Influence of preformed α-helix and α-helix induction on the activity of cationic antimicrobial peptides

One prominent class of cationic antibacterial peptides comprises the α-helical class, which is unstructured in free solution but folds into an amphipathic α-helix upon insertion into the membranes of target cells. To investigate the importance of α-helicity and its induction on interaction with membranes, a series of peptides was constructed based on a hybrid of moth cecropin (amino acids 1-8) and bee melittin (amino acids 1-18) peptides. The new peptides were predicted to have a high tendency to form α-helices or to have preformed α-helices by virtue of construction of a lactam bridge between glutamate and lysine side-chains at positions i and i+ 4 at various locations along the primary sequence. In two examples where the use of lactam bridge constraints induced and stabilized α-helical structure in benign (aqueous buffer) and/or hydrophobic medium, there was a decrease in antibacterial activity relative to the linear counterparts. Thus the preformation of α-helix in solution was not necessarily beneficial to antimicrobial activity. In the one case where the lactam bridge did result in increased antibacterial activity (lower minimal inhibitory concentration values) it did not increase α-helical content in benign or hydrophobic medium. Broadly speaking, good activity of the peptides against Pseudomonas aeruginosa correlated best (r²= 0.88) with a helican parameter which was calculated as the induction of α-helix in α membrane-mimicking environment divided by the α-helix formation under benign conditions. Interestingly, the activity of the lactam bridge peptide constructs correlated in part with alterations in bacterial outer or cytoplasmic membrane permeability.     

Structure-activity relationship study: short antimicrobial peptides

Many short antimicrobial peptides (< 18mer) have been identified for the development of therapeutic agents. However, Structure-activity relationship (SAR) studies about short antimicrobial peptides have not been extensively performed. To investigate the relationship between activity and structural parameters such as an α-helical structure, a net positive charge and a hydrophobicity, we synthesized and characterized diastereomers, scramble peptides and substituted peptides of the short antimicrobial peptide identified by combinatorial libraries. Circular dichroism (CD) spectra and in vitro activity indicated that an α-helical structure correlated with the antimicrobial activity and a β-sheet structure also satisfied a structural requirement for antimicrobial activity. Most peptides consisting of l -amino acids lost antifungal activity in the presence of heat-inactivated serum, while active diastereomers and a scramble peptide with the β-sheet structure retained antifungal activity in the same condition.     

Comparison of the binding of α-helical and β-sheet peptides to a hydrophobic surface

The induction and stabilisation of secondary structure for a series of amphipathic α-helical and β-sheet peptides upon their binding to lipid-like surfaces has been characterised by reversed phase high-performance liquid chromatography (RP-HPLC). In addition, a series of peptides which have been shown to switch from β-sheet to α-helical conformation upon transfer from a polar to a non-polar solution environment also have been studied. Binding parameters related to the hydrophobic contact area and affinity for immobilised C18 chains were determined at temperatures that ranged from 5 to 85°C, allowing conformational transitions for the peptides during surface adsorption to be monitored. The results demonstrated that all peptides which adopt secondary structure in solution also exhibited large changes in their interactive properties. Overall, this study demonstrates that the hydrophobic face of each amphipathic peptide dominates the binding process and that hydrophobic interactions are a major factor controlling the surface induction of secondary structure.     

Design of potent, non-toxic anticancer peptides based on the structure of the antimicrobial peptide, temporin-1CEa

Recent advances in the search for novel anticancer agents have indicated that the positively charged antimicrobial peptides have emerged as promising agents offering several advantages over the conventional anticancer drugs. As a naturally occurring, cationic, α-helical antimicrobial peptide, temproin-1CEa has been proved to exhibit a potent anticancer effect and a moderate hemolytic activity. In order to reduce the hemolytic activity of temporin-1CEa and improve its anticancer potency towards a range of human breast cancer cells, in the present study, six analogs of temporin-1CEa were rationally designed and synthesized. The amphipathicity levels and α-helical structural patterns of peptides were reserved, while their cationic property and hydrophobicity were changed. The results of MTT and hemolysis assay indicated that the analog peptides displayed an improved anticancer activity and showed an overall optimized therapeutic index. The hydrophobicity of peptides was positively correlated with their hemolytic and antitumor activities. Moreover, the data suggest a strategy of increasing the cationicity while maintaining the moderate hydrophobicity of naturally occurring amphipathic α-helical peptides to generate analogs with improved cytotoxicity against tumor cells but decreased activity against non-neoplastic cells such as human erythrocytes. This work highlights the potential for rational design and synthesis of improved antimicrobial peptides that have the capability to be used therapeutically for treatment of cancers.     

Anticancer activity of undecapeptide analogues derived from antimicrobial peptide, Brevinin-1EMa

In spite of great advances in cancer therapy, cancer remains the major cause of death throughout the world. The increasing resistance of cancer cells towards current anticancer drugs requires development of anticancer agents with a new mode of action. Some antimicrobial peptides have become therapeutic candidates as new anticancer agents. As part of an effort to develop new antimicrobial and/or anticancer agents from natural peptides with low molecular weights, we have investigated the shortest bioactive analogues, which were derived from a 24-residue antimicrobial peptide, Brevinin-1EMa. Recently, we found four bioactive undecapeptides derived from a cationic, amphipathic α-helical, 11-residue peptide (named herein GA-W2: FLGWLFKWASK-NH(2)) (Won et al., 2011). In order to identify the potential of these peptides as anticancer agents, we investigated the anticancer activity of four undecapeptides against seven tumor cell lines such as A498 (kidney), A549 (lung), HCT116 (colon), MKN45 (stomach), PC-3 (prostate), SK-MEL-2 (skin) and SK-OV-3 (ovary). GA-K4 (FLKWLFKWAKK-NH(2)), which had the most potent antimicrobial activity of the four undecapeptides, also exhibited the most potent anticancer activity and synergistic effect in combination with doxorubicin. Therefore, GA-K4 peptide may be a potentially useful candidate as an anticancer peptide agent.     

Design and synthesis of cationic antimicrobial peptides with improved activity and selectivity against Vibrio spp

Extensive use of classical antibiotics has led to the growing emergence of many resistant strains of pathogenic bacteria. Evidence has suggested that cationic antimicrobial peptides (AMPs) are of greatest potential to represent a new class of antibiotics. The largest group of AMPs comprises peptides that fold into an amphipathic alpha-helical conformation when interacting with the target microorganism. In the current study, a series of cationic AMPs of 20 amino acids was designed and synthesised based on four structural parameters, including charge, polar angle, hydrophobicity and hydrophobic moment. The effect of these parameters on antimicrobial activity and selectivity was assessed by structural and biological analyses. Our results indicated that high hydrophobicity and amphipathicity (hydrophobic moment) were correlated with increased haemolytic activity, whilst antimicrobial activity was found to be less dependent on these factors. Three of the synthetic AMPs (GW-Q4, GW-Q6 and GW-H1) showed higher antimicrobial activity and selectivity against a broad spectrum of Gram-positive and Gram-negative bacteria compared with the naturally occurring AMPs magainin 2a and pleurocidin. This study also demonstrates that these rationally designed cationic and amphipathic helical AMPs exhibited high selectivity against several Vibrio spp. and are potential agents for future use in the treatment of these marine pathogens.     

Design,synthesis and antibacterial activity of cecropin-like model peptides

In order to investigate structure-activity relationships of cecropins, model peptides that mimic certain structural features of the cecropin molecules were designed and synthesized. The conformational analysis of cecropins and the design of the model peptides were based on Chou-Fasman calculations. The peptides were synthesized by solid-phase methods and purified by reverse-phase liquid-chromatography on C₁₈-silica columns. Their secondary structures were studied by circular dichroism measurements. Antibacterial activities against seven test organisms were determined and compared to the activities of the natural cecropins A and B. These results were discussed on the basis of structural features of the model peptides and on model mechanisms. It was concluded that high antibacterial activity for this class of compounds requires a basic helical amphipathic N-terminal segment that is connected to a hydrophobic helical C-terminal segment by a flexible non-helical hinge region.     

Solution conformation of N-terminal fragments of trichosanthin small domain (TCS 182-200)

Three peptides, T14, T18 and TDK, derived from the N-terminus of trichosanthin small domain (TCS 182-200) have been investigated by circular dichroism. Secondary structure and structural transitions of the above peptides under different conditions were studied. Alcohol prompts a transition of the T18 peptide from a β-sheet to an α-helical structure. It also increases the α-helicities of T14 and TDK. The β-sheet of T18 peptide appears more hydrophobic than the α-helix of T14 or TDK. The effects of polypeptide sequence and solvent on secondary structure formation of these model peptides are discussed.     

NMR structural characterization of cecropin A(1–8) – magainin 2(1–12) and cecropin A(1–8) – melittin(1–12) hybrid peptides

Abstract: In order to elucidate the structure–antibiotic activity relationships of the peptides, the three-dimensional structures of two hybrid peptides, CA(1–8) – MA(1–12) and CA(1–8) – ME(1–12) in trifluoroethanol-containing aqueous solution were investigated by NMR spectroscopy. Both CA(1–8) – MA(1–12) and CA(1–8) – ME(1–12) have strong antibacterial activity but only CA(1–8) – ME(1–12) has hemolytic activity against human erythrocytes. CA(1–8) – MA(1–12) has a hydrophobic 3₁₀-helix of only two turns combined with one short helix in the N-terminus with a flexible hinge section in between. CA(1–8) – MA(1–12) has a severely bent structure in the middle of the peptide. These structural features as well as the low hydrophobicity of CA(1–8) – MA(1–12) seem to be crucial for the selective lysis against the membrane of prokaryotic cells. CA(1–8) – ME(1–12) has an α-helical structure of about three turns in the melittin domain and a flexible structure with one turn in the cecropin domain connected with a flexible hinge section in between, and these might be the structural features required for membrane distruption against prokaryotic and eukaryotic cells. The central hinge region (Gly9-Ile10-Gly11) in an amphipathic antibacterial peptide is considered to play an important role in providing the conformational flexibility required for ion channel formation of the C-terminal hydrophobic α-helix on cell membrane.     

Probing the α-Helical Structural Stability of Stapled p53 Peptides: Molecular Dynamics Simulations and Analysis

Reactivation of the p53 cell apoptosis pathway through inhibition of the p53-hDM2 interaction is a viable approach to suppress tumor growth in many human cancers and stabilization of the helical structure of synthetic p53 analogs via a hydrocarbon cross-link (staple) has been found to lead to increased potency and inhibition of protein–protein binding (J. Am. Chem. Soc. 129: 5298). However, details of the structure and dynamic stability of the stapled peptides are not well understood. Here, we use extensive all-atom molecular dynamics simulations to study a series of stapled α-helical peptides over a range of temperatures in solution. The peptides are found to exhibit substantial variations in predicted α-helical propensities that are in good agreement with the experimental observations. In addition, we find significant variation in local structural flexibility of the peptides with the position of the linker, which appears to be more closely related to the observed differences in activity than the absolute α-helical stability. These simulations provide new insights into the design of α-helical stapled peptides and the development of potent inhibitors of α-helical protein–protein interfaces.     

Solution structure of a cathelicidin‐derived antimicrobial peptide,CRAMP as determined by NMR spectroscopy

Abstract: CRAMP was identified from a cDNA clone derived from mouse femoral marrow cells as a member of cathelicidin‐derived antimicrobial peptides. This peptide shows potent antimicrobial activity against gram‐positive and gram‐negative bacteria but no hemolytic activity against human erythrocytes. CRAMP was known to cause rapid permeabilization of the inner membrane of Escherichia coli. In this study, the structure of CRAMP in TFE/H₂O (1 : 1, v/v) solution was determined by CD and NMR spectroscopy. CD spectra showed that CRAMP adopts a mainly α‐helical conformation in TFE/H₂O solution, DPC micelles, SDS micelles and liposomes, whereas it has a random structure in aqueous solution. The tertiary structure of CRAMP in TFE/H₂O (1 : 1, v/v), as determined by NMR spectroscopy, consists of two amphipathic α‐helices from Leu⁴ to Lys¹⁰ and from Gly¹⁶ to Leu³³. These two helices are connected by a flexible region from Gly¹¹ to Gly¹⁶. Previous analysis of series of fragments composed of various portion of CRAMP revealed that an 18‐residue fragment with the sequence from Gly¹⁶ to Leu³³ was found to retain antibacterial activity. Therefore, the amphipathic α‐helical region from Gly¹⁶ to Leu³³ of CRAMP plays important roles in spanning the lipid bilayers as well as its antibiotic activity. Based on this structure, novel antibiotic peptides having strong antibiotic activity, with no hemolytic effect will be developed.     

Antimicrobial peptides: to membranes and beyond

Background: Antimicrobial peptides (AMP) are widely recognized as promising alternatives to the current use of antibiotics and fungicides. Amino-acid sequences of a vast majority of AMP share cationic and amphipathic biophysical properties that allow their insertion into lipid bilayers and can lead to alteration of biological membrane functions. Initial characterization studies linked these properties to antimicrobial killing activity. However, further data indicate that this is not the sole mode of action and that more subtle mechanisms might mediate the interaction with and effect to target microbes, as well as the specificity and toxicity of peptides. As such, AMP are increasingly viewed as powerful multifunctional drugs. Objective: This review summarizes findings on these alternative non-lytic modes of antimicrobial action that go beyond membrane disruption, with an emphasis on the specific interaction with microbial cell wall/membrane components, signaling of AMP exposure and intracellular targets of peptide action. We also explore how novel technologies can help to reveal, characterize and exploit these antimicrobial properties. Conclusion: Detailed knowledge on non-lytic modes of action of AMP will help in the design and discovery of novel antibacterial and antifungal compounds.     

Effects of Hydrophobicity on the Antifungal Activity of α‐Helical Antimicrobial Peptides

We utilized a series of analogs of D‐V13K (a 26‐residue amphipathic α‐helical antimicrobial peptide, denoted D1) to compare and contrast the role of hydrophobicity on antifungal and antibacterial activity to the results obtained previously with Pseudomonas aeruginosa strains. Antifungal activity for zygomycota fungi decreased with increasing hydrophobicity (D‐V13K/A12L/A20L/A23L, denoted D4, the most hydrophobic analog was sixfold less active than D1, the least hydrophobic analog). In contrast, antifungal activity for ascomycota fungi increased with increasing hydrophobicity (D4, the most hydrophobic analog was fivefold more active than D1). Hemolytic activity is dramatically affected by increasing hydrophobicity with peptide D4 being 286‐fold more hemolytic than peptide D1. The therapeutic index for peptide D1 is 1569‐fold and 62‐fold better for zygomycota fungi and ascomycota fungi, respectively, compared with peptide D4. To reduce the hemolytic activity of peptide D4 and improve/maintain the antifungal activity of D4, we substituted another lysine residue in the center of the non‐polar face (V16K) to generate D5 (D‐V13K/V16K/A12L/A20L/A23L). This analog D5 decreased hemolytic activity by 13‐fold, enhanced antifungal activity to zygomycota fungi by 16‐fold and improved the therapeutic index by 201‐fold compared with D4 and represents a unique approach to control specificity while maintaining high hydrophobicity in the two hydrophobic segments on the non‐polar face of D5.     

Three-dimensional structure of membrane protein stomatin and function of stomatin-specific protease

Stomatin is a major integral membrane protein of human erythrocytes, the absence of which is associated with a form of hemolytic anemia known as hereditary stomatocytosis. It is reported that stomatin regulates the gating of acid-sensing ion channels in mammalian neurons. However, the function of stomatin is not fully understood. In the genomic sequence of the hyperthermophilic archaeon Pyrococcus horikoshii, the putative operon-forming genes PH1511 and PH1510 encode stomatin and its partner protein, respectively. The N-terminal region of PH1510p (1510-N) is a serine protease, and specifically cleaves the C-terminal hydrophobic region of stomatin PH1511p. We have determined the first crystal structure of the core domain of stomatin PH1511p (residues 56-234, designated as PhSto(CD)). This review focuses on the three-dimensional structure of PhSto(CD), and discusses the function of stomatin and its specific protease 1510-N. PhSto(CD) forms a novel homotrimeric structure. Three α/β domains form a triangle of about 50 ? on each side, and three α-helical segments about 60 ? in length extend from the apexes of the triangle. The α/β domain of PhSto(CD) is partly similar in structure to the band-7 domain of mouse flotillin-2. While the α/β domain is relatively rigid, the α-helical segment shows a conformational flexibility, adapting to the neighboring environment. One α-helical segment forms an anti-parallel coiled-coil with another α-helical segment from a symmetry-related molecule. The α-helical segment shows a heptad repeat pattern, and mainly hydrophobic residues form a coiled-coil interface. The coiled-coil fold observed in the crystal probably contributes to the self-association.     

Peptides with indirect in vivo activity against an intracellular pathogen: selective lysis of infected macrophages

Abstract: A collection of natural peptides, simplified analogs of natural peptides, de novo amphipathic peptides and de novo amphipathic peptides composed of 50–80% α,α‐dialkylated glycines (α,α‐Dags) were synthesized on solid‐phase resin as the C‐terminus amides using N‐α‐fluorenylmethyloxycarbonyl protection. The synthesis of the peptides rich in α,α‐Dags used acid fluoride coupling methods. The peptides show antimicrobial activity against Escherichia coli and Staphylococcus aureus but no direct antimicrobial activity against Brucella abortus at 100 µm in vitro. However, in vivo treatment with several of these peptides results in significant reductions of B. abortus in chronically infected immune BALB/c mice relative to infected control animals. The chronically infected mice were susceptible to peptide toxicity at much lower peptide doses than control animals. The highest nonlethal dose for infected mice was only 25 µg for melittin, whereas 500 µg doses were nonlethal for many of the other peptides. Several of the α,α‐Dag‐rich peptides selectively destroy B. abortus‐infected murine macrophages in vitro. Thus, these peptides apparently reduce the bacterial load in vivo by destroying a portion of the infected macrophages and exposing the sequestered bacteria to the immune response in the mice.     

Conformational differences of ovine and human corticotrophin releasing hormone A CD,IR, NMR and dynamic light scattering study

The differences in the conformational properties of ovine (o) and human (h) CRH in aqueous solution, structure-inducing TFE and in the presence of detergent micelles and lipid vesicles have been investigated by circular dichroism, Fourier transform infrared spectroscopy, NMR and dynamic light scattering. o-CRH was found to exist as a monomer with little regular structure in dilute aqueous solution. Association at concentrations higher than 10^?3 mol/L results predominantly in dimers. The induction of a substantial amount of intermolecular β-structure seems to be the result of interactions of the C-terminal hexapeptide and the N-terminal region 6-12 of o-CRH chains in antiparallel orientation. In contrast, h-CRH exhibits a high tendency of association which is highly sensitive to the pH. The formation of tetramers at millimolar peptide concentration is related to a helical content of ca. 50%. The potentially helical, highly hydrophobic region 6-20 enlarged by more hydrophobic residues in position 23 and 25 is proposed to stabilize the h-CRH associates. In the presence of structure inducing TFE (<40%v) both CRH peptides exist as monomers. o-CRH reveals about 72% helicity, in h-CRH the formation of about 85% helix is observed. The differences in helicity of the two CRH molecules are located in the C-terminal heptapeptide, as concluded on the basis of NMR studies. Both peptides bind to detergent micelles at pH 4 as well as 7.4 associated with an increase in the α-helical content. Interaction of the two peptides with DMPC vesicles was found exclusively at pH 4. Above the phase transition temperature of DMPC the α-helical content in h-CRH increases slightly; however, o-CRH reveals a substantial amount of β-type structure. The intramolecular type of β-structure is associated with a deeper insertion of the o-CRH region 6-12 into the hydrophobic region of the lipid bilayer, whereas the corresponding region of h-CRH is kept in the bilayer surface. The higher helicity of h-CRH might explain to some extent its higher affinity to the CRH receptor, CRH antibodies and the CRH binding protein. © Munksgaard 1996.     

Chemical synthesis and characterization of peptides and oligomeric proteins designed to form transmembrane ion channels

A strategy for the synthesis of peptides and oligomeric proteins designed to form transmembrane ion channels is described. A folding motif that exhibits a functional ionic pore encompasses amphipathic α-helices organized as a four-helix bundle around a central hydrophilic pore. The channel-forming activity of monomeric amphipathic peptides may be examined after reconstitution in lipid bilayers in which peptides self-assemble into conductive oligomers. The covalent attachment of channel-forming peptides to the lysine ε-amino groups of a template molecule (KKKPGKEKG) specifies oligomeric number and facilitates the study of ionic permeation and channel blockade. Here we describe detailed protocols for the total synthesis of peptides and template-assembled four-helix bundle proteins, exemplified with the sequence of M2δ (EKM-STAISVLLAQAVFLLLTSQR), considered involved in lining the pore of the nicotinic acetylcholine receptor channel. For comparison, the synthesis of a second four-helix bundle, T₄CaIVS3 with the sequence of predicted transmembrane segment S3 (DPWNVFDFLIVIGSIIDVILSE) of the fourth repeat of the l -type voltage-gated calcium channel, is included. Peptides and proteins are synthesized step-wise by solid-phase methods, purified by reversed-phase HPLC, and homogeneity ascertained by analytical HPLC, capillary zone electrophoresis, SDS/PAGE, amino acid analysis and sequencing. Optimization of synthetic procedures for hydrophobic molecules include reducing resin substitution to avoid steric hindrance and aggregation of the final product. Protocols for the preparation of the samples prior to HPLC purification as well as the conditions and columns required for successful purification are presented. The methods developed are generally applicable for the chemical synthesis, purification and characterization of amphipathic peptides and template directed helical bundle proteins.     

Structure−function studies on the amphibian peptide brevinin 1E: translocating the cationic segment from the C‐terminal end to a central position favors selective antibacterial activity

Abstract: Brevinin 1E, which has the sequence FLPLLAGLAANFLPKIFCKITRKC , is an antimicrobial peptide isolated from the skin secretions of the European frog Rana esculenta. Both the linear and the disulfide‐bridged forms have relatively broad‐spectrum antibacterial as well as hemolytic activities. The antibacterial and hemolytic activities and biophysical properties of synthetic peptides corresponding to brevinin 1E and its analog in which the segment CKITRKC has been transposed to a central location resulting in the sequence FLPLLAGLCKITRKC AANFLPKIF have been investigated. Our studies indicate that the analog peptide has antibacterial activity comparable with brevinin 1E, but with considerably reduced hemolytic activity. The linear variant of the analog has no hemolytic activity, unlike the linear form of brevinin 1E. The biological activities can be explained on the basis of relative affinities for anionic and zwitterionic lipids. A cluster of cationic amino acids flanked on one side by a hydrophobic stretch of amino acids and another side composed of apolar amino acids appears to favor preferential antibacterial activity.     

Lipid membrane-binding properties of tryptophan analogues of linear amphipathic beta-sheet cationic antimicrobial peptides using surface plasmon resonance

Using a surface plasmon resonance (SPR) system, we investigated the lipid membrane-binding properties of four analogues of the 18-residue linear amphipathic beta-sheet cationic antimicrobial peptide (KIGAKI)3-NH2, each of which contains a single isoleucine-to-tryptophan substitution. The results of the SPR study revealed significant differences in the binding characteristics of the peptides depending upon the position of tryptophan residues. These peptides showed higher binding affinity to membranes containing acidic phospholipids than zwitterionic phospholipids. The addition of dimethylsulfoxide to the running buffer was effective in maintaining the solubility of these peptide solutions and obtaining concentration-dependent sensorgrams for the kinetic analysis in this study. The kinetic binding data of SPR correlated closely with both the ability of the peptides to lyse liposomes with the same phospholipid composition and bactericidal activity. The results demonstrate that SPR may be a valuable tool to predict the membrane lytic properties of antimicrobial peptides.