity which is comparable to leucine and phenylalanine, the heptad with valine (VRP) exhibited appreciably reduced cytotoxicity as compared to that of LRP and FRP. The results indicate the involvement of other parameters rather than only mean hydrophobicity of the peptides in controlling the cytotoxic activity of these peptides. Antimicrobial Activity of LRP, FRP, VRP, and ARP. Antimicrobial activities of these designed peptides were tested against three Gram +ve, three Gram −ve, one methilicine resistant bacterium (methilicine resistant Staphylococcus aureus; MRSA), and six fungal strains (Table 1). VRP showed to some extent higher activity against the Gram (+)ve bacteria in comparison to the other peptides except MRSA. LRP exhibited the highest activity against MRSA among these peptides. LRP, FRP, and ARP showed comparable activities against other Gram (+)ve bacteria excluding MRSA. VRP also showed somewhat higher activity against the tested Gram (−)ve bacteria as compared to the other peptides. Nevertheless, LRP, FRP, and ARP exhibited similar MIC values against the Gram

Table 1. Antimicrobial Activities of the Peptides against Different Microorganisms As Markeda minimum inhibitory (MIC) conc in μM peptides bacterial and fungal strains Gram +ve Strain Staphylococcus aureus (ATCC 9144) Bacillus subtilis (ATCC 6633) Staphylococcus epidermidis (NRRL, B4268) Staphylococcus aureus (MR) Gram −ve Strain Escherichia coli (ATCC 10536) Pseudomonas aeruginosa (ATCC BAA427) Klebsiella pneumoniae (ATCC 27736) Fungal Strain Candida albicans Cryptococcus neoformans Candida parapsilosis Sporothrix schenckii Trichophyton mentagrophytes Aspergillus fumigatus a

LRP

VRP

FRP

ARP

6.25 10.5 12.1

5.1 6.8 6.0

6.25 11.30 13.2

6.25 20.0 8.0

6.25

12.5

25.0

6.25 11.2

3.0 3.2

6.0 25.0

6.2

3.2

6.0

25

3.1 6.1 3.0 1.56 1.56 3.1

9.6 8.4 1.5 6.0 6.0 8.4

3.1 6.1 3.0 3.0 3.0 3.0

>25 >25 25 25 >25 >25

>25 4.5 6.25

S. aureus (MR) denotes the methilicine resistant strain.

negative bacteria examined here. ARP showed the least activity against the fungal strains. Although VRP exhibited very significant activity against the fungal strains, LRP and FRP were to some extent more active than VRP against the fungal strains studied here. VRP and ARP More Selectively Bind to Escherichia coli, but LRP and FRP Bind Efficiently to hRBC Also. To explore the binding of these peptides containing heptad repeats of different hydrophobic amino acids toward the mammalian cell and bacteria, their localization onto hRBCs and Escherichia coli 10536 was studied by confocal microscopy by employing their NBD-labeled versions. Prominent green fluorescence was observed on hRBCs when binding experiment was performed with NBD-labeled LRP and FRP (Figure 3A,C), suggesting their strong binding to hRBCs. However, when the same experiment was performed with NBD-VRP, intensity of green color onto the hRBCs was significantly less (Figure 3B), indicating its weaker binding to these cells. The intensity of green fluorescence onto hRBCs with NBD-ARP turned out as almost invisible in the setting of the same parameters used for visualizing the images (Figure 3D) of the cells with other peptides. The data suggest NBD-ARP possesses the weakest binding affinity for hRBCs among these peptides. However, in similar experiments for detecting the binding of the peptides onto bacterial cell, we observed a significant and comparable staining of E. coli 10536 with all these NBD-labeled peptides (Figure 3E−H), suggesting an appreciable and comparable binding of all four peptides onto the bacteria. Overall, the data match with the varying cytotoxic and comparable bactericidal activity of these peptides. In Contrast to Leucine/Phenylalanine Heptadic Peptide (LRP/FRP), Valine/Alanine Heptadic Peptide (VRP/ ARP) More Preferably Damages the Bacterial Membrane Organization than hRBCs. The damage of hRBC/mammalian cell membrane leads to the exposure of phosphatidylserine (PS), which normally present predominantly in inner leaflet of 926

dx.doi.org/10.1021/jm301407k | J. Med. Chem. 2013, 56, 924−939

Journal of Medicinal Chemistry

Article

Figure 3. Detection of localization of the NBD-labeled peptides (LRP, VRP, FRP, and ARP) onto hRBCs and E. coli 10536 cells by confocal microscopy. Each cell type has been marked on top of the images, and NBD-labeled peptides that have been used to treat the cells are shown on the left-hand side. For each of the peptide treatment, DIC and fluorescence images of each cell type have been shown. Peptide concentration of each of the NBD-labeled peptides for treatment with hRBCs was ∼9.0 μM whereas for treatment with E. coli was ∼3.0 μM.

recovery at different peptide concentrations as described in the Experimental Section. In the case of bacterial cell membranes, all heptadic peptides exhibited similar extent of depolarization, whereas the extent of membrane depolarization of hRBCs in the presence of these peptides was very distinct. LRP and FRP induced significantly higher depolarization onto hRBC membrane as compared to VRP and ARP, which is evident from the fluorescence recovery data (Figure 5A). In other words, VRP and ARP selectively perturbed/depolarized bacterial membrane (Figure 5B), whereas LRP and FRP depolarized appreciably both bacterial (S. aureus) and mammalian (hRBCs) membranes (Figure 5A,B). In Contrast to LRP and FRP, VRP and ARP Selectively Perturbed Bacterial Membrane Mimetic Lipid Vesicles. The ability of the antimicrobial peptides to destabilize the phospholipid bilayer is believed to be associated with the mechanism of their antimicrobial and cytotoxic activities. Therefore, to understand the basis of cytotoxic and antimicrobial activities of these designed peptides, membrane permeability were examined by determining their efficacy to dissipate the diffusion potential across the phospholipid membrane of different lipid composition. Zwitterionic, PC/ Chol, and negatively charged PE/PG lipid vesicles were employed as mimetics of mammalian and bacterial membranes, respectively. As evident from the fluorescence recovery data, LRP and FRP permeabilized the zwitterionic, mammalian

the membrane. The selective probing of PS with membrane impermeable dye annexinV−FITC indicates the membrane damaging property of the peptides. LRP and FRP more extensively damaged the organization of hRBCs membrane, whereas VRP and ARP showed insignificant membrane damaging property toward mammalian cells (Figure 4). Selective staining of hRBCs by annexin V-FITC following the treatment of peptides indicated the varying propensity of these peptides to damage the membrane of hRBCs (upper panels, Figure 4). The peptide-induced damage of bacterial cell membrane was probed by propidium iodide (PI) staining of E. coli. (ATCC 10536) following the peptide treatments. This dye binds to the nucleic acids, which is possible only after the damage of bacterial membrane. LRP, VRP, FRP, and ARP exhibited comparable staining of E. coli (lower panels, Figure 4), which were consistent with their relative bactericidal activities. Unlike LRP and FRP, VRP, and ARP Selectively Depolarized Bacterial Cell (Staphylococcus aureus) Membrane but Not to Mammalian Cell (hRBCs). To further look into the mode of action of these designed peptides, membrane depolarization of mammalian and bacterial cells in their presence were studied. Peptide-induced depolarization of mammalian cell membrane (hRBCs) and bacterial cell membrane (S. aureus) was determined by employing a potential sensitive dye diS-C3−5 and by measuring its fluorescence 927

dx.doi.org/10.1021/jm301407k | J. Med. Chem. 2013, 56, 924−939

Journal of Medicinal Chemistry

Article

Figure 4. (upper panel) Peptides induced damage of hRBCs membrane as detected by FITC-annexin V staining. Upper left quadrant of each panel depicts unstained cells, whereas the upper right quadrant depicts the stained cells. Concentrations of the peptides were ∼25.0 μM. (lower panel) Peptides induced membrane damage of E. coli ATCC10536 as detected by PI staining. Upper left quadrant of each panel depicts unstained cells whereas the upper right quadrant depicts the stained cells. Concentrations of the peptides were ∼5.0 μM. 10000 events were recorded for each sample and control panels show staining of the cells in the absence of any peptide.

Figure 5. Dose dependent peptides-induced trans-membrane depolarization of hRBCs and S. aureus (ATCC 9144) membranes and mammalian and bacterial membrane mimetic PC/Chol (8:1, w/w) and PE/PG (7:3, w/w) lipid vesicles. (A−D) Plot of the percentage of fluorescence recovery vs peptide concentrations (or peptide/lipid molar ratios in the case of panels C and D) for peptides-induced depolarization of hRBC, S. aureus, PC/ Chol and PE/PG lipid vesicles, respectively. Symbols: square, LRP; upright triangle, FRP; circle, VRP and inverted triangle, ARP. Each data point is an average of three independent experiments and error bar represents the standard deviation.

membrane mimetic, PC/Chol vesicles to a significantly higher extent than that of VRP and ARP (Figure 5C). However, all the

heptadic peptides induced nearly similar permeability in negatively charged, PE/PG, lipid vesicles (Figure 5D), 928

dx.doi.org/10.1021/jm301407k | J. Med. Chem. 2013, 56, 924−939

Journal of Medicinal Chemistry

Article

Figure 6. Determination of environment of the tryptophan residues of these peptides in their membrane-bound states by recording their emission maxima and by Stern−Volmer plots for acrylamide quenching of tryptophan fluorescence in the presence of different lipid vesicles. Fluorescence spectra of the peptides in zwitterionic, PC/Chol (A) and negatively charged, PE/PG (B) lipid vesicles. Peptides (∼1.0 μM) were added to PBS with subsequent addition of ∼450 μM of either lipid vesicles. Stern−Volmer plots for the acrylamide quenching of tryptophan fluorescence of LRP, VRP, FRP, and ARP in the presence of PC/Chol (C) and PE/PG (D) lipid vesicles. Symbols: square, LRP; upright triangle, FRP; circle, VRP and inverted triangle, ARP. (E), Stern−Volmer constant KSV in the presence of PC/Chol and PE/PG lipid vesicles and buffer; symbols have been shown in the right upper corner of panel E.

signified that tryptophan residues of all these peptides were localized in a similar environment of the PE/PG lipid bilayer. Distinct Tryptophan Quenching by Acrylamide Was Observed When the Peptides Are Bound to Zwitterionic Lipid Vesicles but Not in Negatively Charged Lipid Vesicles. To further look into the accessibility of tryptophan residue of these peptides in their membrane-bound states, quenching of the tryptophan fluorescence of these peptide− lipid complexes in the presence of acrylamide was studied. Acrylamide is a neutral water-soluble quencher, hence it does not show any electrostatic interaction with the negatively charged headgroup of anionic phospholipids.32 The Trp fluorescence quenching by acrylamide was studied in the absence as well as in the presence of different lipid vesicles and are depicted as Stern−Volmer plots in Figure 6C,D. Distinct differences were observed in the quenching of tryptophan fluorescence of the peptides when they were bound to zwitterionic and negatively charged lipid vesicles. For example, tryptophan fluorescence of both LRP and FRP exhibited much lesser acrylamide quenching as compared to that of VRP and ARP when they were bound to the mammalian membrane mimetic, PC/Chol, lipid vesicles (Figure 6C). Stern−Volmer plots suggest (Figure 6C) that fluorescence of ARP and LRP exhibited the highest and lowest acrylamide quenching respectively when they were bound to PC/Chol lipid vesicles. Altogether, the results indicate that tryptophan residues of both LRP and FRP were located more toward the inside of the bilayer of zwitterionic, PC/Chol vesicles and therefore not accessible by acrylamide. However, tryptophan residues of both VRP and ARP were possibly located toward the surface of zwitterionic membrane and thus accessible by acrylamide, which resulted in a significant quenching of their fluorescence.

suggesting their comparable ability to permeabilize bacterial membrane mimetic membrane. The data of peptide-induced permeabilization of different membrane-mimetic lipid vesicles match with cell-selective bactericidal activity of VRP and ARP and both cytotoxic and bactericidal activity of LRP and FRP. Unlike LRP/FRP, VRP/ARP More Selectively Bound to Negative Charged Lipid Vesicle than Zwitterionic Lipid Vesicles As Evidenced by Tryptophan Fluorescence Studies. Typically, tyrptophan fluorescence emission maximum falls in shorter wavelength when it is in the hydrophobic milieu of the phospholipid membrane and it shifts toward the longer wavelength in the case where it is in the more polar environment of the membrane. The sensitivity of the fluorescence emission of the Trp residue to its environment allows us to monitor the binding/localizations of peptides onto the phospholipid vesicles. We observed that all four peptides exhibited an emission maximum at ∼357 nm in buffer, a typical wavelength indicating that the Trp residue is in a polar environment (data not shown). However, after the addition of PC/Chol lipid vesicles a large blue-shift was observed for LRP and FRP (Figure 6A). Interestingly, tryptophan emission maxima of VRP and ARP showed much lesser shifts toward the shorter wavelength (Figure 6A). The result suggests that the tryptophan residues of LRP and FRP were probably localized in the hydrophobic region of the lipid bilayer of mammalian membrane mimetic, PC/Chol, whereas the tryptophan residues of VRP and ARP were located closer to its surface. However, in the presence of bacterial membrane mimetics, PE/PG lipid vesicles, similar extent of blue-shifts of Trp emission maxima were observed for all the heptadic analogues (Figure 6B). The pattern of emission maxima 929

dx.doi.org/10.1021/jm301407k | J. Med. Chem. 2013, 56, 924−939

Journal of Medicinal Chemistry

Article

Figure 7. Determination of secondary structures of LRP, VRP, FRP, and ARP in different environments and their self-association property in PBS containing 1.5 M NaCl. CD spectra of the peptides in PBS (A) and in the presence of ∼500 μM of PC/Chol (B) or PE/PG (C) lipid vesicles. Concentrations of the peptides were ∼36 μM. (D) CD [mdeg] at 84 μM concentrations of different peptides. Tryptophan emission spectra of these peptides at ∼25 μM (E). Symbols for either CD or fluorescence spectra: square, LRP; upright triangle, FRP; circle, VRP; inverted triangle, ARP.

in PBS containing 1.5 M NaCl to look into the self-association properties of these peptides. Higher concentrations of salt were used to stabilize the self-assembly of cationic peptides. The result of circular dichroism studies indicate that with increasing concentration secondary structures of both LRP and FRP increased progressively. However, there was no significant enhancement of helical structure of VRP and ARP with increase in concentration in the same buffer (Figure S1 in the Supporting Information). Figure 7D shows the CD spectra of all four peptides at a particular concentration, which clearly indicates the differences among the peptides in their secondary structures and self-association properties. Similarly, in fluorescence studies, at higher peptide concentration LRP and FRP showed maxima of tryptophan fluorescence at much shorter wavelengths, while VRP and ARP at the same concentration showed emission maxima at much longer wavelengths (Figure 7E). Fluorescence studies indicate that for LRP and FRP with increase in peptide concentration, a relocation of their tryptophan residues in more hydrophobic environment took place whereas the tryptophan residues of VRP and ARP remained more or less in polar environment at higher concentrations (Figure 7E). Altogether, results of CD and fluorescence studies suggest that LRP and FRP were more selfassembled in the aqueous environment as compared to VRP and ARP. LRP and FRP Inhibited LPS Induced NO Production in Macrophage Cells More Efficiently than VRP and ARP. Some antimicrobial peptides can inhibit endotoxin LPS and LTA induced pro-inflammatory response in macrophage cells.33−35 To evaluate the antiendotoxin property of these designed peptides, LPS-induced NO production was evaluated in macrophage cell, RAW 264.7, in the absence and presence of these peptides by Griess reagent. The result of dose dependent response showed that LRP inhibited ∼90% NO level at 3 μM, FRP ∼80% at 4.5 μM, VRP and ARP ∼50% and 13%,

Interestingly, a different picture was observed when the peptides were bound to negatively charged lipid vesicles. Tryptophan fluorescence of all four peptides showed much lesser acrylamide quenching when they were bound to PE/PG vesicles, which suggested that probably (Figure 6D) the tryptophan residues of all four peptides were located more inside the bilayer of negatively charged lipid vesicles and hence not accessible to acrylamide. Stern−Volmer constants (KSV) for LRP and FRP that were appreciably less than that ARP and VRP when the peptides were bound to zwitterionic lipid vesicles (Figure 6E). On the other hand, KSV values of all four peptides were relatively less and quite comparable when they were bound to the negatively charged lipid vesicles (Figure 6E). Furthermore, KSV values for all these peptides were appreciably higher when the peptides were in the aqueous buffer (Figure 6E). Taken together, the results of acrylamide quenching studies of these peptides are supportive of the studies on tryptophan emission maxima in the presence of lipid vesicles with varying lipid compositions (Figure 6A,B). Significant Differences among the Peptides in Their Secondary Structures in Zwitterionic Lipid Vesicles Milieu. To look into secondary structures of the designed peptides, LRP, FRP, VRP, and ARP, circular dichroism studies of the peptides were performed in the presence of PBS (pH 7.4), zwitterionic (PC/Chol, 8:1 w/w), and negatively charged (PE/PG, 7:3 w/w) lipid vesicles. Among these, LRP and FRP appreciably adopted helical structure in PBS (Figure 7A) and in the presence of PC/Chol lipid vesicles their helix contents increased further (Figure 7B). However, in the presence of PE/ PG lipid vesicles, LRP, FRP, as well as VRP adopted appreciable secondary structures (Figure 7C). Unlike VRP and ARP, LRP and FRP Showed Appreciable Self-Association in PBS. The effects of changes in peptide concentrations on their tryptophan fluorescence as well as on the alteration in their secondary structures were studied 930

dx.doi.org/10.1021/jm301407k | J. Med. Chem. 2013, 56, 924−939

Journal of Medicinal Chemistry

Article

Figure 8. Neutralization of LPS-induced pro-inflammatory response in macrophage, RAW 264.7 cells by LRP, VRP, FRP, and ARP. (A) The amount of nitric oxide produced in LPS stimulated RAW 264.7 cells in the presence and absence of the peptides; ctrl and LPS stand for untreated control and LPS (1 μg/mL) treated cells, respectively, while LRP, FRP, VRP, and ARP represent the amount of NO produced by the LPS treated cells in the presence of these peptides (4.5 μM). The μM values of nitric oxides were determined by using a standard sodium nitrite curve. (B) Percentage inhibition of NO production in LPS-stimulated cells in the presence of different peptides at 4.5 μM concentrations. (C) Dose dependent response of peptides onto LPS-induced NO production. Symbols: square, LRP; circle, FRP; upright triangle, VRP; inverted triangle, ARP. (D) Evaluation of expression level of iNOS-2 and TNF-α in LPS-induced macrophage RAW264.7 cells in the presence of these peptides at 4.5 μM concentration. Western blot results represent the effect of treatments of designed peptides on the expression level of iNOS-2 and TNF-α in LPS-stimulated cells in 24 h. β-Actin used as internal control. (E,F) Densitometry analysis for the expression of iNOS-2 and TNF-α, respectively, which were normalized to the levels of β-actin for each sample. (G) β-Actin level, used as internal loading control, for each sample. (H,I) Percentage inhibition of levels of LPS induced secretions of TNF-α and IL-6, respectively, in the presence of LRP, FRP, VRP, and ARP by ELISA experiments. Peptide concentrations for both (H) and (I) are marked in the upper right corner of these panels. Each data point is an average of three independent experiments, and error bar represents the standard deviation.

respectively, when both were at 6 μM concentration (Figure 8A−C). The results of inhibition of LPS-induced NO production in RAW 264.7 cells indicate that different hydrophobic amino acids at “a” and “d” positions of the heptad repeats differently affect the antiendotoxin activity of the peptides (Figure 8). Inhibition of LPS Induced iNOS-2 and TNF-α by the Peptides. The over secretion of nitric oxide is proportional to overexpression of iNOS-2 (inducible nitric oxide synthase-2). To determine the inhibitory effect of these peptides on LPSinduced overexpression of iNOS-2 in RAW 264.7 cells, Western blotting experiments with the cell lysates were performed as described in the Experimental Section. The result shows that among these peptides LRP most prominently down-regulated the LPS-induced intracellular expression of iNOS-2 (Figure 8D,E). FRP was to some extent less active than

LRP in inhibiting the overexpression of iNOS-2 in LPSstimulated RAW 264.7 cells, while VRP and ARP were even lesser active in this regard in the same experimental conditions (Figure 8D,E). Similarly, expression of TNF-α, an important cytokine in systemic inflammation also markedly enhances in presence of LPS in macrophage cells. To detect the effect of these designed peptides on LPS-induced pro-inflammatory response in RAW 264.7 cells, cell lysates were subjected to Western blotting experiments with TNF-α antibody. The inhibition of LPSinduced intracellular expression of TNF-α in RAW 264.7 cells by these heptadic peptides showed a similar trend (Figure 8D,F) as it was observed with iNOS-2 (Figure 8D,E). Similar to Intracellular, Secretary Pro-Inflammatory Mediators Were Inhibited by LRP and FRP. Along with increment at intracellular level, pro-inflammatory mediators are 931

dx.doi.org/10.1021/jm301407k | J. Med. Chem. 2013, 56, 924−939

Journal of Medicinal Chemistry

Article

Figure 9. (A) Determination the effect of LPS onto peptides conformation. CD spectra of designed peptides (36 μM) recorded (marked by different names) at two different concentrations of LPS (3.8 and 6.25 μM). (Symbol: solid line, peptide in aqueous environment; line with square, peptide with 3.8 μM LPS; line with upright triangle, peptide with 6.25 μM LPS). (B) Dissociation of FITC-LPS aggregates in the presence of increasing concentrations of designed peptides. Increase in fluorescence (in arbitrary units) of FITC-LPS has been plotted with respect to peptide concentration in μM. The change in FITC emission after each treatment was monitored until emission reached equilibrium. FITC-LPS concentration (LPS-FITC 0.5 μg/mL) was the same for each experiment, and the collected data with a particular peptide has been marked in the Xaxis of each plot. (C) Binding of the designed peptides to LPS was determined by quantitative chromogenic limulus amoebocyte lysate (LAL) assay. Inhibition in substrate color production represents the inhibition of LAL enzyme’s activity which results from the binding of peptides to LPS. Thus the binding of the peptides to LPS was estimated from the inhibition of substrate color production and presented as the percentage LPS binding. Peptide concentrations are shown in the right upper corner of (C).

reported that peptide−LPS interaction plays an important role in antiendotoxin activity of antimicrobial peptides.36−38 Therefore, to study the interaction between these designed peptides and LPS, CD experiments were performed. The CD spectra of the peptides in presence of increasing concentrations of LPS showed that the most active peptide, LRP adopted the maximum helical structure among these peptides which was followed by FRP (Figure 9A). VRP and ARP adopted lesser secondary structures in comparison to LRP and FRP (Figure 9A) in the same experiment. The maximum helical structure of LRP in comparison to other peptides (Figure 9A) at the same peptide and LPS concentrations could also be indicative of its higher self-association property in LPS among the four peptides as we observed for melittin previously.22 Altogether, the extent of secondary structure adopted by the peptides in presence of LPS follow the trend of their anti-LPS property.

also secreted out in presence of inducers of inflammation like LPS from the macrophage cells. Therefore, to investigate the effect of these peptides on LPS-induced secretion of proinflammatory mediators, ELISA experiments were performed with the cell culture supernatant when RAW 264.7 cells were treated with LPS alone or in combination of LPS and different peptides. We observed that LRP and FRP efficiently inhibited the secretion of pro-inflammatory cytokines (TNF-α and IL-6) from LPS-stimulated RAW 264.7 cells, whereas VRP and ARP were less active in inhibiting the secretion of these proinflammatory cytokines from these cells (Figure 8E,F; the raw absorbance data are shown in Figure S2 in the Supporting Information). Peptides Adopted Relatively Distinct Helical Structures in Milieu of LPS. LPS-induced pro-inflammatory response is exhibited through cell surface receptor MD-2− TLR complex of monocytes and macrophages. It has been 932

dx.doi.org/10.1021/jm301407k | J. Med. Chem. 2013, 56, 924−939

Journal of Medicinal Chemistry

Article

LRP and FRP Disintegrate LPS More Efficiently than VRP and ARP. LPS-induced pro-inflammatory responses depend on its physical state. In previous studies,22,36,39 it has been well correlated that the dissociation of highly aggregated LPS by peptides directs their extent of neutralization of inflammatory response. Therefore, to investigate the basis of anti-inflammatory response of these peptides, peptide-induced disintegration of LPS aggregate was determined by monitoring the dequenching of FITC-tagged LPS fluorescence in their presence. We observed that the most active anti-inflammatory peptide, LRP, induced the disintegration of FITC-LPS most profoundly among these peptides (Figure 9B). FRP also induced appreciable dequenching of FITC-LPS (Figure 9B) fluorescence; however, VRP and ARP induced lesser disintegration of LPS-aggregates which signifies the lower antiendotoxin properties of these peptides (Figure 9B). LRP Showed the Maximum Efficacy to Bind to LPS. The binding of these heptadic peptides to LPS was studied by performing the chromogenic limulus amebocyte lysate (LAL) assay by employing endotoxin detection kit. Binding of the peptides to LPS was determined by measuring their efficacy to inhibit the LPS-induced activation of LAL enzyme. LRP was found to be the most efficient peptide in binding to LPS followed by FRP (Figure 9C) as evident from the relative inhibition of LPS-induced activation of LAL enzyme in the presence of these peptides. VRP also showed to some extent binding to LPS as evidenced by the partial inhibition of the activation of LAL enzyme the binding of ARP to LPS was even lower. At 4.5, 9, and 18 μM peptide concentrations, LRP showed nearly 72%, 90%, and absolute inhibition of LPSinduced LAL enzyme while FRP exhibited nearly 32, 54, and 80%, VRP showed 8, 22, and 29%, and ARP showed 3, 7, and 10% inhibition of activation of LAL enzyme (Figure 9C). The gradual decrease in LPS binding from LRP to FRP to VRP to ARP as evidenced by LAL assay is supportive of their in vitro LPS neutralizing abilities.

lian cells (hRBCs) as compared to LRP and FRP (Figure 3A− D). In the line of their comparable bactericidal activities, these heptadic peptides showed comparable binding to the bacterial cells (E. coli) (Figure 3E−H). Further, the flow cytometric data on PI staining showed that LRP, VRP, FRP, and ARP damaged E. coli membrane appreciably and comparably (Figure 4 lower panel). However, the extents of hRBCs membrane damage were contrasting, as evidenced by the staining of FITCannexinV; LRP and FRP damaged hRBC membrane maximally and VRP and ARP insignificantly (Figure 4, upper panel). Most of the AMPs primarily act through damaging the membrane organization18 by interacting with its lipids. To understand the importance of lipid−peptide interaction40 in the activity of these peptides, their interactions with mammalian membrane-mimetic, zwitterionic PC/Chol (8:1, w/w) and bacterial membrane-mimetic, negatively charged PE/PG (7:3, w/w) lipid vesicles were studied (Figure 5). The results of peptides-induced damage of membrane organization (Figure 4) of bacterial and mammalian cells, trans-membrane depolarization of mammalian and bacterial cells (Figure 5A,B), and permeabilization/perturbation of mimetic model membranes (Figure 5C,D) suggest that these peptides could target the membrane of different cells to exhibit their cytotoxic and antimicrobial activities. The intrinsic tryptophan fluorescence maxima of the peptides indicate that the tryptophan residues of LRP, VRP, FRP, and ARP were localized very similarly into the hydrophobic bilayer of the negatively charged lipid vesicles (Figure 6B), which was also supported by the least acrylamide quenching (Figure 6D) of their tryptophan fluorescence when these peptides were bound to the PE/PG lipid vesicles. However, differences were observed among these peptides when they were bound to the zwitterionic lipid vesicles. Both tryptophan emission maxima (Figure 6A) and acrylamide quenching (Figure 6C) of tryptophan fluorescence suggest that while tryptophan residues of both LRP and FRP were located more inside the hydrophobic bilayer of zwitterionic lipid vesicles, the tryptophan residues of the lesser toxic peptides VRP and ARP did not penetrate the hydrophobic region of the zwitterionic lipid bilayer and were most likely localized onto the surface of this kind of membrane. Probably, the localizations of LRP and FRP more inside the bilayer of both zwitterionic and negatively charged lipid vesicles is an indicator of their noncell-selective lytic activity against both mammalian cells and bacteria. The lack of penetration into the hydrophobic bilayer of zwitterionic lipid vesicles by VRP and ARP could be the basis of their lesser cytotoxicity as compared to LRP and FRP. Adoptions of secondary structures in the model membrane milieu with different lipid compositions have been employed often to explain the antimicrobial activity and cytotoxicity of antimicrobial peptides.40,41 Although LRP and FRP showed helical structures in aqueous environment and their helical contents further increased in the presence of zwitterionic lipid vesicles, VRP and ARP neither attained appreciable helical structure in PBS nor in the zwitterionic model membrane (Figure 7A,B). However, interestingly, all these peptides adopted higher helical structures in the presence of PE/PG lipid vesicles as compared to that in PBS (Figure 7C). The results reported in the literature along with our studies indicate that probably the peptides require definite secondary structure and/or aggregated assembly to permeabilize mammalian cell membrane and thus important for their cytotoxicity.18,19,28−30

■

DISCUSSION In this study, we used an amphipathic heptad repeat sequence as the template to design novel 15-residue antimicrobial peptides by placing different hydrophobic amino acids viz. leucine, valine, phenylalanine, and alanine (designated as LRP, VRP, FRP, and ARP, respectively) residues at “a” and “d” positions of the heptads. The primary objective of this design was to have a comparative picture on the effect of different hydrophobic amino acids at the “a” and “d” positions of a heptad repeat sequence on their antimicrobial, cytotoxic, and antiendotoxin activities. The present study revealed that the heptad repeat with leucine residues (LRP), i.e., typically a leucine zipper peptide and the heptad of phenyalanine (FRP) residues, are more cytotoxic against mammalian cells than heptad repeats of either valine or alanine residue (Figure 2A,B). However, all these peptides exhibited appreciable and more or less comparable antimicrobial activity (Table 1). Analysis of the antimicrobial and cytotoxic activities of these peptides indicates that VRP exhibits both potent antimicrobial and appreciably low cytotoxicity. Therefore, heptad repeat sequence with valine at “a” and “d” positions could be further explored for designing of novel cell-selective antimicrobial peptides. Localization or binding of fluorescent labeled peptides onto hRBCs or bacteria (E. coli) by confocal microscopy and flow cytometry (data not shown) showed that the VRP and ARP possess significantly lesser and negligible binding affinity respectively for mamma933

dx.doi.org/10.1021/jm301407k | J. Med. Chem. 2013, 56, 924−939

Journal of Medicinal Chemistry

Article

inhibition of expression level of iNOS-2 in the presence of these heptadic peptides appreciably correlate with their inhibitory effects on LPS induced nitric oxide production in these cells. The immunoblotting experiments for cytokine, TNF-α, showed similar responses (Figure 8D,F) by these peptides as we observed in case of iNOS-2 (Figure 8D,E). The inhibition of secretion of proinflammatory cytokine, TNF-α in the presence of different peptides as observed by ELISA experiments (Figure 8H) closely matched with the respective peptide induced inhibition of intracellular level expression of the cytokine (Figure 8D) also. In addition, we observed inhibitory effects of the designed peptides on LPS-induced secretion of the other pro-inflammatory cytokine IL-6 (Figure 8I) from RAW 264.7 cells. To further confirm the ability of peptides and their relative activity in inhibiting the proinflammatory responses in LPS-induced-macrophage cells, experiments were performed with rat primary macrophage cell (Figure S5 in the Supporting Information). LRP most efficiently inhibited secretion of cytokines TNF-α and IL-6 in LPS-stimulated rat bone marrow derived macrophage cells followed FRP and VRP and ARP. Thus the data with rat primary macrophage cells support and strengthen the antiendotoxin property of these peptides along with their relative activity in macrophage cells. Reports suggest that LPS-detoxifying peptides interact with LPS first and then disintegration of the LPS core region takes place which is primarily constituted of lipid A.36 The gradual decrease in the ability of the peptides to attain a definite secondary structure in LPS from LRP to FRP to VRP to ARP is a consequential evidence of their increasing inability to interact with LPS (Figure 9A). The results obtained from LAL assay which not only indicates the binding of a molecule to LPS, sometimes it is considered as LPS neutralization assay in aqueous environment, clearly demonstrate the higher efficacy of LRP to bind LPS followed by FRP, VRP, and ARP, respectively (Figure 9C). Thus the relative efficacy of the peptides to bind to LPS follows the same pattern as the neutralization of LPSinduced pro-inflammatory response in macrophage cells by the same peptides. Consequently, the results suggest that physical interaction of these designed peptides to LPS probably plays a prominent role in their antiendotoxin properties like other peptides as reported in the literature.22,36,39 It seems that the peptides which tend to get aggregated are more capable of breaking the organization of the lipid A part of LPS.22,42,43 We found a remarkable difference among these peptides in their self-association properties in an aqueous environment (Figure 7D) and in the presence of LPS (Figure 9A). LRP was the maximum active peptide in disintegrating FITC-LPS aggregate, as evident from the dequenching of its fluorescence followed by FRP among these peptides. VRP and ARP showed to some extent disintegration of the LPS core in the similar experiment but definitely not up to the efficiency levels of LRP and FRP (Figure 9B). This was also in correlation with their respective abilities to neutralize LPS-induced pro-inflammatory responses in macrophages. The significant and maximum activity of LRP probably indicates that the structural properties of leucine zipper sequence in LRP plays a crucial part in neutralizing LPSinduced pro-inflammatory response which is supportive to our recent study on melittin.22 Altogether, the results presented here show the characterization of short peptides designed on the basis of heptad repeat sequence with leucine, phenylalanine, valine, and alanine at the “a” and “d” positions and indicate the influence of helical self-

The present study shows a crucial information for the literature that despite having very similar hydrophobicity heptadic peptide, VRP, with valine residues at “a” and “d” positions does not self-assemble in an aqueous environment (Figure 7D,E) which could contribute in the poor permeabilization of zwitterionic lipid vesicles or mammalian cell membrane in its presence and thus in its lower cytotoxicity. Further, to investigate the effect of substitution of valine at heptadic positions of a highly cytotoxic, natural antimicrobial peptide, melittin, an analogue (MelVal) was designed in which four leucine residues located at “a” and “d” positions (6 and 13 are “a” positions, while 9 and 16 are “d” positions) were replaced by four valine residues (Figure S3 in the Supporting Information). We observed that substitution of these leucine residues by valine residues drastically reduced the hemolytic activity of melittin. While melittin exhibited ∼90% hemolysis of hRBCs at ∼6.4 μM, MelVal exhibited ∼2% hemolytic activity at more than 50 μM concentration. Similarly, in MTT assay, it was observed that at nearly 7 μM concentration melittin killed almost all the 3T3 cells, however, ∼70% viable cells remained even at more than 16 μM concentration of this analogue (Figure S3 in the Supporting Information). The results show that the leucine residues at the “a” and “d” positions of melittin’s leucine zipper sequence play a very specific role in maintaining its cytotoxicity which cannot be restored by replacing these residues by equally hydrophobic valine residues. The result of antimicrobial activity showed that substitution of all heptadic leucine residues by valine residues did not significantly impair the growth inhibitory activity of melittin (Figure S3 in the Supporting Information). To examine the effect of valine substitution on self-association property of melittin, circular dichroism (CD) and tryptophan fluorescence emission spectra of melittin and MelVal were recorded at high ionic strength in phosphate buffer (Figure S4 in the Supporting Information), which shows that melittin dose dependently adopted more and more helical structure in PBS at high salt concentration (1.5 M NaCl) as previously reported.18 However, the substitution of heptadic leucine residues with valine residues drastically diminished the helical self-assembly of melittin (Figure S4 in the Supporting Information), as evident by the shape of the spectra and comparatively smaller increase in CD values of MelVal as compared to that of melittin. Further, tryptophan emission maximum of melittin showed a significantly higher blue-shift as compared to that of MelVal. The results signified that the substitution of heptadic leucine residues by valine residues impaired the self-association property of melittin. Considering the properties of VRP and this valine-substituted melittin analogue, the results altogether demonstrated a new methodology for designing antimicrobial peptides with reduced cytotoxicity without altering their mean hydrophobicity. The other objective of the present investigation was to look into the influence of different hydrophobic amino acids in the heptad repeat sequence on its LPS-neutralization property. The results presented here clearly demonstrate the differences among the four heptadic peptides toward the neutralization of pro-inflammatory response in LPS-stimulated RAW 264.7 cells (Figure 8). LRP was the most efficient molecule among these peptides followed by FRP, VRP, and ARP in neutralization of LPS induced production of nitric oxide in RAW 264.7 cells (Figure 8A−C). The expression level of iNOS-2 (inducible nitric oxide synthase) in LPS-stimulated RAW 264.7 cells altered with the treatments of peptides (Figure 8D,E) and the 934

dx.doi.org/10.1021/jm301407k | J. Med. Chem. 2013, 56, 924−939

Journal of Medicinal Chemistry

Article

viz., sulfanilamide (S92510) and p-naphthylethylenediamine dihydrochloride (N 9125) were purchased from Sigma. The rests of the reagents were of analytical grade and procured locally; buffers were prepared in Milli Q (USF-ELGA) water. Antibodies. For protein expression study by immunoblotting, we have used mouse anti iNOS-2 (610328 BD transduction), rabbit antiTNF-α polyclonal (no. 654300 Calbiochem USA), IL-1β rabbit polyclonal (sc-7884), COX-2 mouse monoclonal (sc-19999), and mouse anti-β-actin monoclonal (CP01) was procured from Calbiochem. For immunoblotting and ELISA experiments, alkaline phosphatase conjugated antimouse (401212) and antirabbit (401352), and peroxidase conjugated antimouse (401215) and antirabbit (401315) were taken from Calbiochem, USA. Cell Culture. Mammalian cell lines murine 3T3 (mouse fibroblast cells), macrophage RAW 264.7, and rat bone marrow derived macrophages were grown in DMEM/IMDM supplemented with 10% fetal calf serum and antibiotics at 37 °C in a humidified atmosphere at 5% CO2 and 95% air. The cell line was maintained in Innova CO2 incubator. Cells were counted with the help of an Olympus microscope for the experiments. Peptide Synthesis, Their Fluorescent Labeling, and Purification. This set of novel designed peptides was synthesized manually via the solid phase method on Rink amide MBHA resin utilizing standard Fmoc chemistry.44−46 Labeling of fluorescent probe like NBD and rhodamine at the N-terminus of peptides, cleavage of the labeled and unlabeled peptides from the resin, and their precipitation were done by standard procedures.18,19 The peptides were purified by reverse phase HPLC on an analytical Waters Symmetry C18 column (300 Å, 5.0 μm, 4.6 mm × 250 mm) using a linear gradient of 20−80% acetonitrile for 40 min with a flow rate of 0.6 mL/min. Both acetonitrile and water contained 0.1% trifluoroacetic acid. The purity of the peptides was further detemined to be ≥95 by reverse phase analytical chromatography.9,47 Experimental molecular mass of the peptides were evaluated by ESI-MS analysis which were very close to their calculated mass. The electrospray mass spectra were recorded on a Micromass Quattro II triple-quadrupole mass spectrometer (Micromass, UK) equipped with an electrospray ionization source. Assay of Hemolytic and Cytotoxic Activity of the Peptides. Hemolytic activity of these designed novel peptides against human red blood cells (hRBCs) in PBS was performed to examine their cytotoxic activity13,21 against a human cell. A standard procedure was followed that has been described earlier. Peptide-induced release of hemoglobin was monitored by measuring the absorbance (Asample) of the supernatant at 540 nm. For negative and positive controls, hRBC in PBS (Ablank) and in 0.2% (final concentration v/v) Triton X-100 (Atriton) were used, respectively. The percentage of hemolysis was calculated according to the following equation.

association property of these peptides in determining their cytotoxic and antiendotoxin properties but not significantly in their antimicrobial activities. The present study also suggests that heptad repeat sequence can be utilized for designing novel LPS-neutralizing peptides by placing either leucine or phenylalanine at the “a” and “d” positions, while the placement of valine at these positions could yield relatively weaker efficacy; yet the peptide comprising valine residues has the advantage of significantly lower cytotoxicity.

■

CONCLUSION The present study demonstrated that alone mean hydrophobicity of the peptides designed on the basis of heptad repeat sequence cannot determine the cytotoxic property of these peptides, rather their self-assembly properties could play a prominent role. Though valine possesses similar mean hydrophobicity to leucine and phenylalanine, heptadic peptide (VRP) with valine at “a” and “d” positions are significantly nontoxic to peptides with leucine (LRP) and phenylalanine (FRP) at these positions; further, VRP showed appreciable activity against both bacteria and fungi. The results could be generalized to a new methodology for designing potent antimicrobial peptides with reduced cytotoxicity by employing the VRP amino acid sequence as a lead template because a drastic loss of cytotoxicity but no alteration in antimicrobial properties of highly cytotoxic melittin was also observed as a result of replacement of leucine residues with valine residues at the “a” and “d” positions of its leucine zipper sequence. The present results also showed antiendotoxin properties of the peptides designed on the heptad repeat sequence within their nontoxic concentration with LRP showing the highest activity followed by FRP, VRP, and ARP. The relative activity of these peptides suggests that probably their ability to adopt helical self-assembly in LPS could be crucial in determining their efficacy to neutralize LPS-induced pro-inflammatory response in macrophages. Overall, the data presented here could be of significant importance for designing antimicrobial peptides with reduced cytotoxicity and antimicrobial peptides with antiendotoxin properties.

■

EXPERIMENTAL SECTION

Materials. Rink amide MBHA resin (loading capacity: 0.4−0.8 mmol/g) and all the N-α Fmoc and necessary side chain protected amino acids were purchased from Novabiochem, Switzerland. Coupling reagents for peptide synthesis like 1-hydroxybenzotriazole (HOBT), di-isopropylcarbodiimide (DIC), 1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), and N,N′-diisopropylethylamine (DIPEA) were purchased from Sigma, USA. Dichloromethane, N,N′-dimethylformamide (DMF), and piperidine were of standard grades and procured from reputed local companies. Acetonitrile (HPLC grade) was procured from Merck, India. Trifluoroacetic acid (TFA), trifluoroethanol (TFE), N-[2-hydroxymethyl] piperazine-N′[2-ethanesulfonic acid] (HEPES), sodium dodecyl sulfate (SDS), calcein, FITC-dextrans, FITC-annexin V, valinomycin, Sephadex G-50, dimethyl sulfoxide (DMSO), and cholesterol (Chol) were purchased from Sigma. Egg phosphatidylcholine (PC), egg phosphatidylglycerol (PG), and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (PE) were obtained from Northern Lipids Inc., Canada while 3,3′dipropylthiadicarbocyanine iodide (diS-C3-5), NBD-fluoride (4fluoro-7-nitrobenz-2-oxa-1,3-diazole), and tetramethylrhodamine succinimidyl ester were procured from Invitrogen, India. E.coli 0111:B4 lipopolysaccharide (L3012), FITC-LPS E.coli 0111:B4 (F3665), sodium nitroprusside (228710) were from Sigma, while p-phenylenediamine (151830) and paraformaldehyde (150146) were procured from MP biomedicals. Components of Griess reagents,

percentage of hemolysis = [(A sample − A blank )/(A triton − A blank )] × 100 Further, cytotoxic activity of the peptides was examined against murine 3T3 cells by a standard procedure as described earlier48 by measuring the viability of the cells in the presence of these peptides. MTT assay was performed for this purpose with cells (10000 per well) seeded in 96-well plates as reported elsewhere.20 Antibacterial Activity Assay of the Peptides. Bacterial growth inhibitory assay of all peptides of this set was done in sterile 96-well plates against different Gram-positive and Gram-negative bacteria.20,40 In brief, bacteria were grown at 37 °C (∼180 rpm, in orbital shaker incubator) in appropriate growth medium (M-H broth) in aerobic conditions to the midlog phase as determined by the optical density at 600 nm, subsequently diluted in same media, and then 50 μL of bacterial culture (∼106 cfu/mL) of diluted were added to 50 μL of water containing 2-fold serially diluted different peptides in each well and incubated for ∼18 h at 37 °C. The peptides’ antibacterial activities, expressed as their MICs (the peptide concentration at which ∼100% inhibition of microbial growth take place), were assessed by measuring the absorbance at 600 nm. 935

dx.doi.org/10.1021/jm301407k | J. Med. Chem. 2013, 56, 924−939

Journal of Medicinal Chemistry

Article

Antifungal Activity Assay. The in vitro antifungal activity of these designed peptides was evaluated against Candida albicans, Cryptococcus neoformans, Sporothrix schenckii, Trichophyton mentagrophytes, Aspergillus fumigatus, and Candida parapsilosis (ATCC 22019). In this methodology, the minimum inhibitory concentration (MIC) of the peptides was determined according to the standard microbroth dilution technique as per NCCLS guidelines.49 Briefly, testing was performed in flat-bottomed 96-well tissue culture plates in RPMI 1640 medium buffered with MOPS (3-[N-morpholino] propanesulfonic acid) for fungal strains. The concentration range of test compounds was 50−0.36 and 32−0.0018 lg/mL for standard compounds. Initial inocula of fungal were maintained at 1−5 × 103 cells/mL. These plates were incubated in a moist chamber at 35 °C, and an absorbance at 492 nm was recorded on a VersaMax microplate reader after 48 h for C. albicans and C. parapsilosis, 72 h for A. fumigatus, S. schenckii, and C. neoformans, and 96 h for T. mentagrophytes. The MICs were determined as 90% inhibition of growth with respect to the growth control as observed using SOFTmax Pro 4.3 Software. Localization of Peptides onto Mammalian and Bacterial Membranes. Localization and binding of the peptides onto mammalian and bacterial membranes was studied using the rhodamine-labeled versions of peptides. Fresh hRBCs (3% in PBS) as used in peptides’ hemolytic activity assays were incubated with NBDlabeled peptides for 10−30 min depending on peptide’s toxic activity at 37 °C. Cells were washed and fixed with 2% paraformaldehyde (10 min.) after extensive washing with PBS and then confocal microscopic images of cells were taken with argon ion laser set for Rho-excitation at 561 nm. Setting of the photomultiplier was constant during the whole experiments.20 Localization and binding of the peptides onto the bacterial cells was also examined with the help of NBD-labeled peptides by employing a confocal microscope (Zeiss confocal microscope LSM-510 Meta. Bacteria (∼106 CFU/mL) in LB medium were incubated in the presence of rhodamine-labeled peptides for half an hour and then centrifuged, washed, and analyzed by the confocal microscope as described above. Preparation of Small Unilamellar Vesicles (SUVs). SUVs were prepared by a standard procedure19,50−52 with required amounts of either of the PC/cholesterol (8:1 w/w) or PE/PG (7:3 w/w) by employing a bath type sonicator (Laboratory Supplies Company, New York) as reported earlier. Detection of Peptide-Induced Membrane Damage of hRBCs and Bacterial Cells. Peptide-induced phospholipid asymmetry or damage of phospholipid membrane organization of hRBCs was determined by staining the cells (∼3.0 × 107 cells/mL) with FITCannexin V53,54 after the treatment with the peptides at room temparature for 5 min. Extent of staining was measured by analyzing peptide treated cells with respect to peptide untreated control using Becton Dickinson FACSCalibur flow cytometer and CellQuest Pro software. To check the peptide induced damage to membrane integrity of E. coli ATCC10536 and S. aureus ATCC 9144, the cells at midlog phase were incubated with peptides for 30 min at 37 °C with constant shaking. The cells were centrifuged, washed two times with PBS, and incubated further with propidium iodide at 4 °C for 30 min, followed by removal of the unbound dye through washing with an excess of PBS and resuspended in buffer. Peptide-induced damage of bacterial cells was then analyzed by flow cytometer as mentioned above. Assay of Peptide-Induced Depolarization of hRBC and Bacterial Membrane. Peptide-induced depolarization of the hRBC and bacterial membrane was detected by its efficiency to devastate the potential across these cell membranes as measured by employing a potential sensitive dye diS-C3-5 as reported before.19,20,50,55 Membrane depolarization as measured by the fluorescence recovery (Ft) was defined by the equation19,55

concentration either to hRBCs or to bacterial suspensions, which were already incubated with diS-C3-5 probe for 1 h and I0, was the steady-state fluorescence level of the cell suspensions after 1 h incubation with the probe. Assay of Peptide Induced Dissipation of Diffusion Potential. Peptides induced disorder of membrane bilayer was measured by their propensity to dissipate the diffusion potential across the lipid vesicles composed of zwitterionic PC/Chol (8:1, w/w) or negatively charged PE/PG (7:3 w/w) as described in earlier reports.20,56,57 The peptideinduced dissipation of diffusion potential was measured in terms of percentage of fluorescence recovery (Ft) by the same equation as shown in the previous section of assay of peptide-induced depolarization of hRBC and bacteria. Here It = the observed fluorescence after the addition of a peptide at time t (∼5 min after the addition of the peptide), I0 = the fluorescence after the addition of valinomycin, and If = the total fluorescence observed before the addition of valinomycin. Tryptophan Blue-Shift Assay. For the measurement of vesicleinduced changes in the emission spectra of tryptophan, the fluorescence emission spectrum of Trp-containing peptides (designed peptides LRP, FRP, VRP, and ARP) was monitored in PBS and in the presence of small unilamellar vesicles (SUVs) composed of either PC/ Chol (8:1, w/w) or PE/PG (7:3, w/w). The tryptophan was excited at 280 nm, and the emission was scanned from 300 to 400 nm. Each of the peptides (∼1.1 μM) were added to PBS with subsequent stepwise addition of vesicles up to 400 μM for PC/PG or PE/PG vesicles and 400 μM for PC/Chol lipid vesicles. Quenching of Trp Emission by Acrylamide. To know the contrasting cytotoxicity, incorporation of peptides into membranes evaluated through monitoring the change in fluorescence of tryptophan in the presence of its quencher acrylamide. In this experiment, excitation of Trp at 295 nm instead of 280 nm was used to reduce the absorbance of acrylamide.58 Aliquots of the 3.0 M solution of this water-soluble quencher were added to the peptide in the absence or presence of liposomes at a peptide/lipid molar ratio of 1:100. The values obtained were corrected for dilution, and the scatter contribution was derived from acrylamide titration of a vesicle blank. The data were analyzed according to the Stern−Volmer equation,59 F0/F = 1 + KSV[Q ] Where F0 and F represent the fluorescence intensities in the absence and the presence of the quencher (Q), respectively, and KSV is the Stern−Volmer quenching constant, which is a measure of the accessibility of Trp to acrylamide. On the premise that acrylamide does not significantly partition into the membrane bilayer (22), the value for KSV can be considered to be a reliable reflection of the bimolecular rate constant for collisional quenching of the Trp residue present in the aqueous phase. Accordingly, KSV is determined by the amount of nonvesicle- associated free peptide as well as the fraction of the peptide residing in the surface of the bilayer. Circular Dichroism (CD) Studies. The circular dichroism (CD) spectra of the peptides were recorded on Jasco J-710 spectropolarimeter in phosphate buffered saline (PBS, pH 7.4), zwitterionic PC/Chol (8:1, w/w), and negatively charged PE/PG (7:3 w/w) lipid vesicles. The spectropolarimeter was calibrated routinely with 10-camphor sulfonic acid. The samples were scanned at room temperature (∼30 °C) with the help of a capped quartz cuvettes of 0.2 cm path length at a wavelength range of 250−190 nm. An average of 4−6 scans was taken for each sample with a scan speed of 20 nm/min and data interval of 0.5 nm. Assay for NO Neutralizing Activity. RAW 264.7 cells were plated at 5 × 105 cells/well in 24-well plates and then incubated with LPS (1 μg/mL) in the presence of 4.5 μM peptides (LRP, FRP, VRP, and ARP). The cells with and without LPS addition were taken for maximum and basal level of nitric oxide production, respectively. The nitric oxide production was measured by using Griess reagent by reading absorbance at 548 nm as reported earlier.22 Similarly, in another experiment, the effect of varying dose of peptides on LPSinduced NO production was determined.

Ft = [(It − I0)/(If − I0)] × 100% Where If, the total fluorescence, was the fluorescence levels of cell suspensions just after addition of diS-C3-5; It was the observed fluorescence after the addition of a peptide at a particular 936

dx.doi.org/10.1021/jm301407k | J. Med. Chem. 2013, 56, 924−939

Journal of Medicinal Chemistry

Article

Immunoblotting Experiments for iNOS-2 and TNF-α. RAW 264.7 macrophage cells (approx 2 × 105) were stimulated with 1 μg/ mL LPS in the presence of peptides (4.5 μM) in 24-well plates for 24 h. LPS treated and untreated cells were taken as positive and negative control, respectively, representing the stimulated and unstimulated levels of protein expressions. For immunoblotting, cells were harvested, washed with ice-cold PBS pH 7.4, and lysed in Laemmli buffer. Lysates were resolved by SDS-PAGE on 8% gel for iNOS-2 and 12% for TNF-α and then transferred to nitrocellulose membrane (Immobilins, Milipore). Signals were developed with alkaline phosphatase conjugated secondary antibodies with the help of substrate NBT/5-bromo-4-chloro-3-indolyl phosphate (calbiochem) like previously reported method.22 β-Actin served as loading controls for western experiment. Measurement of Cytokine Expression Levels in Supernatant. Enzyme linked immunosorbent assays were performed to estimate the secreted TNF-α and IL-6 in LPS treated cells in the presence of peptide (LRP, FRP, VRP, and ARP) after 12 h incubation. Levels of these cytokines in culture supernatant of untreated and LPS treated cells were taken as minima and maxima to calculate percentage inhibition by peptides as mentioned above.10,22,42 Concentrations of TNF-α and IL-6 in the samples were evaluated using mouse enzymelinked immunosorbent assay kits for TNF-α (BD Biosciences cat.no.558534) and IL-6 (BD Biosciences cat. no. 555240) according to manufacturers’ protocol. The experiments were repeated twice, and the average values of the determined cytokine concentrations are included here. Endotoxin Neutralization Assay (LAL assay). The LPS binding ability of LRP, FRP, VRP, and ARP was assessed by using a quantitative chromogenic limulus amoebocyte lysate (LAL) with QCL-1000 (LONZA 50−647U) kit. Endotoxin neutralization experiments were carried out by following the protocols recommended by the manufacturer. Stock solutions of these peptides were prepared in pyrogen-free water provided with the kit. Peptides at concentrations of 4.5, 9, and 18 μM were incubated with 1 EU/mL concentration of LPS in a flat-bottom nonpyrogenic 96-well tissue culture plate at 37 °C for 30 min to allow peptide binding to LPS. A total of 50 μL of this mixture was then added to equal volume of LAL reagent (50 μL), and the mixture was further incubated for 10 min followed by the addition of 100 μL of LAL chromogenic substrate (Ac-Ile-Ala-Arg-p-nitroaniline). The reaction was terminated by the addition of 25% acetic acid, and the yellow color that developed due to cleavage of the substrate was measured spectrophotometrically at 410 nm. The reduction of absorbance at 410 nm as a function of peptide concentrations is directly proportional to the inhibition of activation of LPS-induced LAL enzyme by the peptide which results from the binding of peptide to LPS. All assays were repeated twice, and average values are reported.22,39,60,61

■

Author Contributions

J.K.G. conceived the idea. S.A. did the major part of the experiments. N.N.M. did antifungal and also some of the antibacterial assays with the peptides which were supervised by P.K.S. S.S. performed experiments on anti-inflammatory properties of these peptides and analyzed these data with J.K.G. and S.A. J.K.T. took part in peptide synthesis. S.A. and J.K.G. analyzed the results and wrote the manuscript; all authors consulted on the final version. J.K.G. arranged the funding for this work. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS J.K.G. dedicates this study to the loving memory of his younger brother whose sudden and untimely death is an irrevocable loss in his life. This work was supported by a Council of Scientific and Industrial Research (CSIR) network project NWP0005 and partly by CSIR network project “BioDiscovery”. S.A. and S.S. acknowledge the receipt of SRFs from CSIR, J.K.T. from UGC, and N.N.M. from ICMR, Government of India. We are thankful to A. L. Vishwakarma for recoding the flow cytometry profiles. Manish Singh, Electron Microscopy Unit, CDRI, is acknowledged for assistance in recording the confocal microscopy images. Animal House facility, CDRI, is thankfully acknowledged for the supply of Wistar rats for the collection of macrophage cells. Mass facilities of SAIF, CDRI, is acknowledged for recording the mass of our peptides. The CSIRCentral Drug Research Institute communication number of this manuscript is 8382.

■

ABBREVIATIONS USED E. coli, Escherichia coli; MTT, dimethyl thiazolyl diphenyl tetrazolium salt; TNF-α, tumor necrosis factor-α; NO, nitric oxide; hRBCs, human red blood cells; MIC, minimum inhibitory concentration; PI, propidium iodide; NBD, 4fluoro-7-nitrobenz-2-oxa-1,3-diazole; PC, egg phosphatidylcholine; Chol, cholesterol; PE, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine; PG, egg phosphatidylglycerol; Trp, tryptophan; PBS, phosphate buffer saline; CD, circular dichroism; LPS, lipopolysaccharides; FITC-LPS, fluorescein isothiocyanate-lipopolysaccharides

■

ASSOCIATED CONTENT

S Supporting Information *

Study of self-assembly of designed peptides LRP, FRP, VRP, and ARP; amino acid sequences of Melittin and its novel analogue, MelVal, their antibacterial and cytotoxic activities; detection of self-assembly of Melittin and MelVal and inhibitory action of designed peptides, LRP, FRP, VRP, and ARP on LPS induced production of inflammatory cytokines TNF-α and IL-6 in rat bone marrow derived primary macrophage cells. This material is available free of charge via the Internet at http://pubs.acs.org.

■

REFERENCES

(1) Zasloff, M. Antimicrobial peptides of multicellular organisms. Nature 2002, 415, 389−395. (2) Brogden, K. A. Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nature Rev. Microbiol. 2005, 3, 238−250. (3) Hultmark, D. Drosophila immunity: paths and patterns. Curr. Opin. Immunol. 2003, 15, 12−19. (4) Zanetti, M. The role of cathelicidins in the innate host defenses of mammals. Curr. Issues Mol. Biol. 2005, 7, 179−196. (5) Levy, O. Antimicrobial proteins and peptides: anti-infective molecules of mammalian leukocytes. J. Leukocyte Biol. 2004, 76, 909− 925. (6) Hancock, R. E.; Sahl, H. G. Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies. Nature Biotechnol. 2006, 24, 1551−1557. (7) Huang, H. N.; Pan, C. Y.; Rajanbabu, V.; Chan, Y. L.; Wu, C. J.; Chen, J. Y. Modulation of immune responses by the antimicrobial peptide, epinecidin (Epi)-1, and establishment of an Epi-1-based inactivated vaccine. Biomaterials 2011, 32, 3627−3636. (8) Gao, G.; Lange, D.; Hilpert, K.; Kindrachuk, J.; Zou, Y.; Cheng, J. T.; Kazemzadeh-Narbat, M.; Yu, K.; Wang, R.; Straus, S. K.; Brooks,

AUTHOR INFORMATION

Corresponding Author

*Phone: 091-522- 2612411-18 (ext 4451). Fax: 091-5222623405/ 2623938. E-mail: [email protected]; jk_ghosh@ cdri.res.in. 937

dx.doi.org/10.1021/jm301407k | J. Med. Chem. 2013, 56, 924−939

Journal of Medicinal Chemistry

Article

D. E.; Chew, B. H.; Hancock, R. E.; Kizhakkedathu, J. N. The biocompatibility and biofilm resistance of implant coatings based on hydrophilic polymer brushes conjugated with antimicrobial peptides. Biomaterials 2011, 32, 3899−3909. (9) Soliman, W.; Wang, L.; Bhattacharjee, S.; Kaur, K. Structure− activity relationships of an antimicrobial peptide plantaricin s from two-peptide class IIb bacteriocins. J. Med. Chem. 2011, 54, 2399−2408. (10) Carotenuto, A.; Malfi, S.; Saviello, M. R.; Campiglia, P.; GomezMonterrey, I.; Mangoni, M. L.; Gaddi, L. M.; Novellino, E.; Grieco, P. A different molecular mechanism underlying antimicrobial and hemolytic actions of temporins A and L. J. Med. Chem. 2008, 51, 2354−2362. (11) Qin, C.; Zhong, X.; Bu, X.; Ng, N. L.; Guo, Z. Dissociation of antibacterial and hemolytic activities of an amphipathic peptide antibiotic. J. Med. Chem. 2003, 46, 4830−4833. (12) Yeaman, M. R.; Yount, N. Y. Mechanisms of antimicrobial peptide action and resistance. Pharmacol. Rev. 2003, 55, 27−55. (13) Hunt, T. K.; Knighton, D. R.; Thakral, K. K.; Goodson, W. H., III; Andrews, W. S. Studies on inflammation and wound healing: angiogenesis and collagen synthesis stimulated in vivo by resident and activated wound macrophages. Surgery 1984, 96, 48−54. (14) Matera, M. G.; Calzetta, L.; Cazzola, M. TNF-alpha inhibitors in asthma and COPD: we must not throw the baby out with the bath water. Pulm. Pharmacol. Ther. 2010, 23, 121−128. (15) Martin, G. S.; Mannino, D. M.; Eaton, S.; Moss, M. The epidemiology of sepsis in the United States from 1979 through 2000. N. Engl. J. Med. 2003, 348, 1546−1554. (16) Firestein, G. S. Evolving concepts of rheumatoid arthritis. Nature 2003, 423, 356−361. (17) Odingo, J. O. Inhibitors of PDE4: a review of recent patent literature. Expert Opin. Ther. Pat. 2005, 15, 14. (18) Asthana, N.; Yadav, S. P.; Ghosh, J. K. Dissection of antibacterial and toxic activity of melittin: a leucine zipper motif plays a crucial role in determining its hemolytic activity but not antibacterial activity. J. Biol. Chem. 2004, 279, 55042−55050. (19) Ahmad, A.; Yadav, S. P.; Asthana, N.; Mitra, K.; Srivastava, S. P.; Ghosh, J. K. Utilization of an amphipathic leucine zipper sequence to design antibacterial peptides with simultaneous modulation of toxic activity against human red blood cells. J. Biol. Chem. 2006, 281, 22029−22038. (20) Ahmad, A.; Asthana, N.; Azmi, S.; Srivastava, R. M.; Pandey, B. K.; Yadav, V.; Ghosh, J. K. Structure−function study of cathelicidinderived bovine antimicrobial peptide BMAP-28: design of its cellselective analogs by amino acid substitutions in the heptad repeat sequences. Biochim. Biophys. Acta 2009, 1788, 2411−2420. (21) Ahmad, A.; Azmi, S.; Srivastava, R. M.; Srivastava, S.; Pandey, B. K.; Saxena, R.; Bajpai, V. K.; Ghosh, J. K. Design of nontoxic analogues of cathelicidin-derived bovine antimicrobial peptide BMAP-27: the role of leucine as well as phenylalanine zipper sequences in determining its toxicity. Biochemistry 2009, 48, 10905−10917. (22) Srivastava, R. M.; Srivastava, S.; Singh, M.; Bajpai, V. K.; Ghosh, J. K. Consequences of alteration in leucine zipper sequence of melittin in its neutralization of lipopolysaccharide-induced proinflammatory response in macrophage cells and interaction with lipopolysaccharide. J. Biol. Chem. 2012, 287, 1980−1995. (23) Javadpour, M. M.; Barkley, M. D. Self-assembly of designed antimicrobial peptides in solution and micelles. Biochemistry 1997, 36, 9540−9549. (24) Landschulz, W. H.; Johnson, P. F.; McKnight, S. L. The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science 1988, 240, 1759−1764. (25) Kouzarides, T.; Ziff, E. The role of the leucine zipper in the fos− jun interaction. Nature 1988, 336, 646−651. (26) Dubay, J. W.; Roberts, S. J.; Brody, B.; Hunter, E. Mutations in the leucine zipper of the human immunodeficiency virus type 1 transmembrane glycoprotein affect fusion and infectivity. J. Virol. 1992, 66, 4748−4756. (27) Luo, Z.; Matthews, A. M.; Weiss, S. R. Amino acid substitutions within the leucine zipper domain of the murine coronavirus spike

protein cause defects in oligomerization and the ability to induce cellto-cell fusion. J. Virol. 1999, 73, 8152−8159. (28) Dennison, S. R.; Wallace, J.; Harris, F.; Phoenix, D. A. Amphiphilic alpha-helical antimicrobial peptides and their structure/ function relationships. Protein Pept. Lett. 2005, 12, 31−39. (29) Ghosh, J. K.; Shaool, D.; Guillaud, P.; Ciceron, L.; Mazier, D.; Kustanovich, I.; Shai, Y.; Mor, A. Selective cytotoxicity of dermaseptin S3 toward intraerythrocytic Plasmodium falciparum and the underlying molecular basis. J. Biol. Chem. 1997, 272, 31609−31616. (30) Oren, Z.; Lerman, J. C.; Gudmundsson, G. H.; Agerberth, B.; Shai, Y. Structure and organization of the human antimicrobial peptide LL-37 in phospholipid membranes: relevance to the molecular basis for its non-cell-selective activity. Biochem. J. 1999, 341 (Pt 3), 501− 513. (31) Pandey, B. K.; Srivastava, S.; Singh, M.; Ghosh, J. K. Inducing toxicity by introducing a leucine-zipper-like motif in frog antimicrobial peptide, magainin 2. Biochem. J. 2011, 436, 609−620. (32) Sood, R.; Domanov, Y.; Pietiainen, M.; Kontinen, V. P.; Kinnunen, P. K. Binding of LL-37 to model biomembranes: insight into target vs host cell recognition. Biochim. Biophys. Acta 2008, 1778, 983−996. (33) Radek, K.; Gallo, R. Antimicrobial peptides: natural effectors of the innate immune system. Semin. Immunopathol. 2007, 29, 27−43. (34) Scott, M. G.; Hancock, R. E. Cationic antimicrobial peptides and their multifunctional role in the immune system. Crit. Rev. Immunol. 2000, 20, 407−431. (35) Bowdish, D. M.; Hancock, R. E. Anti-endotoxin properties of cationic host defence peptides and proteins. J. Endotoxin Res. 2005, 11, 230−236. (36) Rosenfeld, Y.; Papo, N.; Shai, Y. Endotoxin (lipopolysaccharide) neutralization by innate immunity host-defense peptides. Peptide properties and plausible modes of action. J. Biol. Chem. 2006, 281, 1636−1643. (37) Nagaoka, I.; Hirota, S.; Niyonsaba, F.; Hirata, M.; Adachi, Y.; Tamura, H.; Tanaka, S.; Heumann, D. Augmentation of the lipopolysaccharide-neutralizing activities of human cathelicidin CAP18/LL-37-derived antimicrobial peptides by replacement with hydrophobic and cationic amino acid residues. Clin. Diagn. Lab. Immunol. 2002, 9, 972−982. (38) Japelj, B.; Pristovsek, P.; Majerle, A.; Jerala, R. Structural origin of endotoxin neutralization and antimicrobial activity of a lactoferrinbased peptide. J. Biol. Chem. 2005, 280, 16955−16961. (39) Bhunia, A.; Mohanram, H.; Bhattacharjya, S. Lipopolysaccharide bound structures of the active fragments of fowlicidin-1, a cathelicidin family of antimicrobial and antiendotoxic peptide from chicken, determined by transferred nuclear Overhauser effect spectroscopy. Biopolymers 2009, 92, 9−22. (40) Oren, Z.; Shai, Y. Selective lysis of bacteria but not mammalian cells by diastereomers of melittin: structure−function study. Biochemistry 1997, 36, 1826−1835. (41) Hancock, R. E.; Rozek, A. Role of membranes in the activities of antimicrobial cationic peptides. FEMS Microbiol. Lett. 2002, 206, 143− 149. (42) Mangoni, M. L.; Epand, R. F.; Rosenfeld, Y.; Peleg, A.; Barra, D.; Epand, R. M.; Shai, Y. Lipopolysaccharide, a key molecule involved in the synergism between temporins in inhibiting bacterial growth and in endotoxin neutralization. J. Biol. Chem. 2008, 283, 22907−22917. (43) Rosenfeld, Y.; Lev, N.; Shai, Y. Effect of the hydrophobicity to net positive charge ratio on antibacterial and anti-endotoxin activities of structurally similar antimicrobial peptides. Biochemistry 2010, 49, 853−861. (44) Fields, G. B.; Noble, R. L. Solid phase peptide synthesis utilizing 9-fluorenylmethoxycarbonyl amino acids. Int. J. Pept. Protein Res. 1990, 35, 161−214. (45) Wild, C.; Oas, T.; McDanal, C.; Bolognesi, D.; Matthews, T. A synthetic peptide inhibitor of human immunodeficiency virus replication: correlation between solution structure and viral inhibition. Proc. Natl. Acad. Sci. U. S. A. 1992, 89, 10537−10541. 938

dx.doi.org/10.1021/jm301407k | J. Med. Chem. 2013, 56, 924−939

Journal of Medicinal Chemistry

Article

(46) Bouchayer, E.; Stassinopoulou, C. I.; Tzougraki, C.; Marion, D.; Gans, P. NMR and CD conformational studies of the C-terminal 16peptides of Pseudomonas aeruginosa c551 and Hydrogenobacter thermophilus c552 cytochromes. J. Pept. Res. 2001, 57, 39−47. (47) Isaksson, J.; Brandsdal, B. O.; Engqvist, M.; Flaten, G. E.; Svendsen, J. S.; Stensen, W. A synthetic antimicrobial peptidomimetic (LTX 109): stereochemical impact on membrane disruption. J. Med. Chem. 2011, 54, 5786−5795. (48) Nordahl, E. A.; Rydengard, V.; Morgelin, M.; Schmidtchen, A. Domain 5 of high molecular weight kininogen is antibacterial. J. Biol. Chem. 2005, 280, 34832−34839. (49) Lakshmi, V.; Srivastava, S.; Mishra, S. K.; Shukla, P. K. Antifungal activity of bivittoside-D from Bohadschia vitiensis (Semper). Nat. Prod. Res. 2011, 26, 913−918. (50) Pandey, B. K.; Ahmad, A.; Asthana, N.; Azmi, S.; Srivastava, R. M.; Srivastava, S.; Verma, R.; Vishwakarma, A. L.; Ghosh, J. K. Cellselective lysis by novel analogues of melittin against human red blood cells and Escherichia coli. Biochemistry 2010, 49, 7920−7929. (51) Verma, R.; Malik, C.; Azmi, S.; Srivastava, S.; Ghosh, S.; Ghosh, J. K. A synthetic S6 segment derived from KvAP channel selfassembles, permeabilizes lipid vesicles, and exhibits ion channel activity in bilayer lipid membrane. J. Biol. Chem. 2011, 286, 24828−24841. (52) Verma, R.; Ghosh, J. K. Phospholipid membrane interaction of a peptide from S4 segment of KvAP K(+) channel and the influence of the positive charges and an identified heptad repeat in its interaction with a S3 peptide. Biochimie 2011, 93, 1001−1011. (53) Kuypers, F. A.; Lewis, R. A.; Hua, M.; Schott, M. A.; Discher, D.; Ernst, J. D.; Lubin, B. H. Detection of altered membrane phospholipid asymmetry in subpopulations of human red blood cells using fluorescently labeled annexin V. Blood 1996, 87, 1179−1187. (54) Yadav, S. P.; Ahmad, A.; Pandey, B. K.; Verma, R.; Ghosh, J. K. Inhibition of lytic activity of Escherichia coli toxin hemolysin E against human red blood cells by a leucine zipper peptide and understanding the underlying mechanism. Biochemistry 2008, 47, 2134−2142. (55) Papo, N.; Shai, Y. New lytic peptides based on the D,Lamphipathic helix motif preferentially kill tumor cells compared to normal cells. Biochemistry 2003, 42, 9346−9354. (56) Sims, P. J.; Waggoner, A. S.; Wang, C. H.; Hoffman, J. F. Studies on the mechanism by which cyanine dyes measure membrane potential in red blood cells and phosphatidylcholine vesicles. Biochemistry 1974, 13, 3315−3330. (57) Loew, L. M.; Rosenberg, I.; Bridge, M.; Gitler, C. Diffusion potential cascade. Convenient detection of transferable membrane pores. Biochemistry 1983, 22, 837−844. (58) De Kroon, A. I.; Soekarjo, M. W.; De Gier, J.; De Kruijff, B. The role of charge and hydrophobicity in peptide−lipid interaction: a comparative study based on tryptophan fluorescence measurements combined with the use of aqueous and hydrophobic quenchers. Biochemistry 1990, 29, 8229−8240. (59) Eftink, M. R.; Ghiron, C. A. Exposure of tryptophanyl residues in proteins. Quantitative determination by fluorescence quenching studies. Biochemistry 1976, 15, 672−680. (60) Schulke, S.; Waibler, Z.; Mende, M. S.; Zoccatelli, G.; Vieths, S.; Toda, M.; Scheurer, S. Fusion protein of TLR5-ligand and allergen potentiates activation and IL-10 secretion in murine myeloid DC. Mol. Immunol. 2010, 48, 341−350. (61) Bhunia, A.; Mohanram, H.; Domadia, P. N.; Torres, J.; Bhattacharjya, S. Designed beta-boomerang antiendotoxic and antimicrobial peptides: structures and activities in lipopolysaccharide. J. Biol. Chem. 2009, 284, 21991−22004.

939

dx.doi.org/10.1021/jm301407k | J. Med. Chem. 2013, 56, 924−939