Solution structure of the antimicrobial peptide gaegurin 4 by H and 15N nuclear magnetic resonance spectroscopy.

Gaegurin 4 (GGN4) is a 37-residue antimicrobial peptide isolated from the skin of a Korean frog, Rana rugosa. This peptide shows a broad range of activity against prokaryotic cells but shows very little hemolytic activity against human red blood cells. The solution structure of GGN4 was studied by using circular dichroism (CD) and NMR spectroscopy. CD investigations revealed that GGN4 adopts mainly an alpha-helical conformation in trifluoroethanol/water solution, in dodecylphosphocholine and in SDS micelles, but adopts random structure in aqueous solution. By using both homonuclear and heteronuclear NMR experiments, complete 1H and 15N resonance assignments were obtained for GGN4 in 50% trifluoroethanol/water solution. The calculated structures of GGN4 consist of two amphipathic alpha-helices extending from residues 2-10 and from residues 16-32. These two helices are connected by a flexible loop spanning between the residues 11 and 15. By using enzyme digestion and matrix-assisted laser desorption/ionization mass spectroscopy, we confirmed that GGN4 contains a disulfide bridge formed between the residues Cys31 and Cys37 in its C-terminus. The effect of disulfide bridge on the structure and the activity of GGN4 was investigated. The reduced form of GGN4 revealed a similar activity and conformation to native GGN4, suggesting that the disulfide bridge does not strongly affect the conformation and the antimicrobial activity of GGN4.     

NMR spectroscopic assessment of the structure and dynamic properties of an amphibian antimicrobial peptide (Gaegurin 4) bound to SDS micelles

The structure and dynamics of a 37-residue antimicrobial peptide gaegurin 4 (GGN4) isolated from the skin of the native Korean frog, Rana rugosa, was determined in SDS micelles by NMR spectroscopy. The solution structure of the peptide in SDS micelles was determined from 352 NOE-derived distance constraints and 22 backbone torsion angle constraints. Dynamic properties for the amide backbone were characterized by (1)H-(15)N heteronuclear NOE experiments. The structural study revealed two amphipathic helices spanning residues 2-10 and 16-32 and that the helices were connected by a flexible loop. An intraresidue disulfide bridge was formed between residues Cys31 and Cys37 near the C-terminus. The loop region (11-15) connecting the two helices are were slightly more flexible than these helices themselves. From the fact that since there is no contact NOEs between two helices, it is implied that the GGN4 peptide shows an independent motion of both helices which has an angle of about 60 degrees -120 degrees from each other.     

Systematic peptide engineering and structural characterization to search for the shortest antimicrobial peptide analogue of gaegurin 5

As part of an effort to develop new, low molecular mass peptide antibiotics, we searched for the shortest bioactive analogue of gaegurin 5 (GGN5), a 24-residue antimicrobial peptide. Thirty-one kinds of GGN5 analogues were synthesized, and their biological activities were analyzed against diverse microorganisms and human erythrocytes. The structural properties of the peptides in various solutions were characterized by spectroscopic methods. The N-terminal 13 residues of GGN5 were identified as the minimal requirement for biological activity. The helical stability, the amphipathic property, and the hydrophobic N terminus were characterized as the important structural factors driving the activity. To develop shorter antibiotic peptides, amino acid substitutions in an inactive 11-residue analogue were examined. Single tryptophanyl substitutions at certain positions yielded some active 11-residue analogues. The most effective site for the substitution was the hydrophobic-hydrophilic interface in the amphipathic helical structure. At this position, tryptophan was the most useful amino acid conferring favorable activity to the peptide. The introduced tryptophan played an important anchoring role for the membrane interaction of the peptides. Finally, two 11-residue analogues of GGN5, which exhibited strong bactericidal activity with little hemolytic activity, were obtained as property-optimized candidates for new peptide antibiotic development. Altogether, the present approach not only characterized some important factors for the antimicrobial activity but also provided useful information about peptide engineering to search for potent lead molecules for new peptide antibiotic development.     

Importance of Tryptophan in Transforming an Amphipathic Peptide into a Pseudomonas aeruginosa-Targeted Antimicrobial Peptide

Here, we found that simple substitution of amino acids in the middle position of the hydrophobic face of an amphipathic peptide RI16 with tryptophan (T9W) considerably transformed into an antimicrobial peptide specifically targeting Pseudomonas aeruginosa. Minimal inhibitory concentration (MIC) results demonstrated that T9W had a strong and specifically antimicrobial activity against P. aeruginosa, including antibiotic-resistant strains, but was not active against Escherichia coli, Salmonella typhimurium, Staphylococcus aureus and Staphyfococcus epidermidis. Fluorescent spectroscopic assays indicated that T9W interacted with the membrane of P. aeruginosa, depolarizing the outer and the inner membrane of bacterial cells. Salt susceptibility assay showed that T9W still maintained its strong anti-pseudomonas activity in the presence of salts at physiological concentrations, and in hemolytic and MTT assays T9W also showed no toxicity against human blood cells and macrophages. In vivo assay demonstrated that T9W also displayed no toxicity to Chinese Kun Ming (KM) mice. Furthermore, the strong antibiofilm activity was also observed with the peptide T9W, which decreased the percentage of biomass formation in a dose-dependent manner. Overall, these findings indicated that design of single-pathogen antimicrobial agents can be achieved by simple amino acid mutation in naturally occurring peptide sequences and this study suggested a model of optimization/design of anti-pseudomonas drugs in which the tryptophan residue was a conserved element.     

Design of potent, non-toxic antimicrobial agents based upon the structure of the frog skin peptide, pseudin-2

Pseudin-2, a naturally occurring 24 amino-acid-residue antimicrobial peptide first isolated from the skin of the South American paradoxical frog Pseudis paradoxa, has weak hemolytic and cytolytic activity but also relatively low potency against microorganisms. In a membrane-mimetic environment, the peptide exists in an amphipathic alpha-helical conformation. Analogs of the peptide with increased cationicity and alpha-helicity were chemically synthesized by progressively substituting neutral and acidic amino acid residues on the hydrophilic face of the alpha-helix by lysine. Analogs with up to three L-lysine substitutions showed increased potency against a range of gram-negative and gram-positive bacteria (up to 16-fold) whilst retaining low hemolytic activity. The analog [D-Lys3, D-Lys10, D-Lys14]pseudin-2 showed potent activity against gram-negative bacteria (minimum inhibitory concentration, MIC=5 microM against several antibiotic-resistant strains of Escherichia coli) but very low hemolytic activity (HC50>500 microM) and cytolytic activity against L929 fibroblasts (LC50=215 microM). Increasing the number of l-lysines to four and five did not enhance antimicrobial potency further but increased hemolytic activity towards human erythrocytes. Time-kill studies demonstrated that the analog [Lys3, Lys10, Lys14, Lys21]pseudin-2 at a concentration of 1 x MIC was bacteriocidal against E. coli (99.9% cell death after 96 min) but was bacteriostatic against S. aureus. Increasing the hydrophobicity of pseudin-2, while maintaining the amphipathic character of the molecule, by substitution of neutral amino acids on the hydrophobic face of the alpha-helix by L-phenylalanine, had only minor effects on antimicrobial and hemolytic activities.     

Novel short AMP: design and activity study

In a previous study, we reported that truncation of HP (2-20) (derived from the N-terminal region of Helicobacter pylori Ribosomal Protein L1 (RPL1)) at the N- (residues 2-3) and C-terminal (residues 17-20) truncated fragments to give HP (4-16) induces increased antibiotic activity against several bacterial strains without hemolysis. In this study, to develop novel short antibiotic peptides useful as therapeutic drugs, an analogue was designed to possess increased hydrophobicity by Trp substitution in position 2 region of HP (4-16). Synthetic HP (4-16)-W showed an enhanced antimicrobial and antitumor activity. The antimicrobial activity of this peptide and others was measured by their growth inhibitory effect upon S. aureus, B. subtilis, S. epidermidis, E. coli, S. typimurium, P. aeruginosa, C. albicans, T. beigelii and S. cerevisiae. None of the peptides exhibited hemolytic activity against human erythrocyte cells except melittin as a positive control. Its antibiotic activity suggests that HP (4-16)-W is an excellent candidate as a lead compound for the development of novel antibiotic agents.     

Role of C-terminal heptapeptide in pore-forming activity of antimicrobial agent, gaegurin 4.

Gaegurin 4 (GGN4) is an antimicrobial peptide of 37 amino acids isolated from the skin of a frog, Rana rugosa. GGN4 has a disulfide bond between the residues 31 and 37, which is highly conserved among the antimicrobial peptides isolated from skin of the genus, Rana. However, the role of this C-terminal heptapeptide motif is not well understood. In this work, we compared the membrane effects of the full-length GGN4 (C37) and GGN4 1-30 (C30), which is devoid of the C-terminal seven amino acids to elucidate the function of the C-terminal motif. C37 induced significantly larger membrane conductance (>10x) in the model lipid bilayers formed with acidic and neutral phospholipids and larger K+ efflux from gram-positive (>30x) and gram-negative bacteria. However, the pores induced by C37 and C30 were not different in their permeability to K+ over Cl- (permeability ratio of K+ to Cl- = 4.8-7.1). In addition, the pore-forming effect of C37 or C30 in acidic membranes was not different from that in neutral membranes. Furthermore, C37-induced K+ efflux was not significantly decreased by the reducing agent, dithiothreitol. The results indicate that C-terminal heptapeptide sequence plays an important role in maintaining the high pore-forming activity of GGN4, but does not participate in forming GGN4-induced pore structure. The disulfide bond in this region does not appear critical for such high ionophoric activity of GGN4.     

Gaegurin 4, a peptide antibiotic of frog skin, forms voltage-dependent channels in planar lipid bilayers.

Gaegurin 4 (GGN4) is a cationic peptide of 37 amino acids (MW 3748) isolated from the skin of Rana rugosa. It has shown a broad spectrum antimicrobial activity in vitro against Gram-negative and -positive bacteria, fungi and protozoa. To understand its mechanism of antimicrobial action, we examined the effect of GGN4 on the membrane conductance and the electrical properties of GGN4-induced pores in planar lipid bilayers under voltage clamp. Natural and synthetic GGN4 (0.01-1 microg/mL) increased the membrane conductance in a concentration-dependent manner, but GGN4 (1-23), an N-terminal fragment of the peptide with little antimicrobial activity, failed to increase the conductance. At symmetrical 100 mM KCI, unitary conductances of about 120 pS were frequently observed. Their current-voltage relations were linear and open state probabilities were close to 1, but longer closing events were seen more frequently at negative voltages. In addition, GGN4-induced pores were selective for cation over anion, the permeability ratio of K+ to Cl- being 6: 1 in neutral and 7: 1 in acidic lipid bilayers. In conclusion, our results indicate that GGN4 forms voltage-dependent and cation-selective pores in planar lipid bilayers. The ionophoric property of GGN4 is likely to contribute to its antimicrobial activity.     

Dissociation of antimicrobial and hemolytic activities in cyclic peptide diastereomers by systematic alterations in amphipathicity

We have investigated the role of amphipathicity in a homologous series of head-to-tail cyclic antimicrobial peptides in efforts to delineate features resulting in high antimicrobial activity coupled with low hemolytic activity (i.e. a high therapeutic index). The peptide GS14, cyclo(VKLKVd-YPLKVKLd-YP), designed on the basis of gramicidin S (GS), exists in a preformed highly amphipathic beta-sheet conformation and was used as the base compound for this study. Fourteen diastereomers of GS14 were synthesized; each contained a different single enantiomeric substitution within the framework of GS14. The beta-sheet structure of all GS14 diastereomers was disrupted as determined by CD and NMR spectroscopy under aqueous conditions; however, all diastereomers exhibited differential structure inducibility in hydrophobic environments. Because the diastereomers all have the same composition, sequence, and intrinsic hydrophobicity, the amphipathicity of the diastereomers could be ranked based upon retention time from reversed-phase high performance liquid chromatography. There was a clear correlation showing that high amphipathicity resulted in high hemolytic activity and low antimicrobial activity in the diastereomers. The latter may be the result of increased affinity of highly amphipathic peptides to outer membrane components of Gram-negative microorganisms. The diastereomers possessing the most favorable therapeutic indices possessed some of the lowest amphipathicities, although there was a threshold value below which antimicrobial activity decreased. The best diastereomer exhibited 130-fold less hemolytic activity compared with GS14, as well as greatly increased antimicrobial activities, resulting in improvement in therapeutic indices of between 1,000- and 10,000-fold for a number of microorganisms. The therapeutic indices of this peptide were between 16- and 32-fold greater than GS for Gram-negative microorganisms and represents a significant improvement in specificity over GS. Our findings show that a highly amphipathic nature is not desirable in the design of constrained cyclic antimicrobial peptides and that an optimum amphipathicity can be defined by systematic enantiomeric substitutions.     

利用精氨酸和缬氨酸设计新型α-螺旋抗菌肽

摘要：【目的】抗菌肽是生命体的自身免疫系统的重要组成部分。其中两性的α-螺旋抗菌肽在抗菌肽家族中又占有重要的地位,发挥着重要的作用。为了得到具有更高抗菌活性同时具有很低细胞毒性的抗菌肽,根据α-螺旋二级结构衍生出来的螺旋轮模型,设计了一条在疏水一侧含有8个缬氨酸和亲水一侧含有5个精氨酸的新型16残基抗菌肽。【方法】测定了设计得到的新型抗菌肽的最小抑菌浓度、对于红细胞和哺乳动物肾细胞的细胞毒性以及杀菌动力学。 【结果】抗菌活性检测表明,新型抗菌肽VGR16显示了强并快速的杀菌作用,其最小抑菌浓度在16－64     

Structure-activity relationships of antimicrobial peptides from the skin of Rana esculenta inhabiting in Korea

The anuran (frogs and toads) skin is a rich source of antimicrobial peptides that can be developed therapeutically. We searched the skin secretions of Korean Rana esculenta for antimicrobial peptides, and isolated two cationic peptides with antimicrobial activity and little hemolytic activity: a 46-residue peptide of the esculentin-1 family and a 24-residue peptide of the brevinin-1 family. Their sequences showed some differences from the esculentins-1 and brevinins-1 of European Rana esculenta, indicating that sequence diversification of anuran skin antimicrobial peptides can arise from differences in habitat as well as from species differences. The 46-residue peptide named esculentin-1c had broad antimicrobial activity, while the 24-residue peptide named brevinin-1Ed exhibited limited activity. The solution structure of brevinin-1Ed was in good agreement with that of other brevinin-1-like peptides, with an amphipathic alpha-helix spanning residues 3-20, stabilized in membrane-mimetic environments. The weak bioactivity of brevinin-1Ed was attributable to the unusual presence of an anionic amino acid in the middle of the helical hydrophilic face. This report contributes to world-wide investigations of the structure-activity relationships and evolutional diversification of anuran-skin antimicrobial peptides.     

Structures and mode of membrane interaction of a short alpha helical lytic peptide and its diastereomer determined by NMR, FTIR, and fluorescence spectroscopy.

The interaction of many lytic cationic antimicrobial peptides with their target cells involves electrostatic interactions, hydrophobic effects, and the formation of amphipathic secondary structures, such as alpha helices or beta sheets. We have shown in previous studies that incorporating approximately 30%d-amino acids into a short alpha helical lytic peptide composed of leucine and lysine preserved the antimicrobial activity of the parent peptide, while the hemolytic activity was abolished. However, the mechanisms underlying the unique structural features induced by incorporating d-amino acids that enable short diastereomeric antimicrobial peptides to preserve membrane binding and lytic capabilities remain unknown. In this study, we analyze in detail the structures of a model amphipathic alpha helical cytolytic peptide KLLLKWLL KLLK-NH2 and its diastereomeric analog and their interactions with zwitterionic and negatively charged membranes. Calculations based on high-resolution NMR experiments in dodecylphosphocholine (DPCho) and sodium dodecyl sulfate (SDS) micelles yield three-dimensional structures of both peptides. Structural analysis reveals that the peptides have an amphipathic organization within both membranes. Specifically, the alpha helical structure of the L-type peptide causes orientation of the hydrophobic and polar amino acids onto separate surfaces, allowing interactions with both the hydrophobic core of the membrane and the polar head group region. Significantly, despite the absence of helical structures, the diastereomer peptide analog exhibits similar segregation between the polar and hydrophobic surfaces. Further insight into the membrane-binding properties of the peptides and their depth of penetration into the lipid bilayer has been obtained through tryptophan quenching experiments using brominated phospholipids and the recently developed lipid/polydiacetylene (PDA) colorimetric assay. The combined NMR, FTIR, fluorescence, and colorimetric studies shed light on the importance of segregation between the positive charges and the hydrophobic moieties on opposite surfaces within the peptides for facilitating membrane binding and disruption, compared to the formation of alpha helical or beta sheet structures.     

Design of model amphipathic peptides having potent antimicrobial activities.

Induced amphipathic alpha-helical conformations play an important role in the biological activity of peptides. By using reversed-phase high-performance liquid chromatography (RP-HPLC) as a means to study the secondary structure of peptides at aqueous/lipid interfaces, a sequence (Ac-LKLLKKLLKKLKKLLKKL-NH2) was found to readily adopt an amphipathic alpha-helical conformation upon interacting with the lipid groups of the stationary phase during RP-HPLC. This peptide exhibited potent antimicrobial activities against both Gram-positive and Gram-negative bacteria. We have prepared a complete set of omission, as well as of leucine and lysine substitution, analogs of this sequence. These analogs were used to investigate the effects of such alterations on the parent sequence's antimicrobial and hemolytic activities relative to each analog's behavior during RP-HPLC. The potential for the formation of ion channels through cell membranes by this amphipathic model peptide was also evaluated through preparation of analogs which varied in length from 8 to 22 residues, while maintaining their amphipathicity.     

De novo generation of short antimicrobial peptides with simple amino acid composition

As potential therapeutic agents, antimicrobial peptides with shorter length and simpler amino acid composition can be better candidates for clinical and commercial development. Here, we attempted de novo design of short (5- to 11-residue) antimicrobial peptides with three kinds of amino acids. Amphipathic helical properties were conferred by using leucines and lysines and two tryptophan residues were positioned at the critical amphipathic interface between the hydrophilic ending side and the hydrophobic starting side. According to this specified rule, 12 model peptides were generated and their helical propensity was confirmed by circular dichroism spectroscopy. Antimicrobial and hemolytic activities were compared with those of the known 12-residue peptide agent, omiganan, which is currently under therapeutic and commercial development. Antimicrobial activities against Gram-negative and Gram-positive bacteria, including a multi-drug resistant strain, were observed for certain 7- to 11-residue models. Among them, the most potent activity was found for a 9-residue peptide (L₅K₂W₂), although it also had severe hemolytic activity. Alternatively, an 11-residue peptide (L₄K₅W₂) with little hemolytic activity was potentially the most useful agent, as it showed higher antibacterial activity than omiganan. These results not only suggest useful candidates for novel antibiotic development, but also provide an efficient strategy to design such peptides.     

Effects of net charge and the number of positively charged residues on the biological activity of amphipathic alpha-helical cationic antimicrobial peptides

In our previous study, we utilized a 26-residue amphipathic alpha-helical antimicrobial peptide L-V13K (Chen et al., Antimicrob Agents Chemother 2007, 51, 1398-1406) as the framework to study the effects of peptide hydrophobicity on the mechanism of its antimicrobial action. In this study, we explored the effects of net charge and the number of positively charged residues on the hydrophilic/polar face of L-V13K on its biological activity (antimicrobial and hemolytic) and biophysical properties (hydrophobicity, amphipathicity, helicity, and peptide self-association). The net charge of V13K analogs at pH 7 varied between -5 and +10 and the number of positively charged residues varied from 1 to 10. The minimal inhibitory concentrations (MIC) against six strains of Pseudomonas aeruginosa as well as other gram-negative and gram-positive bacteria were determined along with the maximal peptide concentration that produces no hemolysis of human red blood cells (MHC). Our results show that the number of positively charged residues on the polar face and net charge are both important for both antimicrobial activity and hemolytic activity. The most dramatic observation is the sharp transition of hemolytic activity on increasing one positive charge on the polar face of V13K i.e., the change from +8 to +9 resulted in greater than 32-fold increase in hemolytic activity (250 microg/ml to <7.8 microg/ml, respectively).     

Structure-activity analysis of SMAP-29, a sheep leukocytes-derived antimicrobial peptide

SAMP-29 is a cathelecidin-derived antimicrobial peptide deduced from sheep myeloid mRNA. To elucidate the structural-activity relationship of SMAP-29, several analogues were synthesized and their antibiotic activity was investigated. Compared to parental SMAP-29, SMAP-29(1-17) and [K(22,25,27)]-SMAP-29 retained relatively effective antimicrobial activity (MIC: 1.0-8.0 microM), but resulted in a complete loss of hemolytic activity. Pro-19 --> Ala substitution ([A19]-SMAP-29) in SMAP-29 induced a significant reduction in antibacterial activity. These results suggested that the N-terminal amphipathic alpha-helical region and the C-terminal hydrophobic region of SMAP-29 are responsible for antimicrobial activity and hemolytic activity, respectively, and the central Pro-19 in SMAP-29 plays a critical role in showing improved antibacterial activity. In particular, [K(2,7,13)]-SMAP-29(1-17) showed potent antimicrobial activity under high salt conditions without hemolytic activity. Thus, this short peptide could serve as an attractive candidate for the development of therapeutic antimicrobial drugs. Structural analysis by circular dichroism suggested that SMAP-29 seems to adopt a helix-bend/turn-extended random conformation.     

The spectroscopic analysis for binding of amphipathic and antimicrobial model peptides containing pyrenylalanine and tryptophan to lipid bilayer

The binding of basic amphipathic fluorescent peptides to lipid bilayers was studied in relation to their antimicrobial activity. Four fluorescent peptides containing pyrenylalanine or tryptophan in an amphipathic basic peptide (4(4] consisting of four repeated units of tetrapeptide, -L-Leu-L-Ala-L-Arg-L-Leu-, were found to have antimicrobial activities against Gram-positive bacteria and to take conformations with fairly high alpha-helical content both in aqueous solutions and liposomes. The fluorescence spectroscopic data suggested that the pyrenylalanine-peptide existed as a monomer in methanol or liposomes but as an oligomer in aqueous solutions to form an excimer between pyrenylalanyl residues. Upon binding with liposomes, the fluorescence spectra of the tryptophan-containing peptide shifted to a shorter wavelength, indicating the change in the state of tryptophan from hydrophilic environment to hydrophobic one. The analytical data for the quenching of tryptophan fluorescence by I- anion suggest that the tryptophan residue in the peptide is not deeply buried in the hydrophobic core of the bilayers. Based on these findings, it is suggested that the peptides may interact with liposomes in such a manner that they lie parallel to the surface of the lipid bilayers with their hydrophobic regions shallowly in the amphipathic moiety of the bilayers.     

Lipopolysaccharide bound structures of the active fragments of fowlicidin-1, a cathelicidin family of antimicrobial and antiendotoxic peptide from chicken, determined by transferred nuclear Overhauser effect spectroscopy

Cathelicidins comprise a major family of host-defense antimicrobial peptides in vertebrates. The C-terminal part of the cathelicidins is bestowed with antimicrobial and lipopolysaccharide (LPS) neutralizing activities. In this work, we repot high resolution solution structures of two nontoxic active fragments, residues 1-16 or RG16 and residues 8-26 or LK19, of fowlicidin-1, a cathelicidin family of peptide from chicken, as a complex with LPS using two-dimensional transferred nuclear Overhauser effect (Tr-NOE) spectroscopy. Both peptides are highly flexible and do not assume any preferred conformations in their free states. Upon complexation with endotoxin or LPS, peptides undergo structural transitions towards folded conformations. Structure calculations reveal that the LK19 peptide adopts a well defined helical structure with a bend at the middle. By contrast, the first seven amino acids of RG16 are found to be flexible followed by a helical conformation for the residues L8-A15. In addition, a truncated version of LK19 encompassing residues A15-K26 or AK12 displays an amphipathic helical structure in LPS. Saturation transfer difference (STD) NMR studies demonstrate that all peptides, RG16, LK19, and AK12, are in close proximity with LPS, whereby the aromatic residues showed the strongest STD effects. Fluorescence studies with fluorescein isothiocyanate (FITC) labeled LPS in the presence of full-length fowlicidin-1, LK19, RG16, and AK12 indicated that LPS-neutralization property of these peptides may result from plausible dissociation of LPS aggregates. The helical structures of peptide fragments derived from fowlicidin-1 in LPS could be utilized to develop nontoxic antiendotoxic compounds.     

Activity optimization of an undecapeptide analogue derived from a frog-skin antimicrobial peptide

While natural antimicrobial peptides are potential therapeutic agents, their physicochemical properties and bioactivity generally need to be enhanced for clinical and commercial development. We have previously developed a cationic, amphipathic α-helical, 11-residue peptide (named herein GA-W2: FLGWLFKWASK-NH₂) with potent antimicrobial and hemolytic activity, which was derived from a 24-residue, natural antimicrobial peptide isolated from frog skin. Here, we attempted to optimize peptide bioactivity by a rational approach to sequence modification. Seven analogues were generated from GA-W2, and their activities were compared with that of a 12-residue peptide, omiganan, which is being developed for clinical and commercial applications. Most of the modifications reported here improved antimicrobial activity. Among them, the GA-K4AL (FAKWAFKWLKK-NH₂) peptide displayed the most potent antimicrobial activity with negligible hemolytic activity, superior to that of omiganan. The therapeutic index of GA-K4AL was improved more than 53- and more than 31-fold against Gram-negative and Gram-positive bacteria, respectively, compared to that of the starting peptide, GA-W2. Given its relatively shorter length and simpler amino acid composition, our sequence-optimized GA-K4AL peptide may thus be a potentially useful antimicrobial peptide agent.     

Antimicrobial properties of the frog skin peptide, ranatuerin-1 and its [Lys-8]-substituted analog

The predicted conformation of ranatuerin-1 (SMLSVLKNLG(10)KVGLGFVACK(20)INK QC), an antimicrobial peptide first isolated from the skin of the bullfrog Rana catesbeiana, comprises three structural domains: alpha-helix (residues 1-8), beta-sheet (residues 11-16) and beta-turn (residues 20-25). Circular dichroism studies confirm significant alpha-helical character in 50% trifluoroethanol. Replacement of Cys-19 and Cys-25 by serine resulted only in decreased antimicrobial potency but deletion of either the cyclic heptapeptide region [residues (19-25)] or the N-terminal domain [residues (1-8)] produced inactive analogs. Substitution of the glycine residues in the central domain of the [Ser-19, Ser-25] analog by lysine produced inactive peptides despite increased alpha-helical content and cationicity. The substitution Asn-8-->Lys gave a ranatuerin-1 analog with increased alpha-helicity and cationicity and increased potency against a range of Gram-positive and Gram-negative bacteria and against C. albicans but only a small increase (21%) in hemolytic activity. In contrast, increasing alpha-helicity and hydrophobicity by the substitution Asn-22-->Ala resulted in a 3.5-fold increase in hemolytic activity. Effects on antimicrobial potencies of substitutions of neutral amino acids at positions 4, 18, 22, and 24 by lysine were less marked. Strains of pathogenic E. coli from different groups showed varying degrees of sensitivity to ranatuerin-1 (MIC between 5 and 40 microM) but [Lys-8] ranatuerin-1 showed increased potency (between 2- and 8-fold; P < 0.01) against all strains. The data demonstrate that [Lys-8] ranatuerin-1 shows potential as a candidate for drug development.