Selective acylation enhances membrane charge sensitivity of the antimicrobial peptide mastoparan-x

The partitioning of the wasp venom peptide mastoparan-X (MPX) into neutral and negatively charged lipid membranes has been compared with two new synthetic analogs of MPX where the N^α-terminal of MPX was acylated with propanoic acid (PA) and octanoic acid (OA). The acylation caused a considerable change in the membrane partitioning properties of MPX and it was found that the shorter acylation with PA gave improved affinity and selectivity toward negatively charged membranes, whereas OA decreased the selectivity. Based on these findings, we hypothesize that minor differences in the embedding and positioning of the peptide in the membrane caused by either PA or OA acylation play a critical role in the fine-tuning of the effective charge of the peptide and thereby the fine-tuning of the peptide's selectivity between neutral and negatively charged lipid membranes. This finding is unique compared to previous reports where peptide acylation enhanced membrane affinity but also resulted in impaired selectivity. Our result may provide a method of enhancing selectivity of antimicrobial peptides toward bacterial membranes due to their high negative charge—a finding that should be investigated for other, more potent antimicrobial peptides in future studies.     

General aspects of peptide selectivity towards lipid bilayers and cell membranes studied by variation of the structural parameters of amphipathic helical model peptides.

Model compounds of modified hydrophobicity (Eta), hydrophobic moment (mu) and angle subtended by charged residues (Phi) were synthesized to define the general roles of structural motifs of cationic helical peptides for membrane activity and selectivity. The peptide sets were based on a highly hydrophobic, non-selective KLA model peptide with high antimicrobial and hemolytic activity. Variation of the investigated parameters was found to be a suitable method for modifying peptide selectivity towards either neutral or highly negatively charged lipid bilayers. Eta and mu influenced selectivity preferentially via modification of activity on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) bilayers, while the size of the polar/hydrophobic angle affected the activity against 1-palmitoyl-2-oleoylphosphatidyl-DL-glycerol (POPG). The influence of the parameters on the activity determining step was modest in both lipid systems and the activity profiles were the result of the parameters' influence on the second less pronounced permeabilization step. Thus, the activity towards POPC vesicles was determined by the high permeabilizing efficiency, however, changes in the structural parameters preferentially influenced the relatively moderate affinity. In contrast, intensive peptide accumulation via electrostatic interactions was sufficient for the destabilization of highly negatively charged POPG lipid membranes, but changes in the activity profile, as revealed by the modification of Phi, seem to be preferentially caused by variation of the low permeabilizing efficiency. The parameters proved very effective also in modifying antimicrobial and hemolytic activity. However, their influence on cell selectivity was limited. A threshold value of hydrophobicity seems to exist which restricted the activity modifying potential of mu and Phi on both lipid bilayers and cell membranes.     

From "carpet" mechanism to de-novo designed diastereomeric cell-selective antimicrobial peptides

Living organisms of all types produce a large repertoire of gene-encoded, net positively charged, antimicrobial peptides as part of their innate immunity to microbial invasion. Despite significant variations in composition, length and secondary structure most antimicrobial peptides are active in micromolar concentrations, suggesting a common general mechanism for their mode of action. Many antimicrobial peptides bind bacterial phospholipid membranes up to a threshold concentration, followed by membrane permeation/disintegration (the "carpet" mechanism). Recent data suggest that the details of the permeation pathways may vary for different peptides and are assigned to different modes of action. Accumulating data reveal that the molecular basis for cell selectivity is the ability of peptides to specifically bind the negatively charged bacterial membrane, as well as their oligomeric state in solution and in the membrane. Based on the "carpet" mechanism and the role of the peptide oligomeric state, a novel group of diastereomeric (containing D- and L-amino acids) antimicrobial peptides were developed. These peptides may serve as promising templates for the future designs of antimicrobial peptides.     

General aspects of peptide selectivity towards lipid bilayers and cell membranes studied by variation of the structural parameters of amphipathic helical model peptides

Model compounds of modified hydrophobicity (H), hydrophobic moment (μ) and angle subtended by charged residues (Φ) were synthesized to define the general roles of structural motifs of cationic helical peptides for membrane activity and selectivity. The peptide sets were based on a highly hydrophobic, non-selective KLA model peptide with high antimicrobial and hemolytic activity. Variation of the investigated parameters was found to be a suitable method for modifying peptide selectivity towards either neutral or highly negatively charged lipid bilayers. H and μ influenced selectivity preferentially via modification of activity on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) bilayers, while the size of the polar/hydrophobic angle affected the activity against 1-palmitoyl-2-oleoylphosphatidyl-DL-glycerol (POPG). The influence of the parameters on the activity determining step was modest in both lipid systems and the activity profiles were the result of the parameters’ influence on the second less pronounced permeabilization step. Thus, the activity towards POPC vesicles was determined by the high permeabilizing efficiency, however, changes in the structural parameters preferentially influenced the relatively moderate affinity. In contrast, intensive peptide accumulation via electrostatic interactions was sufficient for the destabilization of highly negatively charged POPG lipid membranes, but changes in the activity profile, as revealed by the modification of Φ, seem to be preferentially caused by variation of the low permeabilizing efficiency. The parameters proved very effective also in modifying antimicrobial and hemolytic activity. However, their influence on cell selectivity was limited. A threshold value of hydrophobicity seems to exist which restricted the activity modifying potential of μ and Φ on both lipid bilayers and cell membranes.     

Cell selectivity and mechanism of action of antimicrobial model peptides containing peptoid residues

To develop a useful method for designing cell-selective antimicrobial peptides and to investigate the effect of incorporating peptoid residues into an alpha-helical model peptide on structure, function, and mode of action, we synthesized a series of model peptides incorporating Nala (Ala-peptoid) into different positions of an amphipathic alpha-helical model peptide (KLW). Incorporation of one or two Nala residues into the hydrophobic helix face of KLW was more effective at disrupting the alpha-helical structure and bacterial cell selectivity than incorporation into the hydrophilic helix face or hydrophobic/hydrophilic interface. Tryptophan fluorescence studies of peptide interaction with model membranes indicated that the cell selectivity of KLW-L9-a and KLW-L9,13-a is closely correlated with their selective interactions with negatively charged phospholipids. KLW-L9,13-a, which has two Nala residues in its hydrophobic helix face, showed a random structure in membrane-mimicking conditions. KLW-L9,13-a exhibited the highest selectivity toward bacterial cells, showing no hemolytic activity and no or less cytotoxicity compared with other peptides against four mammalian cell lines. Unlike other model peptides, KLW-L9,13-a caused no or little membrane depolarization in Staphylococcus aureus or lipid flip-flop in negatively charged vesicles. In addition, KLW-L9,13-a caused very little fluorescent dye leakage from negatively charged vesicles. Furthermore, confocal laser-scanning microscopy and DNA-binding assays showed that KLW-L9,13-a probably exerts its antibacterial action by penetrating the bacterial membrane and binding to cytoplasmic compounds (e.g., DNA), resulting in cell death. Collectively, our results demonstrate that the incorporation of two Nala residues into the central position of the hydrophobic helix face of noncell-selective alpha-helical peptides is a promising strategy for the rational design of intracellular, cell-selective antimicrobial peptides.     

Membrane association and selectivity of the antimicrobial peptide NK‐2: a molecular dynamics simulation study

In an effort to better understand the initial mechanism of selectivity and membrane association of the synthetic antimicrobial peptide NK‐2, we have applied molecular dynamics simulation techniques to elucidate the interaction of the peptide with the membrane interfaces. A homogeneous dipalmitoylphosphatidylglycerol (DPPG) and a homogeneous dipalmitoylphosphatidylethanolamine (DPPE) bilayers were taken as model systems for the cytoplasmic bacterial and human erythrocyte membranes, respectively. The results of our simulations on DPPG and DPPE model membranes in the gel phase show that the binding of the peptide, which is considerably stronger for the negatively charged DPPG lipid bilayer than for the zwitterionic DPPE, is mostly governed by electrostatic interactions between negatively charged residues in the membrane and positively charged residues in the peptide. In addition, a characteristic distribution of positively charged residues along the helix facilitates a peptide orientation parallel to the membrane interface. Once the peptides reside close to the membrane surface of DPPG with the more hydrophobic side chains embedded into the membrane interface, the peptide initially disturbs the respective bilayer integrity by a decrease of the order parameter of lipid acyl chain close to the head group region, and by a slightly decrease in bilayer thickness. We found that the peptide retains a high content of helical structure on the zwitterionic membrane‐water interface, while the loss of α‐helicity is observed within a peptide adsorbed onto negatively charged lipid membranes. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Peptide induced demixing in PG/PE lipid mixtures: A mechanism for the specificity of antimicrobial peptides towards bacterial membranes?

Antimicrobial peptides attract a lot of interest as potential candidates to overcome bacterial resistance. So far, nearly all the proposed scenarios for their mechanism of action are associated with perforating and breaking down bacterial membranes after a binding process. In this study we obtained additional information on peptide induced demixing of bacterial membranes as a possible mechanism of specificity of antimicrobial peptides. We used DSC and FT-IR to study the influence of a linear and cyclic arginine- and tryptophan-rich antimicrobial peptide having the same sequence (RRWWRF) on the thermotropic phase transitions of lipid membranes. The cyclization of the peptide was found to enhance its antimicrobial activity and selectivity ( Dathe, M. Nikolenko, H. Klose, J. Bienert, M. Biochemistry 43 (2004) 9140-9150). A particular preference of the binding of the peptides to DPPG headgroups compared to other headgroups of negatively charged phospholipids, namely DMPA, DPPS and cardiolipin was observed. The main transition temperature of DPPG bilayers was considerably decreased by the bound peptides. The peptides caused a substantial down-shift of the transition of DPPG/DMPC. In contrast, they induced a demixing in DPPG/DPPE bilayers and led to the appearance of two peaks in the DSC curves indicating a DPPG-peptide-enriched domain and a DPPE-enriched domain. These results could be confirmed by FT-IR-spectroscopic measurements. We therefore propose that the observed peptide-induced lipid demixing in PG/PE-membranes could be a further specific effect of the antimicrobial peptides operating only on bacterial membranes, which contain appreciable amounts of PE and PG, and which could in principle also occur in liquid-crystalline membranes.     

rBPI21 interacts with negative membranes endothermically promoting the formation of rigid multilamellar structures

rBPI₂₁ belongs to the antimicrobial peptide and protein (AMP) family. It has high affinity for lipopolysaccharide (LPS), acting mainly against Gram-negative bacteria. This work intends to elucidate the mechanism of action of rBPI₂₁ at the membrane level. Using isothermal titration calorimetry, we observed that rBPI₂₁ interaction occurs only with negatively charged membranes (mimicking bacterial membranes) and is entropically driven. Differential scanning calorimetry shows that membrane interaction with rBPI₂₁ is followed by an increase of rigidity on negatively charged membrane, which is corroborated by small angle X-ray scattering (SAXS). Additionally, SAXS data reveal that rBPI₂₁ promotes the multilamellarization of negatively charged membranes. The results support the proposed model for rBPI₂₁ action: first it may interact with LPS at the bacterial surface. This entropic interaction could cause the release of ions that maintain the packed structure of LPS, ensuring peptide penetration. Then, rBPI₂₁ may interact with the negatively charged leaflets of the outer and inner membranes, promoting the interaction between the two bacterial membranes, ultimately leading to cell death.     

Cell selectivity and interaction with model membranes of Val/Arg‐rich peptides

Antimicrobial peptides are major components of the innate self‐defence system and a large number of peptides have been designed to study the mechanism of action. In the present study, a small combinatorial library was designed to study whether the biological activity of Val/Arg‐rich peptides is associated with targeted cell membranes. The peptides were produced by segregating hydrophilic residues on the polar side and hydrophobic residues on the opposite side. The peptides displayed strong antimicrobial activity against Gram‐negative and Gram‐positive bacteria, but weak haemolysis even at a concentration of 256 µM. CD spectra showed that the peptides formed α‐helical‐rich structure in the presence of negatively charged membranes. The tryptophan fluorescence and quenching experiments indicated that the peptides bound preferentially to negatively charged phospholipids over zwitterionic phospholipids, which corresponds well with the biological activity data. In the in vivo experiment, the peptide G6 decreased the bacterial counts in the mouse peritoneum and increased survival after 7 days. Overall, a high binding affinity with negatively charged phospholipids correlated closely with the cell selectivity of the peptides and some peptides in this study may be likely candidates for the development of antibacterial agents. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Biophysical characterization and antitumor activity of synthetic Pantinin peptides from scorpion's venom

Antimicrobial peptides have been extensively described as bioactive agents, mainly considering their selective toxicity towards bacteria but not to healthy mammalian cells. In past years, this class of compounds has been classified as an attractive and novel family of anticancer agents. Pantinin peptides isolated from scorpion Pandinus imperator presented antimicrobial activity. In this study, we have explored the in vitro antitumor activity of antimicrobial pantinin peptides against the tumor cell lines MDA-MB-231 (breast adenocarcinoma) and DU − 145 (prostate adenocarcinoma) and healthy fibroblasts HGF − 1. To further improve our mechanistic understanding for this class of compounds, we have also performed a biophysical characterization of these peptides in lipid model membranes. Cell viability assays revealed that all peptides were more effective on tumor cells when compared to fibroblasts, indicating selectivity towards cancer cells. Furthermore, flow cytometry analysis revealed that all peptides induced apoptosis in cancer cells in a different way from fibroblasts. Circular dichroism spectroscopy showed that all peptides adopted an α-helical structure and an evaluation of the binding constant indicates a higher affinity of the peptides to negatively charged phospholipids. Additionally, permeabilization assays showed that POPG and POPS anionic vesicles were more susceptible to peptide-induced lysis than POPC:Chol and POPC:POPE vesicles. Moreover, we have observed that increasing concentrations of cholesterol inhibits peptide binding process. Therefore, our findings suggest that Pantinin peptides may have chemotherapeutic potential for cancer treatment.     

Cationic amphipathic peptides accumulate sialylated proteins and lipids in the plasma membrane of eukaryotic host cells

Cationic antimicrobial peptides (CAMPs) selectively target bacterial membranes by electrostatic interactions with negatively charged lipids. It turned out that for inhibition of microbial growth a high CAMP membrane concentration is required, which can be realized by the incorporation of hydrophobic groups within the peptide. Increasing hydrophobicity, however, reduces the CAMP selectivity for bacterial over eukaryotic host membranes, thereby causing the risk of detrimental side-effects. In this study we addressed how cationic amphipathic peptides-in particular a CAMP with Lysine-Leucine-Lysine repeats (termed KLK)-affect the localization and dynamics of molecules in eukaryotic membranes. We found KLK to selectively inhibit the endocytosis of a subgroup of membrane proteins and lipids by electrostatically interacting with negatively charged sialic acid moieties. Ultrastructural characterization revealed the formation of membrane invaginations representing fission or fusion intermediates, in which the sialylated proteins and lipids were immobilized. Experiments on structurally different cationic amphipathic peptides (KLK, 6-MO-LF11-322 and NK14-2) indicated a cooperation of electrostatic and hydrophobic forces that selectively arrest sialylated membrane constituents.     

Molecular basis for membrane selectivity of NK-2, a potent peptide antibiotic derived from NK-lysin

Increasing resistance of pathogenic bacteria against antibiotics is a severe problem in health care. Natural antimicrobial peptides and derivatives thereof have emerged as promising candidates for “new antibiotics”. In contrast to classical antibiotics, these peptides act by direct physical destabilization of the target cell membrane. Nevertheless, they exhibit a high specificity for bacteria over mammalian cells. However, the precise mechanism of action and the molecular basis for membrane selectivity are still a matter of debate. We have designed a new peptide antibiotic (NK-2) with enhanced antimicrobial activity based on an effector protein of mammalian immune cells (NK-lysin). Here we describe the interaction of this α-helical synthetic peptide with membrane mimetic systems, designed to mimic the lipid compositions of mammalian and bacterial cytoplasmic membranes. Utilizing fluorescence and biosensor assays, we could show that on one hand, NK-2 strongly interacts with negatively charged membranes; on the other hand, NK-2 is able to discriminate, without the necessity of negative charges, between the zwitterionic phospholipids phosphatidylethanolamine (PE) and phosphatidylcholine (PC), the major constituents of the outer leaflet of the cytoplasmic membranes of bacteria and mammalian cells, respectively.     

Substitution of the leucine zipper sequence in melittin with peptoid residues affects self-association, cell selectivity, and mode of action

Melittin (ME), a non-cell-selective antimicrobial peptide, contains the leucine zipper motif, wherein every seventh amino acid is leucine or isolucine. Here, we attempted to generate novel cell-selective peptides by substituting amino acids in the leucine zipper sequence of ME with peptoid residues. We generated a series of ME analogues by replacing Leu-6, Lue-13 and Ile-20 with Nala, Nleu, Nphe, or Nlys, and we examined their secondary structure, self-association activity, cell selectivity and mode of action. Circular dichroism spectroscopy indicated that the substitutions disrupt the α-helical structure of ME in micelles of sodium dodecyl sulfate and on negatively charged and zwitterionic phospholipid vesicles. Substitution by Nleu, Nphe, or Nlys but not Nala disturbed the self-association in an aqueous environment, interaction with zwitterionic membranes, and toxicity to mammalian cells of ME but did not affect the interaction with negatively charged membranes or antibacterial activity. Notably, peptides with Nphe or Nlys substitution had the highest therapeutic indices, consistent with their lipid selectivity. In addition, all of peptoid residue-containing ME analogues had little or no ability to induce membrane disruption, membrane depolarization and lipid flip-flop. Taken together, our studies indicate that substitution of the leucine zipper motif in ME with peptoid residues increases its selectivity against bacterial cells by impairing self-association activity and changes its mode of antibacterial action from membrane-targeting mechanism to possible intracellular targeting mechanism. Furthermore, our ME analogues especially those with Nleu, Nphe, or Nlys substitutions, may be therapeutically useful antimicrobial peptides.     

Substitution of the leucine zipper sequence in melittin with peptoid residues affects self-association, cell selectivity, and mode of action

Melittin (ME), a non-cell-selective antimicrobial peptide, contains the leucine zipper motif, wherein every seventh amino acid is leucine or isolucine. Here, we attempted to generate novel cell-selective peptides by substituting amino acids in the leucine zipper sequence of ME with peptoid residues. We generated a series of ME analogues by replacing Leu-6, Lue-13 and Ile-20 with Nala, Nleu, Nphe, or Nlys, and we examined their secondary structure, self-association activity, cell selectivity and mode of action. Circular dichroism spectroscopy indicated that the substitutions disrupt the alpha-helical structure of ME in micelles of sodium dodecyl sulfate and on negatively charged and zwitterionic phospholipid vesicles. Substitution by Nleu, Nphe, or Nlys but not Nala disturbed the self-association in an aqueous environment, interaction with zwitterionic membranes, and toxicity to mammalian cells of ME but did not affect the interaction with negatively charged membranes or antibacterial activity. Notably, peptides with Nphe or Nlys substitution had the highest therapeutic indices, consistent with their lipid selectivity. In addition, all of peptoid residue-containing ME analogues had little or no ability to induce membrane disruption, membrane depolarization and lipid flip-flop. Taken together, our studies indicate that substitution of the leucine zipper motif in ME with peptoid residues increases its selectivity against bacterial cells by impairing self-association activity and changes its mode of antibacterial action from membrane-targeting mechanism to possible intracellular targeting mechanism. Furthermore, our ME analogues especially those with Nleu, Nphe, or Nlys substitutions, may be therapeutically useful antimicrobial peptides.     

The structure of the antimicrobial peptide Ac-RRWWRF-NH2 bound to micelles and its interactions with phospholipid bilayers.

The hexapeptide Ac-RRWWRF-NH2 has earlier been identified as a potent antimicrobial peptide by screening synthetic combinatorial hexapeptide libraries. In this study, it was found that this peptide had a large influence on the thermotropic phase behavior of model membranes containing the negatively charged headgroup phosphatidylglycerol, a major component of bacterial membranes. In contrast, differential scanning calorimetry showed that it had little effect on model membranes containing the zwitterionic phosphatidylcholine headgroup, the main component of erythrocyte membranes. This behavior is consistent with its biological activity and with its affinity to these membranes as determined by titration calorimetry, implying that peptide-lipid interactions play an important role in this process. The structure of this peptide bound to membrane-mimetic sodium dodecyl sulfate (SDS) and dodecylphosphocholine micelles has been determined using conventional two-dimensional nuclear magnetic resonance methods. It forms a marked amphipathic structure in SDS with its hydrophobic residues on one side of the structure and with the positively charged residues on the other side. This amphipathic structure may allow this peptide to penetrate deeper into the interfacial region of negatively charged membranes, leading to local membrane destabilization. Knowledge about the importance of electrostatic interactions of Arg and the role of Trp residues as a membrane interface anchor will provide insight into the future design of potent antimicrobial peptidomimetics.     

Deletion of all cysteines in tachyplesin I abolishes hemolytic activity and retains antimicrobial activity and lipopolysaccharide selective binding

Tachyplesin I is a cyclic beta-sheet antimicrobial peptide isolated from the hemocytes of Tachypleus tridentatus. The four cysteine residues in tachyplesin I play a structural role in imparting amphipathicity to the peptide which has been shown to be essential for its activity. We investigated the role of amphipathicity using an analogue of tachyplesin I (TP-I), CDT (KWFRVYRGIYRRR-NH(2)), in which all four cysteines were deleted. Like TP-I, CDT shows antimicrobial activity and disrupts Escherichia coli outer membrane and model membranes mimicking bacterial inner membranes at micromolar concentrations. The CDT peptide does not cause hemolysis up to 200 microg/mL while TP-I showed about 10% hemolysis at 100 microg/mL and about 25% hemolysis at 150 microg/mL. Peptide-into-lipid titrations under isothermal conditions reveal that the interaction of CDT with lipid membranes is an enthalpy-driven process. Binding assays performed using fluorometry demonstrate that the peptide CDT binds and inserts into only negatively charged membranes. The peptide-induced thermotropic phase transition of MLVs formed of DMPC and the DMPC/DMPG (7:3) mixture suggests specific lipid-peptide interactions. The circular dichroism study shows that the peptide exists as an unordered structure in an aqueous buffer and adopts a more ordered beta-structure upon binding to negatively charged membrane. The NMR data suggest that CDT binding to negatively charged bilayers induces a change in the lipid headgroup conformation with the lipid headgroup moving out of the bilayer surface toward the water phase, and therefore, a barrel stave mechanism of membrane disruption is unlikely as the peptide is located near the headgroup region of lipids. The lamellar phase (31)P chemical shift spectra observed at various concentrations of the peptide in bilayers suggest that the peptide may function neither via fragmentation of bilayers nor by promoting nonlamellar structures. NMR and fluorescence data suggest that the presence of cholesterol inhibits the peptide binding to the bilayers. These properties help to explain that cysteine residues may not contribute to antimicrobial activity and that the loss of hemolytic activity is due to lack of hydrophobicity and amphipathicity.     

Investigations into the membrane activity of arenicin antimicrobial peptide AA139

Arenicin-3 is an amphipathic β-hairpin antimicrobial peptide that is produced by the lugworm Arenicola marina. In this study, we have investigated the mechanism of action of arenicin-3 and an optimized synthetic analogue, AA139, by studying their effects on lipid bilayer model membranes and Escherichia coli bacterial cells. The results show that simple amino acid changes can lead to subtle variations in their interaction with membranes and therefore alter their pre-clinical potency, selectivity and toxicity. While the mechanism of action of arenicin-3 is primarily dependent on universal membrane permeabilization, our data suggest that the analogue AA139 relies on more specific binding and insertion properties to elicit its improved antibacterial activity and lower toxicity, as exemplified by greater selectivity between lipid composition when inserting into model membranes i.e. the N-terminus of AA139 seems to insert deeper into lipid bilayers than arenicin-3 does, with a clear distinction between zwitterionic and negatively charged lipid bilayer vesicles, and AA139 demonstrates a cytoplasmic permeabilization dose response profile that is consistent with its greater antibacterial potency against E. coli cells compared to arenicin-3.     

Antimicrobial and anti-inflammatory activities of designed antimicrobial peptide P18 analogues

To develop antimicrobial peptides having higher bacterial selectivity than a novel antimicrobial peptide P18, we synthesized several analogues. The P18 analogues are designed by movement of the N-terminal Trp2 residue in P18 (P18-W6, P18-W8 and P18-W15) and the substitution of the central Pro9 residue with D-Pro or Nala (P18-Nala9 and P18-D-Pro9). These analogues retained potent antibacterial activity but displayed less hemolytic activity than P18. From the viewpoint of their therapeutic index, P18 analogues had approximate 3- to 7-fold higher bacterial selectivity compared to P18. The analogues preferentially bind to bacterial membrane-mimicking negatively charged liposomes as well as does P18. Their high specificity to negatively charged phospholipids corresponds well with their high bacterial selectivity. Furthermore, P18-W6, P18-W8 and P18-Nala9 induced a significant inhibition in NO production from LPS-stimulated macrophage RAW264.7 cells, as well as P18. This result suggests that these peptides appear to have promising therapeutic potential for future development as a novel anti-inflammatory agent as well as antimicrobial agent.     

Aggregation of cateslytin beta-sheets on negatively charged lipids promotes rigid membrane domains. A new mode of action for antimicrobial peptides?

Cateslytin, a positively charged (5+) arginine-rich antimicrobial peptide (bCgA, RSMRLSFRARGYGFR), was chemically synthesized and studied against membranes that mimic bacterial or mammalian systems. Circular dichroism, polarized attenuated total reflection infrared spectroscopy, (1)H high-resolution MAS NMR, and (2)H and (31)P solid state NMR were used to follow the interaction from peptide and membrane points of view. Cateslytin, which is unstructured in solution, is converted into antiparallel beta-sheets that aggregate mainly flat at the surface of negatively charged bacterial mimetic membranes. Arginine residues are involved in the binding to negatively charged lipids. Following the interaction of the cateslytin peptide, rigid and thicker membrane domains enriched in negatively charged lipids are found. Much less interaction is detected with neutral mammalian model membranes, as reflected by only minor percentages of beta-sheets or helices in the peptide secondary structure. No membrane destruction was detected for both bacterial and mammalian model membranes. A molecular model is proposed in which zones of different rigidity and thickness bring about phase boundary defects that ultimately lead to permeability induction and peptide crossing through bacterial membranes.     

Thermodynamics of Antimicrobial Lipopeptide Binding to Membranes: Origins of Affinity and Selectivity

The development of novel antibiotic drugs is one of the most pressing biomedical problems due to the increasing number of antibiotic-resistant pathogens. Antimicrobial peptides and lipopeptides are a promising category of candidates, but the molecular origins of their antimembrane activity is unclear. Here we explore a series of recently developed antimicrobial lipopeptides, using coarse-grained molecular-dynamics simulations and free energy methods to uncover the thermodynamics governing their binding to membranes. Specifically, we quantify C16-KGGK’s binding affinity to the two types of membrane by umbrella sampling. We also examined the origin of C16-KGGK’s selectivity for bacterial versus mammalian membranes by systematically varying the peptide sequence and salt concentration. Our data showed that the C16 hydrophobic tail is the main contributor to its affinity to lipid membrane, whereas the peptide portion is mainly responsible for its selectivity. Furthermore, the electrostatic interaction between the cationic peptide and anionic bacterial membrane plays a significant role in the selectivity.