Antimicrobial HPA3NT3 peptide analogs: placement of aromatic rings and positive charges are key determinants for cell selectivity and mechanism of action.

In an earlier study, we determined that HP(2-20) (residues 2-20 of parental HP derived from the N-terminus of the Helicobacter pylori ribosomal protein L1) and its analog, HPA3NT3, had potent antimicrobial effects. However, HPA3NT3 also showed undesirable cytotoxicity against HaCaT cells. In the present study, we designed peptide analogs including HPA3NT3-F1A (-F1A), HPA3NT3-F8A (-F8A), HPA3NT3-F1AF8A (-F1AF8A), HPA3NT3-A1 (-A1) and HPA3NT3-A2 (-A2) in an effort to investigate the effects of amino acid substitutions in reducing their hydrophobicity or increasing their cationicity, and any resulting effects on their selectivity in their interactions with human cells and pathogens, as well as their mechanism of antimicrobial action. With the exception of HPA3NT3-A1, all of these peptides showed potent antimicrobial activity. Moreover, substitution of Ala for Phe at positions 1 and/or 8 of the HPA3NT3 peptides (-F1A, -F8A and -F1AF8A) dramatically reduced their cytotoxicity. Thus the cytotoxicity of HPA3NT3 appears to be related to its Phe residues (positions 1 and 8), which strongly interact with sphingomyelin in the mammalian cell membrane. HPA3NT3 exerted its bactericidal effects through membrane permeabilization mediated by pore formation. In contrast, fluorescent dye leakage and nucleic acid gel retardation assays showed that -A2 acted by penetrating into the cytoplasm, where it bound to nucleic acids and inhibited protein synthesis. Notably, Staphylococcus aureus did not develop resistance to -A2 as it did with rifampin. These results suggest that the -A2 peptide could potentially serve as an effective antibiotic agent against multidrug-resistant bacterial strains.

Interaction of hydrophobic and amphipathic antimicrobial peptides with lipid bicelles.

Bicelles are model membrane systems that can be macroscopically oriented in a magnetic field at physiological temperature. The macroscopic orientation of bicelles allows to detect, by means of magnetic resonance spectroscopies, small changes in the order of the bilayer caused by solutes interacting with the membrane. These changes would be hardly detectable in isotropic systems such as vesicles or micelles. The aim of this work is to show that bicelles represent a convenient tool to investigate the behavior of antimicrobial peptides (AMPs) interacting with membranes, using electron paramagnetic resonance (EPR) spectroscopy. We performed the EPR experiments on spin-labeled bicelles using various AMPs of different length, charge, and amphipathicity: alamethicin, trichogin GA IV, magainin 2, HP(2-20), and HPA3. We evaluated the changes in the order parameter of the spin-labeled lipids as a function of the peptide-to-lipid ratio. We show that bicelles labeled at position 5 of the lipid chains are very sensitive to the perturbation induced by the AMPs even at low peptide concentrations. Our study indicates that peptides that are known to disrupt the membrane by different mechanisms (i.e., alamethicin vs magainin 2) show very distinct trends of the order parameter as a function of peptide concentration. Therefore, spin-labeled bicelles proved to be a good system to evaluate the membrane disruption mechanism of new AMPs.

The importance of being kinked: role of Pro residues in the selectivity of the helical antimicrobial peptide P5.

Antimicrobial peptides (AMPs) are promising compounds for developing new antibiotic drugs against drug-resistant bacteria. Many of them kill bacteria by perturbing their membranes but exhibit no significant toxicity towards eukaryotic cells. The identification of the features responsible for this selectivity is essential for their pharmacological development. AMPs exhibit few conserved features, but a statistical analysis of an AMP sequence database indicated that many α-helical AMPs surprisingly have a helix-breaking Pro residue in the middle of their sequence. To discriminate among the different possible hypotheses for the functional role of this feature, we designed an analogue of the antimicrobial peptide P5, in which the central Pro was deleted (analogue P5Del). Pro removal resulted in a dramatic increase of toxicity. This was explained by the observation that P5Del binds both charged and neutral membranes, whereas P5 has no appreciable affinity towards neutral bilayers. CD and simulative data provided a rationalization of this behavior. In solution P5, due to the presence of Pro, attains compact conformations, in which its apolar residues are partially shielded from the solvent, whereas P5Del is more helical. These structural differences reduce the hydrophobic driving force for association of P5 to neutral membranes, whereas its binding to anionic bilayers can still take place because of electrostatic attraction. After membrane binding, the Pro residue does not preclude the attainment of a membrane-active amphiphilic helical conformation. These findings shed light on the role of Pro residues in the selectivity of AMPs and provide hints for the design of new, highly selective compounds.

A plausible mode of action of pseudin-2, an antimicrobial peptide from Pseudis paradoxa.

The search for new antibiotic agents is continuous, reflecting the continuous emergence of antibiotic-resistant pathogens. Among the new agents are the antimicrobial peptides (AMPs), which have the potential to become a leading alternative to conventional antibiotics. Studies for the mechanisms of action of the naturally occurring parent peptides can provide the structural and functional information needed for the development of effective new antibiotic agents. We therefore characterized pseudin-2, an AMP isolated from the skin of the South American paradoxical frog Pseudis paradoxa. We found that pseudin-2 organized to an aggregated state in aqueous solution, but that it dissociated into monomers upon binding to lipopolysaccharide (LPS), even though it did not neutralize LPS in Gram-negative bacteria. In addition, pseudin-2 assumed an α-helical structure in the presence of biological membranes and formed pores in both bacterial and fungal membranes, through which it entered the cytoplasm and tightly bound to RNA. Thus, the potent antimicrobial activity of pseudin-2 likely results from both the formation of pores capable of collapsing the membrane potential and releasing intracellular materials and its inhibition of macromolecule synthesis through its binding to RNA.

Bcl-2 expression suppresses mismatch repair activity through inhibition of E2F transcriptional activity.

Bcl-2 stimulates mutagenesis after the exposure of cells to DNA-damaging agents. However, the biological mechanisms of Bcl-2-mediated mutagenesis have remained largely obscure. Here we demonstrate that the Bcl-2-mediated suppression of hMSH2 expression results in a reduced cellular capacity to repair mismatches. The pathway linking Bcl-2 expression to the suppression of mismatch repair (MMR) activity involves the hypophosphorylation of pRb, and then the enhancement of the E2F-pRb complex. This is followed by a decrease in hMSH2 expression. MMR has a key role in protection against deleterious mutation accumulation and in maintaining genomic stability. Therefore, the decreased MMR activity by Bcl-2 may be an underlying mechanism for Bcl-2-promoted oncogenesis.

Antimicrobial lipopeptaibol trichogin GA IV: role of the three Aib residues on conformation and bioactivity.

The lipopeptaibol trichogin GA IV is a natural, non-ribosomally synthesized, antimicrobial peptide remarkably resistant to the action of hydrolytic enzymes. This feature may be connected to the multiple presence in its sequence of the non-coded residue α-aminoisobutyric acid (Aib), which is known to be responsible for the adoption of particularly stable helical structures already at the level of short peptides. To investigate the role of Aib residues on the 3D-structure and bioactivity of trichogin GA IV, we synthesized and fully characterized four analogs where one or two Aib residues are replaced by L-Leu. Our extensive conformational studies (including an X-ray diffraction analysis) and biological assays performed on these analogs showed that the Aib to L-Leu replacements do not affect the resistance to proteolysis, but modulate the bioactivity of trichogin GA IV in a 3D-structure related manner.

Protein nanopore-based, single-molecule exploration of copper binding to an antimicrobial-derived, histidine-containing chimera peptide.

Metal ions binding exert a crucial influence upon the aggregation properties and stability of peptides, and the propensity of folding in various substates. Herein, we demonstrate the use of the α-HL protein as a powerful nanoscopic tool to probe Cu(2+)-triggered physicochemical changes of a 20 aminoacids long, antimicrobial-derived chimera peptide with a His residue as metal-binding site, and simultaneously dissect the kinetics of the free- and Cu(2+)-bound peptide interaction to the α-HL pore. Combining single-molecule electrophysiology on reconstituted lipid membranes and fluorescence spectroscopy, we show that the association rate constant between the α-HL pore and a Cu(2+)-free peptide is higher than that of a Cu(2+)-complexed peptide. We posit that mainly due to conformational changes induced by the bound Cu(2+) on the peptide, the resulting complex encounters a higher energy barrier toward its association with the protein pore, stemming most likely from an extra entropy cost needed to fit the Cu(2+)-complexed peptide within the α-HL lumen region. The lower dissociation rate constant of the Cu(2+)-complexed peptide from α-HL pore, as compared to that of Cu(2+)-free peptide, supports the existence of a deeper free energy well for the protein interaction with a Cu(2+)-complexed peptide, which may be indicative of specific Cu(2+)-mediated contributions to the binding of the Cu(2+)-complexed peptide within the pore lumen.

Trichogin GA IV: a versatile template for the synthesis of novel peptaibiotics.

Trichogin GA IV, isolated from the fungus Trichoderma longibrachiatum, is the prototype of lipopeptaibols, the sub-class of short-length peptaibiotics exhibiting membrane-modifying properties. This peptaibol is predominantly folded in a mixed 3(10)-/α- helical conformation with a clear, albeit modest, amphiphilic character, which is likely to be responsible for its capability to perturb bacterial membranes and to induce cell death. In previous papers, we reported on the interesting biological properties of trichogin GA IV, namely its good activity against Gram positive bacteria, in particular methicillin-resistant S. aureus strains, its stability towards proteolytic degradation, and its low hemolytic activity. Aiming at broadening the antimicrobial activity spectrum by increasing the peptide helical amphiphilicity, in this work we synthesized, by solution and solid-phase methodologies, purified and fully characterized a set of trichogin GA IV analogs in which the four Gly residues at positions 2, 5, 6, 9, lying in the poorly hydrophilic face of the helical structure, are substituted by one (position 2, 5, 6 or 9), two (positions 5 and 6), three (positions 2, 5, and 9), and four (positions 2, 5, 6, and 9) Lys residues. The conformational preferences of the Lys-containing analogs were assessed by FT-IR absorption, CD and 2D-NMR techniques in aqueous, organic, and membrane-mimetic environments. Interestingly, it turns out that the presence of charged residues induces a transition of the helical conformation adopted by the peptaibols (from 3(10)- to α-helix) as a function of pH in a reversible process. The role played in the analogs by the markedly increased amphiphilicity was further tested by fluorescence leakage experiments in model membranes, protease resistance, antibacterial and antifungal activities, cytotoxicity, and hemolysis. Taken together, our biological results provide evidence that some of the least substituted among these analogs are good candidates for the development of new membrane-active antimicrobial agents.

The membrane insertion of helical antimicrobial peptides from the N-terminus of Helicobacter pylori ribosomal protein L1.

The interaction of two helical antimicrobial peptides, HPA3 and HPA3P with planar supported lipid membranes was quantitatively analysed using two complementary optical biosensors. The peptides are analogues of Hp(2-20) derived from the N-terminus of Helicobacter pylori ribosomal protein L1 (RpL1). The binding of these two peptide analogues to zwitterionic dimyristoyl-phosphatidylcholine (DMPC) and negatively charged membranes composed of DMPC/dimyristoylphosphatidylglycerol (DMPG) (4:1) was determined using surface plasmon resonance (SPR) and dual polarisation interferometry (DPI). Using SPR analysis, it was shown that the proline substitution in HPA3P resulted in much lower binding for both zwitterionic and anionic membranes than HPA3. Structural changes in the planar DMPC and DMPC/DMPG (4:1) bilayers induced by the binding of both Hp(2-20) analogues were then resolved in real-time with DPI. The overall process of peptide-induced changes in membrane structure was analysed by the real-time changes in bound peptide mass as a function of bilayer birefringence. The insertion of both HPA3 and HPA3P into the supported lipid bilayers resulted in a decrease in birefringence with increasing amounts of bound peptide which reflects a decrease in the order of the bilayer. The binding of HPA3 to each membrane was associated with a higher level of bound peptide and greater membrane lipid disordering and a faster and higher degree of insertion into the membrane than HPA3P. Furthermore, the binding of both HPA3 and HPA3P to negatively charged DMPC/DMPG bilayers also leads to a greater disruption of the lipid ordering. These results demonstrate the geometrical changes in the membrane upon peptide insertion and the extent of membrane structural changes can be obtained quantitatively. Moreover, monitoring the effect of peptides on a structurally characterised bilayer has provided further insight into the role of membrane structure changes in the molecular basis of peptide selectivity and activity and may assist in defining the mode of antimicrobial action.

C-terminal amidation of PMAP-23: translocation to the inner membrane of Gram-negative bacteria.

PMAP-23 is a member of the cathelicidin family derived from pig myeloid cells and has potent antimicrobial activity. Amidation of the carboxyl terminus (C-terminus) of an antimicrobial peptide generally enhances its structural stability and antimicrobial activity or decreases its cytotoxicity. The aim of the present study was to investigate the effect of amidation on the mode of action in PMAP-23. Irrespective of amidation, PMAP-23 adopts a helix-hinge-helix structure in a membrane-mimetic environment. The antibacterial activities of PMAP-23C, which had a free C-terminus, and PMAP-23N, which had an amidated C-terminus, were similar against Gram-negative bacteria, reflecting a similar ability to neutralize lipopolysaccharide. However, PMAP-23N assumed a perpendicular orientation across the outer to the inner leaflet of the bacterial inner membrane, while PMAP-23C was orientated parallel to the lipid bilayer, as determined by following the blue shift in tryptophan fluorescence, as well as calcein release from liposomes and SYTOX Green uptake assays. These results suggest that N-terminal amidation of PMAP-23 provides structural stability and increases the peptide's cationic charge, facilitating translocation into the bacterial inner membrane.

Investigation of single-molecule kinetics mediated by weak hydrogen bonds within a biological nanopore.

The study of factors essential for protein-peptide interactions and protein pore-mediated peptide transport are of particular relevance in biology. Wild-type α-hemolysin was adopted as a "nanoreactor" in which perturbations of the current through a protein containing a lumen-residing, aryl-capped antimicrobial peptide were seen for the first time and studied at the single-molecule level. Energy and steric considerations hint that Met-aryl interactions between aromatic residues placed at a peptide's extremities and any of the methionines lining the α-hemolysin constriction region may be the primary cause of peptide stabilization within the lumen and may be particularly important to the peptide-α-hemolysin interaction.

Interactions of a synthetic Leu-Lys-rich antimicrobial peptide with phospholipid bilayers.

The interaction of the synthetic antimicrobial peptide P5 (KWKKLLKKPLLKKLLKKL-NH(2)) with model phospholipid membranes was studied using solid-state NMR and circular dichroism (CD) spectroscopy. P5 peptide had little secondary structure in buffer, but addition of large unilamellar vesicles (LUV) composed of dimyristoylphosphatidylcholine (DMPC) increased the ß-sheet content to ~20%. Addition of negatively charged LUV, DMPC-dimyristoylphosphatidylglycerol (DMPG) 2:1, led to a substantial (~40%) increase of the α-helical conformation. The peptide structure did not change significantly above and below the phospholipid phase transition temperature. P5 peptide interacted differently with DMPC bilayers with deuterated acyl chains (d(54)-DMPC) and mixed d(54)-DMPC-DMPG bilayers, used to mimic eukaryotic and prokaryotic membranes, respectively. In DMPC vesicles, P5 peptide had no significant interaction apart from slightly perturbing the upper region of the lipid acyl chain with minimum effect at the terminal methyl groups. By contrast, in the DMPC-DMPG vesicles the peptide increased disorder throughout the entire acyl chain of DMPC in the mixed bilayer. P5 promoted disordering of the headgroup of neutral membranes, observed by (31)P NMR. However, no perturbations in the T(1) relaxation nor the T(2-) values were observed at 30°C, although a slight change in the dynamics of the headgroup at 20°C was noticeable compared with peptide-free vesicles. However, the P5 peptide caused similar perturbations of the headgroup of negatively charged vesicles at both temperatures. These data correlate with the non-haemolytic activity of the P5 peptide against red blood cells (neutral membranes) while inhibiting bacterial growth (negatively charged membranes).

Effect of acyl chain structure and bilayer phase state on binding and penetration of a supported lipid bilayer by HPA3.

The effect of acyl chain structure and bilayer phase state on binding and penetration by the peptide HPA3 was studied using dual polarisation interferometry. This peptide is an analogue of Hp(2-20) derived from the N-terminus of Helicobacter pylori ribosomal protein L1 (RpL1) which has been shown to have antimicrobial and cell-penetrating properties. The binding of HPA3 to zwitterionic 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1-palmitolyl-2-oleyl-sn-glycero-3-phosphocholine (POPC) and negatively charged membranes composed of DMPC and 1,2-dimyristoyl-sn-glycero-3-(phosphor-rac-(1-glycerol)) (DMPG) or POPC and 1-palmitolyl-2-oleyl-sn-glycero-3-(phosphor-rac-(1-glycerol)) (POPG) was determined using dual polarisation interferometry (DPI). Mass and birefringence were measured in real time, enabling the creation of birefringence-mass plots for detailed analysis of the changes in lipid bilayer order during the peptide-binding process. HPA3 bound to all four lipids and the binding progressed as a single phase for the saturated gel phase bilayers DMPC and DMPC-DMPG. However, the binding process involved two or more phases, with penetration of the unsaturated fluid phase POPC and POPC-POPG bilayers. Structural changes in the saturated bilayer were partially reversible whereas binding to the unsaturated bilayer resulted in irreversible changes in membrane structure. These results demonstrate that more disordered unsaturated bilayers are more susceptible to further disorganisation and have a lower capacity to recover from peptide-induced structural changes than saturated ordered bilayers. In addition, this study further establishes DPI as powerful tool for analysis of multiphase peptide-insertion processes associated with complex structural changes in the liquid-crystalline membrane.

Reversed sequence enhances antimicrobial activity of a synthetic peptide.

A class of cationic antimicrobial peptides involved in host defense consists of sequences rich in Lys and Trp. Small peptides, (WK)(3) and (KW)(3) , were designed by the combination of alternating Lys (K) and Trp (W) amino acids, and then their antimicrobial and hemolytic activities were determined. It was noticed that the reversed sequence of (KW)(3) showed more activity against all strains than did (WK)(3) . The non-hemolytic behavior of (WK)(3) is identical to that of the reversed analog of (KW)(3) . CD spectra revealed that these peptides had an unfolded structure in buffer and EYPC:CH (10:1, w/w), but adopted folded conformation in the presence of EYPE:EYPG (7:3, w/w). The reversed-(KW)(3) peptide caused a higher extent of calcein release from EYPE:EYPG (7:3, w/w), though the activity was higher than that of the (WK)(3) . The interaction of the peptides with model lipid vesicles was examined using Trp fluorescence. The reversed-(KW)(3) showed higher interaction with EYPE:EYPG (7:3, w/w) membrane than did (WK)(3) . Both the peptides show less affinities while binding to EYPC:CH (10:1, w/w). This clearly indicated that the reversal of sequence factors is relevant to increased antimicrobial activity and lipid membrane permeability.

Antimicrobial activity, bactericidal mechanism and LPS-neutralizing activity of the cell-penetrating peptide pVEC and its analogs.

pVEC is a cell-penetrating peptide derived from the murine vascular endothelial-cadherin protein. To evaluate the potential of pVEC as antimicrobial peptide (AMP), we synthesized pVEC and its analogs with Trp and Arg/Lys substitution, and their antimicrobial and lipopolysaccharide (LPS)-neutralizing activities were investigated. pVEC and its analogs displayed a potent antimicrobial activity (minimal inhibitory concentration: 4-16 µM) against Gram-positive and Gram-negative bacteria but no or less hemolytic activity (less than 10% hemolysis) even at a concentration of 200 µM. These peptides induced a near-complete membrane depolarization (more than 80%) at 4 µM against Staphylococcus aureus and a significant dye leakage (35-70%) from bacterial membrane-mimicking liposome at a concentration as low as 1 µM. The fluorescence profiles of pVEC and its analogs in dye leakage from liposome and membrane depolarization were similar to those of a frog-derived AMP, magainin 2. These results suggest that pVEC and its analogs kill bacteria by forming a pore or ion channel in the cytoplasmic membrane. pVEC and its analogs significantly inhibited nitric oxide production or tumor necrosis factor-α release in LPS-stimulated mouse macrophage RAW264.7 cells at 10 to 50 µM, in which RAW264.7 were not damaged. Taken together, our results suggest that pVEC and its analogs with potent antimicrobial and LPS-neutralizing activities can serve as AMPs for the treatment of microbial infection and sepsis.

The thin line between cell-penetrating and antimicrobial peptides: the case of Pep-1 and Pep-1-K.

Cell-penetrating peptides (CPPs) are cationic oligopeptides able to translocate across biological membranes without perturbing them, while antimicrobial peptides (AMPs) kill bacteria mainly by disrupting their membranes. The two peptide classes share several characteristics (charge, amphipathicity, helicity, and length), and therefore the molecular properties discriminating between the two different bioactivities are not clear. Pep-1-K (KKTWWKTWWTKWSQPKKKRKV) is a new AMP derived from the widely studied CPP Pep-1 (KETWWETWWTEWSQPKKKRKV), or 'Chariot', known for its ability to carry large cargoes across biological membranes. Pep-1-K was obtained from Pep-1 by substituting the three Glu residues with Lys, to increase its cationic character. Previous studies showed that these modifications endow Pep-1-K with a potent antimicrobial activity, with MICs in the low micromolar range. Here, we characterized the interaction of Pep-1 and Pep-1-K with model membranes to understand the reason for the antimicrobial activity of Pep-1-K. The data show that this peptide causes vesicle aggregation, perturbs membrane order, and induces the leakage of ions, but not of larger solutes, while these effects were not observed for Pep-1. These differences are likely due, at least in part, to the higher affinity of Pep-1-K toward anionic bilayers, which mimick the composition of bacterial membranes.

Synthesis, preferred conformation, protease stability, and membrane activity of heptaibin, a medium-length peptaibiotic.

The medium-length peptaibiotics are characterized by a primary structure of 14-16 amino acid residues. Despite the interesting antibiotic and antifungal properties exhibited by these membrane-active peptides, their exact mechanism of action is still unknown. Here, we present our results on heptaibin, a 14-amino acid peptaibiotic found to exhibit antimicrobial activity against Staphylococcus aureus. We carried out the very challenging synthesis of heptaibin on solid phase and a detailed conformational analysis in solution. The peptaibiotic is folded in a mixed 310-/α-helix conformation which exhibits a remarkable amphiphilic character. We also find that it is highly stable toward degradation by proteolytic enzymes and nonhemolytic. Finally, fluorescence leakage experiments using small unilamellar vesicles of three different compositions revealed that heptaibin, although uncharged, is a selective compound for permeabilization of model membranes mimicking the overall negatively charged surface of Gram-positive bacteria. This latter finding is in agreement with the originally published antimicrobial activity data.

Effect of acidic pH on antibacterial action of peptide isolated from Korean pen shell (Atrina pectinata).

Recently, the rapid emergence of microbial pathogens which are resistant to currently available antibiotics has triggered considerable interest searching for naturally occuring antimicrobial peptides (AMPs). Because AMPs from food organisms are comparatively nontoxic, a number of them are used as sources, purified in new antibiotics. Herein, an antibacterial peptide (heat-stable KPS-1) was isolated from Korean pen shell (Atrina pectinata) by the following procedures: solvent-extraction, heating, ultrafiltration, and RP-HPLC. The molecular weight of KPS-1 (4549.1 Da) was revealed by MALDI-TOF/MS analysis. Interestingly, KPS-1 inhibited in vitro growth of Gram-negative bacteria, including Escherichia coli, E. coli O157, Pseudomonas aeruginosa, Enterobacter sakazakii, and Salmonella typhimurium, at pH 5.2, rather than at pH 7.2. Its minimal inhibitory concentrations (MICs) were ranged from 20 to 80 µg/ml; however, it was not effective against human red blood cells at a concentration of 500 µg/ml. This suggests that this peptide is useful as a clinical agent for some human organs in an acidic environment.

The role of antimicrobial peptides in preventing multidrug-resistant bacterial infections and biofilm formation.

Over the last decade, decreasing effectiveness of conventional antimicrobial-drugs has caused serious problems due to the rapid emergence of multidrug-resistant pathogens. Furthermore, biofilms, which are microbial communities that cause serious chronic infections and dental plaque, form environments that enhance antimicrobial resistance. As a result, there is a continuous search to overcome or control such problems, which has resulted in antimicrobial peptides being considered as an alternative to conventional drugs. Antimicrobial peptides are ancient host defense effector molecules in living organisms. These peptides have been identified in diverse organisms and synthetically developed by using peptidomimic techniques. This review was conducted to demonstrate the mode of action by which antimicrobial peptides combat multidrug-resistant bacteria and prevent biofilm formation and to introduce clinical uses of these compounds for chronic disease, medical devices, and oral health. In addition, combinations of antimicrobial peptides and conventional drugs were considered due to their synergetic effects and low cost for therapeutic treatment.

Cell specificity, anti-inflammatory activity, and plausible bactericidal mechanism of designed Trp-rich model antimicrobial peptides.

To develop novel short Trp-rich antimicrobial peptides (AMPs) with potent cell specificity (targeting bacteria but not eukaryotic cells) and anti-inflammatory activity, a series of 11-meric Trp-rich model peptides with different ratios of Leu and Lys/Arg residues, XXWXXWXXWXX-NH(2) (X indicates Leu or Lys/Arg), was synthesized. K(6)L(2)W(3) displayed an approximately 40-fold increase in cell specificity, compared with the natural Trp-rich AMP indolicidin (IN). Lys-containing peptides (K(8)W(3), K(7)LW(3) and K(6)L(2)W(3)) showed approximately 2- to 4-fold higher cell specificities than did their counterparts, the Arg-containing peptides (R(8)W(3), R(7)LW(3) and R(6)L(2)W(3)), indicating that multiple Lys residues are more important than multiple Arg residues in the design of AMPs with good cell specificity. The excellent resistance of d-enantiomers (K(6)L(2)W(3)-D and R(6)L(2)W(3)-D) and Orn/Nle-containing peptides (O(6)L(2)W(3) and O(6)L(2)W(3)) to trypsin digestion compared with the rapid breakdown of the l-enantiomers (K(6)L(2)W(3) and R(6)L(2)W(3)), highlights the clinical potential of such peptides. K(6)L(2)W(3), R(6)L(2)W(3), K(6)L(2)W(3)-D and R(6)L(2)W(3)-D caused weak dye leakage from bacterial membrane-mimicking negatively charged EYPG/EYPE (7:3, v/v) liposomes. Confocal microscopy showed that these peptides penetrated the cell membrane of Escherichia coli and accumulated in the cytoplasm, as observed for buforin-2. Gel retardation studies revealed that the peptides bound more strongly to DNA than did IN. These results suggested that one possible peptide bactericidal mechanism may relate to the inhibition of intracellular functions via interference with DNA/RNA synthesis. Furthermore, some model peptides, containing K(6)L(2)W(3), K(5)L(3)W(3), R(6)L(2)W(3), O(6)L(2)W(3), O(6)L(2)W(3), and K(6)L(2)W(3)-D inhibited LPS-induced inducible nitric oxide synthase (iNOS) mRNA expression, the release of nitric oxide (NO) following LPS stimulation in RAW264.7 cells and had powerful LPS binding activities at bactericidal concentrations. Collectively, our results indicated that these peptides have potential for future development as novel antimicrobial and anti-inflammatory agents.