Addition and omission analogs of the 13-residue antibacterial and hemolytic peptide PKLLKTFLSKWIG: structural preferences,model membrane binding and biological activities

The consequences of selective addition or deletion of polar amino acids in a 13-residue antibacterial peptide PKLLKTFLSKWIG on structure, membrane binding and biological activities have been investigated. The variants generated are (a) S and T residues replaced by K, (b) S and T residues deleted individually and together, (c) introduction of two additional K and (d) deletion of L and L with T. In the aqueous environment all the peptides were unordered. In trifluoroethanol, the spectra of peptides belonging to groups (a-c) suggest distorted helical conformation. Peptides in group (d) appear to adopt β-sheet conformation. The peptides bind to zwitterionic and negatively charged lipid vesicles, although to different extents. With the exception of peptides in group (d), all the other peptides exhibited comparable antibacterial activity against Escherichia coli and Staphylococcus aureus. However, the changes made in the peptides in groups (a-c) resulted in reduction of hemolytic activity compared to the parent peptide. Extent of binding to lipid vesicles composed of phosphatidylcholine and cholesterol appears to correlate with hemolytic activity. It appears that polar and charged residues play a major role in modulating the biological activities of the 13-residue peptide PKLLKTFLSKWIG. The 11-residue peptide-like PKLLKFLKWIG has selective antibacterial activity. Thus, by judicious engineering it should be possible to generate short peptides with selective antibacterial activity.     

Relationship between the tertiary structures of mastoparan B and its analogs and their lytic activities studied by NMR spectroscopy

Abstract: Mastoparan B (MP‐B), an antimicrobial cationic tetradecapeptide amide isolated from the venom of the hornet Vespa basalis, is an amphiphilic α‐helical peptide. MP‐B possesses a variety of biological activities, such as mast cells degradation histamine release, erythrocyte lysis and inhibition of the growth of Gram‐positive and Gram‐negative bacteria. In order to study the relationship between the structure and the biological activity of MP‐B, we used four analogs by replacing amino acids with alanine. Tertiary structures of MP‐B and its analogs in 2,2,2‐trifluoroethanol (TFE)‐containing aqueous solution have been determined by NMR spectroscopy and molecular modeling. The results indicate that [Ala⁴]MP‐B and [Ala¹²]MP‐B with higher hydrophobicity adopt a higher content of amphiphilic helical structures, and have better antimicrobial and hemolytic activities than MP‐B. However, [Ala³]MP‐B and [Ala⁹]MP‐B with lower hydrophobicity have disordered structures. [Ala³]MP‐B and [Ala⁹]MP‐B have low antimicrobial activity and much less hemolytic activity relative to MP‐B. It is likely that tryptophan residue in MP‐B and appropriate hydrophobicity of MP‐B to induce α‐helical structure is essential for the antibacterial and hemolytic activity of MP‐B. This study can aid understanding of the structure–activity relationship of MP‐B and to design peptides to possess lytic activity.     

Identification of the Molecular Determinants of the Antibacterial Activity of LmutTX,a Lys49 Phospholipase A2 Homologue Isolated from Lachesis muta muta Snake Venom (Linnaeus, 1766)

Snake venom phospholipases A₂ (PLA₂s) are responsible for numerous pathophysiological effects in snakebites; however, their biochemical properties favour antimicrobial actions against different pathogens, thus constituting a true source of potential microbicidal agents. This study describes the isolation of a Lys49 PLA₂ homologue from Lachesis muta muta venom using two chromatographic steps: size exclusion and reverse phase. The protein showed a molecular mass of 13,889 Da and was devoid of phospholipase activity on an artificial substrate. The primary structure made it possible to identify an unpublished protein from L. m. muta venom, named LmutTX, that presented high identity with other Lys49 PLA₂s from bothropic venoms. Synthetic peptides designed from LmutTX were evaluated for their cytotoxic and antimicrobial activities. LmutTX was cytotoxic against C2C12 myotubes at concentrations of at least 200 μg/mL, whereas the peptides showed a low cytolytic effect. LmutTX showed antibacterial activity against Gram‐positive and Gram‐negative bacteria; however, S. aureusATCC 29213 and MRSA strains were more sensitive to the toxin's action. Synthetic peptides were tested on S. aureus, MRSA and P. aeruginosaATCC 27853 strains, showing promising results. This study describes for the first time the isolation of a Lys49 PLA₂ from Lachesis snake venom and shows that peptides from specific regions of the sequence may constitute new sources of molecules with biotechnological potential.     

Structure-antibacterial,antitumor and hemolytic activity relationships of cecropin A-magainin 2 and cecropin A-melittin hybrid peptides

In order to elucidate the structure-antibiotic activity relationship of cecropin A-magainin 2 and cecropin A-melittin hybrid peptides, several truncated peptides and the analogues with amino acid substitutions were synthesized and their antibacterial, antitumor and hemolytic activities of were examined. Cecropin A-magainin 2 hybrid analog, L¹⁶-CA(1–8)-MA(1–12) (termed as L-CA-MA in this study: KWKLFKKIGIGKFLHLAKKF-NH₂), is known to have potent antibacterial and antitumor activity with less hemolytic activity. We found that the C-terminal region of L-CA-MA is more involved in the α-helical structure on cell membrane-like environment than N-terminal one by circular dichroism analysis. Deletion of the Gly-lle-Gly sequence, the central hinge region of L-CA-MA, produced a considerable reduction in antitumor and hemolytic activity rather than an antibacterial one. The insertion of Pro, Gly-lle or Gly-Pro in this hinge region of L-CA-MA caused retention of both antibacterial and antitumor activity while causing a significant decrease in hemolytic activity. However, the substitution with Gly-Pro-Gly instead of the Gly-lle-Gly in CA(1–8)-MA(1–12), CA(1–8)-ME(1–12), CA(1–13)-MA(1–13) and CA(1–13)-ME(1–13) hybrids resulted in a drastic decrease in antibacterial, antitumor and hemolytic activity. The increase of hydrophobicity at position 16 in CA(1–8)-MA(1–12) by substituting Trp or Phe induced a significant increase in hemolytic activity without a considerable change in either antibacterial or antitumor activity. Therefore, these results suggested that the appropriate flexibility in the hinge region of CA-MA and CA-ME hybrid peptides and the appropriate hydrophobicity at position 16 in the hydrophobic region of CA (1–8)-MA(1–12) are important in potent antibacterial and antitumor activity with no hemolytic effect.     

Factors affecting the hemolytic activity of the Oriental hornet (Vespa orientalis) venom and venom sac extracts

Extracts from the venom sacs of the Oriental hornet (V. orientalis) pupae are devoid of either direct or indirect hemolytic activity. However, in venom sac extract obtained during and following eclosion of the imagines, hemolytic activity of both types is present and gradually rises, reaching maximal levels at days 3 or 4. Complete daily evacuation of the venom sac for 5 consecutive days, results in progressive diminution of its hemolytic activity. Soybean trypsin inhibitor and, to a lesser degree, other protease inhibitors, partially suppress the direct hemolytic activity of V. orientalis venom, thus suggesting the possible role of venom proteases as potentiators of the direct hemolytic activity.     

Structure-antitumor and hemolytic activity relationships of synthetic peptides derived from cecropin A-magainin 2 and cecropin A-melittin hybrid peptides

The hybrid peptide (CA-ME) derived from cecropin A(1–8) and melittin(1–12) has potent antibacterial and antimalarial activities. Because the N-terminal sequence 1–12 of magainin 2 is similar to melittin(1–12), CA-MA with CA(1–8) and MA(1–12) and their analogues were designed and synthesized. Antitumor activities of these peptides were evaluated using three small cell lung cancer cell lines. Greater antitumor activity was observed when the residues 16, 18 and 19 of the peptide were hydrophobic (Leu or Val), basic (Lys) and basic (Lys), respectively. The IC₅₀ values of the peptides with the residues were 2 to 4 μm . Residue 12 was related to hemolytic activity rather than antitumor activity. Increase in amphipathicity of P4 enhanced hemolytic activity without significant change in antitumor activity. The α-helicity of the peptides in a 30 mm sodium dodecyl sulfate solution was more closely correlated to hemolytic activity than antitumor activity.     

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.     

Effects of increasing hydrophobicity by N‐terminal myristoylation on the antibacterial and hemolytic activities of the C‐terminal cationic segments of human‐β‐defensins 1–3

Analogs of the cationic C‐terminal segments of human‐β‐defensins HBD1‐3, Phd1‐3 with a single disulfide bond, exhibited comparable antimicrobial activity that was salt sensitive. They did not show hemolytic activity. In this study, N‐terminal myristoylation was carried out on Phd1‐3 to examine whether increasing hydrophobicity would result in improved antibacterial activity. The antibacterial activity of the oxidized myristoylated peptides MPhd1‐3 and their reduced forms MPhd1r‐3r was determined. These peptides showed enhanced antibacterial activity as compared to Phd1‐3, on mid‐log phase and stationary phase of Staphylococcus aureus and Escherichia coli, except MPhd1r‐3r that were inactive on stationary‐phase E. coli. In the presence of 150 mm NaCl, MPhd1‐3 showed activity against S. aureus. MPhd1and two exhibited activity against E. coli but MPhd3 was inactive. Zeta potential measurements indicated that MPhd1‐3 were more effective in surface charge neutralization of bacteria as compared to Phd1‐3. MPhd1‐3 exhibited hemolytic activity to varying extents with MPhd1 being most hemolytic. The data indicate that myristoylation enhances antibacterial activity and modulates hemolytic activity to different extents. Apart from hydrophobicity, distribution of cationic residues in MPhd1‐3 plays important roles for these activities.     

Structure-activity analysis of the antitumor and hemolytic properties of the amphiphilic α-helical peptide,C18G

The in vitro antitumor and hemolytic activities of analogs of peptide C18G were compared in order to elucidate important structural features which affect cytotoxicity. The sequence of C18G, a basic peptide which can form an amphiphilic α-helix, is a derivative of the carboxyl terminus of human platelet factor IV. The results demonstrate that both amphiphilicity and helicity are essential for peptide activity, and that addition of a negatively charged amino acid results in decreased cell lysis. Whereas peptides exhibiting various degrees of potency did not differ with respect to helical content, an increase in peptide hydrophobicity did correlate with an increase in antitumor and hemolytic activity, as well as susceptibility to inhibition by serum. Higher hydrophobicity could be associated with improved ability to insert into the cell membrane. The position or context of specific residues within an amphiphilic peptide can also be important for activity. Furthermore, an increase in tumoricidal activity is not always accompanied by an increase in hemolytic activity or susceptibility to inhibition by serum. Possible reasons for the lower sensitivity of RBCs versus tumor cells to peptide cytotoxicity are discussed. Finally, compared with structurally idealized amphiphilic α-helical peptides, non-idealized peptides can possess higher tumoricidal activity, but are less hemolytic and less susceptible to serum inhibition.     

Two families of antimicrobial peptides from wasp (Vespa magnifica) venom.

The hornet possesses highly toxic venom, which is rich in toxin, enzymes and biologically active peptides. Many bioactive substances were identified from wasp venom. Two families of antimicrobial peptides were purified and characterized from the venom of the wasp, Vespamagnifica (Smith). The primary structures of these peptides are homologous to those of chemotactic peptides and mastoparans isolated from other vespid venoms. They also share similarity to temporins which are amphibian antimicrobial peptides identified from the skin of the frog, Ranaboylii. These peptides show antimicrobial activities against bacteria and fungi. However, they show little hemolytic activity against human blood red cells.     

Conformation and interaction of the cyclic cationic antimicrobial peptides in lipid bilayers

Abstract: To investigate the role of peptide–membrane interactions in the biological activity of cyclic cationic peptides, the conformations and interactions of four membrane‐active antimicrobial peptides [based on Gramicidin S (GS)] were examined in neutral and negatively charged micelles and phospholipid vesicles, using CD and fluorescence spectroscopy and ultracentrifugation techniques. Moreover, the effects of these peptides on the release of entrapped fluorescent dye from unilamellar vesicles of phosphatidylcholine (PC) and phosphatidylethanolamine/phosphatidylglycerol (PE/PG) were studied. The cyclic peptides include GS10 [Cyclo(VKLdY P)₂], GS12 [Cyclo(VKLKdY PKVKLdY P)], GS14 [Cyclo(VKLKVdY PLKVKLdY P)] and [d ‐Lys]⁴GS14 [Cyclo(VKLdK VdY PLKVKLdY P)] (underlined residues are d ‐amino acids), were different in their ring size, structure and amphipathicity, and covered a broad spectrum of hemolytic and antimicrobial activities. Interaction of the peptides with the zwitterionic PC and negatively charged PE/PG vesicles were distinct from each other. The hydrophobic interaction seems to be the dominant factor in the hemolytic activity of the peptides, as well as their interaction with the PC vesicles. A combination of electrostatic and hydrophobic interactions of the peptides induces aggregation and fusion in PE/PG vesicles with different propensities in the order: [d ‐Lys]⁴GS14 > GS14 > GS12 > GS10. GS10 and GS14 are apparently located in the deeper levels of the membrane interfaces and closer to the hydrophobic core of the bilayers, whereas GS12 and [d ‐Lys]⁴GS14 reside closer to the outer boundary of the interface. Because of differing modes of interaction of the cyclic cationic peptides with lipid bilayers, the mechanism of their biological activity (and its relation to peptide–lipid interaction) proved to be versatile and complex, and dependent on the biophysical properties of both the peptides and membranes.     

Phospholipases A and B activities of the oriental hornet (Vespa orientalis) venom and venom apparatus.

Phospholipases A and B activities of the oriental hornet (Vespa orientalis) venom and venom apparatus. Toxicon15, 141–156, 1977.—Oriental hornet venom is a rich source of both phospholipase A (PhA) and phospholipase B (PhB) activities. This was shown by incubating venom with egg yolk or with pure lecithin and lysolecithin. Activity was measured by titrating liberated fatty acids and by phosphorus analyses of separated phospholipids from thin-layer chromatographic plates. Both lecithin and lysolecithin were rapidly hydrolyzed by venom at pH 4 and 8. With egg yolk as substrate, the optimum Ph activity was observed at pH 5 although considerable activity was observed from pH 3·5 to 9·5. In contrast purified substrates showed greater activity at an alkaline pH, whether assay was in the presence of collidine-acetate or Tris buffer or in the presence or absence of ether. Ether dramatically changed the optimal pH for Ph activity, with egg yolk as substrate, from acidic to alkaline. It is not known whether these PhA and PhB activities are dual activities of a single enzyme or activities of two separate enzymes. The venom has neither PhC nor lipase activities.PhA and PhB activities were observed not only in pure venom (V), but also in venom sacs (VS) where the venom is stored, in the acid (venom) glands (H⁺) where the venom is produced, and in the alkaline (Dufours) gland (OH^?) whose function is unknown. The release of free fatty acids from egg yolk at pH 5 was in the ratios of 1 (V): 0·13 (VS): 0·04 (H⁺): 0·30 (OH^?). Measurements of Ph activities in combinations of the above preparations showed that strong activators or inhibitors of the enzyme are not present. The mid-gut, fat body and hemolymph of the hornet showed Ph activity only equal to about one-half of one per cent of that of the OH^? gland.Antisera produced against V, VS, H⁺ and OH^? were cross-reactive and inhibited to varying degrees the Ph activities of each of the above preparations. The highest titre antisera were produced in rabbits injected with V or VS, in contrast to the low titres produced with H⁺ and OH^? glands. Low Ph activity is detectable in the venom sacs 2 days prior to emergence, with 20-fold higher activities being observed at 5 days of age.The oriental hornet may be extremely useful as a rich source of not only PhA but also PhB activities. The effects of this latter enzyme on biological systems have not been thoroughly evaluated. The drastic disruption of phospholipid structure and hydrophobic binding forces between phospholipid and protein, by the combined PhA plus PhB activities, may be responsible for some of the pharmacological actions of hornet venom.     

Pharmacological and toxicological properties of harvester ant, Pogonomyrmex badius, venom

The venom of Pogonomyrmex badius exhibits the highest toxicity to mice of any reported insect venom. Its ld₅₀ (i.p.) of 0·42 μg/g is comparable with the most toxic snake venoms. In mammals the venom exhibits strong central and peripheral neurotoxicity; in insects the venom appears devoid of specific neurotoxicity and is, in fact, only moderately toxic to wax moth larvae (LD₅₀ = 103 μg/g). Death of envenomated insects appears to be a result of the hydrolytic activity of the venom enzymes. The venom possesses a powerful direct hemolysin capable of lysing washed erythrocytes. In addition, it possesses strong indirect hemolytic properties (due to phospholipase A₂). Heparin, when mixed in large doses with the venom, does not reduce the toxicity of the venom; nor is heparin capable of reducing the activity of the direct hemolytic factor in the venom. No precipitation zones, indicative of an acid-base complex between heparin and venom, were observed on Ouchterlony diffusion plates.     

Biological activities of retro and diastereo analogs of a 13‐residue peptide with antimicrobial and hemolytic activities

Abstract: The biological activities of synthetic retro and diastereo analogs of PKLLKTFLSKWIG (SPFK), a 13‐residue peptide with antimicrobial and hemolytic activities, have been investigated. Retro peptides with C‐terminal acid and amide exhibited antibacterial activities comparable with those of SPFK. Their hemolytic activities were, however, only marginally lower. The diastereo analog with C‐terminal acid was not antibacterial and was weakly hemolytic. Amidation of this analog could restore antibacterial activity. Both retro analogs were unordered in aqueous medium but had a propensity for a helical structure in trifluoroethanol. However, diastereo analogs were unordered in both aqueous medium and trifluoroethanol. Thus, reversing the sequence in a short amphiphilic peptide may not always result in the selective loss of biological activity such as hemolytic activity. Also, introduction of enantiomeric amino acids in a short peptide to generate a diastereomer may result in loss of structure as well as antimicrobial and hemolytic activities, unless compensated by an increase in positive charges.     

Structural and charge requirements for antimicrobial and hemolytic activity in the peptide PKLLETFLSKWIG,corresponding to the hydrophobic region of the antimicrobial protein bovine seminalplasmin

Several analogs of the 13-residue antimicrobial and hemolytic peptide PKLLETFLSKWIG (SPF), which is the most hydrophobic region of the 47-residue antimicrobial protein seminalplasmin [Sitaram, N. & Nagaraj, R. (1990) J. Biol. Chem. 265, 10438-104423 have been synthesized. The antimicrobial and hemolytic properties of the peptides were investigated with a view to gain an insight into the structural and charge requirements for these activities of SPF. Peptides in which E was replaced by K exhibited considerably improved antimicrobial activity with no concomitant increase in hemolytic activity. A peptide in which the aromatic amino acids were replaced by leucine exhibited antimicrobial activity like those of the peptides which had aromatic amino acids. Interchange in the positions of E and K and total replacement of K by E resulted in complete loss of activity. The peptides having antimicrobial activities showed appreciable helical content in a hydrophobic environment, whereas inactive peptides did not. Thus, by suitable‘engineering’ the biological activity of a short 13-residue peptide can be altered to yield peptides specifically having only antimicrobial activity with increased potency. © Munksgaard 1995.     

Comprehensive Analysis and Biological Characterization of Venom Components from Solitary Scoliid Wasp Campsomeriella annulata annulata

Venoms of solitary wasps are utilized for prey capture (insects and spiders), paralyzing them with a stinger injection to be offered as food for their larvae. Thus, the identification and characterization of the components of solitary wasp venoms can have biotechnological application. In the present study, the venom components profile of a solitary scoliid wasp, Campsomeriella annulata annulata, was investigated through a comprehensive analysis using LC-MS and -MS/MS. Online mass fingerprinting revealed that the venom extract contains 138 components, and MS/MS analysis identified 44 complete sequences of the peptide components. The peptides are broadly divided into two classes: bradykinin-related peptides, and linear α-helical peptides. Among the components of the first class, the two main peptides, α-campsomerin (PRLRRLTGLSPLR) and β-campsomerin (PRLRRLTGLSPLRAP), had their biological activities evaluated. Both peptides had no effects on metallopeptidases [human neprilysin (NEP) and angiotensin-converting enzyme (ACE)] and acetylcholinesterase (AChE), and had no cytotoxic effects. Studies with PC12 neuronal cells showed that only α-campsomerin was able to enhance cell viability, while β-campsomerin had no effect. It is noteworthy that the only difference between the primary structures from these peptides is the presence of the AP extension at the C-terminus of β-campsomerin, compared to α-campsomerin. Among the linear α-helical peptides, annulatin (ISEALKSIIVG-NH₂) was evaluated for its biological activities. Annulatin showed histamine releasing activity from mast cells and low hemolytic activity, but no antimicrobial activities against all microbes tested were observed. Thus, in addition to providing unprecedented information on the whole components, the three peptides selected for the study suggest that molecules present in solitary scoliid wasp venoms may have interesting biological activities.     

Structure and biological activities of eumenine mastoparan-AF (EMP-AF), a new mast cell degranulating peptide in the venom of the solitary wasp (Anterhynchium flavomarginatum micado).

A new mast cell degranulating peptide, eumenine mastoparan-AF (EMP-AF), was isolated from the venom of the solitary wasp Anterhynchium flavomarginatum micado, the most common eumenine wasp found in Japan. The structure was analyzed by FAB-MS/MS together with Edman degradation, which was corroborated by solid-phase synthesis. The sequence of EMP-AF, Ile-Asn-Leu-Leu-Lys-Ile-Ala-Lys-Gly-Ile-Ile-Lys-Ser-Leu-NH(2), was similar to that of mastoparan, a mast cell degranulating peptide from a hornet venom; tetradecapeptide with C-terminus amidated and rich in hydrophobic and basic amino acids. In fact, EMP-AF exhibited similar activity to mastoparan in stimulating degranulation from rat peritoneal mast cells and RBL-2H3 cells. It also showed significant hemolytic activity in human erythrocytes. Therefore, this is the first example that a mast cell degranulating peptide is found in the solitary wasp venom. Besides the degranulation and hemolytic activity, EMP-AF also affects on neuromuscular transmission in the lobster walking leg preparation. Three analogs EMP-AF-1 approximately 3 were snythesized and biologically tested together with EMP-AF, resulting in the importance of the C-terminal amide structure for biological activities.     

Structure–biological activity relationships of 11-residue highly basic peptide segment of bovine lactoferrin

The antimicrobial peptide, lactoferricin, is generated upon the gastric pepsin cleavage of lactoferrin and has many basic and hydrophobic amino acid residues essential for its biological activity. To investigate the structure-antimicrobial activity relationships, the basic amino acid-rich region of bovine lactoferricin (BLFC), RRWQWRMKKLG, was selected. Using chemically synthesized BLFC and its substituted peptides, the antimicrobial activities of the peptides were tested by determining the minimal inhibitory concentration (MIC) of Escherichia coli and Bacillus subtilis and the disruption of the outer cell membrane of E. coli, and the peptide's toxicities were assayed by hemolysis. The short peptide (B3) composed of only 11 residues had similar antimicrobial activities while losing most of the hemolytic activities as compared with the 25 residue-long ones (B1 and B2). The short peptides (B3, B5 and B7) with double arginines at the N-termini had more potent antimicrobial activity than those (B4 and B6) with lysine. However, no antimicrobial and hemolytic activities were found in B8, in which all basic amino acids were substituted with glutamic acid, and in B9, in which all hydrophobic amino acids were substituted with alanine. The circular dichroism (CD) spectra of the short peptides in 30 MM SDS were correlated with their antimicrobial activities. These results suggested that the 11-residue peptide of BLFC is involved in the interaction with bacterial phospholipid membranes and plays an important role in antimicrobial activity with little or no hemolytic activity. © Munksgaard 1996.     

Consequences of introducing a disulfide bond into an antibacterial and hemolytic peptide

Abstract: The effect of introducing a disulfide bridge between the N- and C-terminal ends on the structure and biological activities of the 13-residue linear peptide PKLLKTFLSKWIG(SPFK), which has both antibacterial and hemolytic activity, have been investigated. The terminal amino acids P and G in SPFK were replaced by cysteines to form a disulfide bridge. The linear peptides C(Acm)KLLKTFLSKWIC(Acm) and C(Acm) KLLKTFLSKWIC(Acm)-amide, where Acm is acetamidomethyl group, showed antibacterial activity but did not possess hemolytic activity unlike SPFK. Introduction of an S–S bridge resulted in enhanced hemolytic activity compared with SPFK. The hemolytic activity was particularly pronounced in the cyclic peptide CKLLKTFLSKWIC-amide. Circular dichroism studies indicate that the cyclic peptides tend to adopt distorted helical structures. The cyclic peptides also have a greater affinity for lipid vesicles, which could be the reason for the effective perturbation of the erythrocyte membrane.     

Increased antibacterial activity of 15‐residue murine lactoferricin derivatives

Abstract: LFM W8 is a synthetic 15‐residue lactoferricin derivative (H₂N‐EKCLRWQWEMRKVGG‐COOH), corresponding to residues 16–30 of the mature murine lactoferrin protein except that the asparagine residue in position 8 of the native peptide is replaced with tryptophan. We have previously reported that the two tryptophan residues in positions 6 and 8 are of crucial importance for the antibacterial activity of many lactoferricin derivatives but, despite fulfilling this requirement, LFM W8 is inactive against Escherichia coli and Staphylococcus aureus. In order to solve this puzzle, a quantitative structure–antibacterial activity relationship study of synthetic LFM W8 derivatives was performed by replacing the glutamate residues in positions 1 and 9 with arginine or alanine, and the valine residue in position 13 with tyrosine. The results from the study were analyzed using multivariate data analysis. The derived mathematical model clustered the peptides into distinct groups which reflected their antibacterial activities, pointed out correlations between different structural parameters, highlighted the structural parameters that were important for antibacterial activity, and enabled us to predict the activity of a 15‐residue bovine lactoferricin derivative. The results showed that net charge and micelle affinity, as determined from the ratio of α‐helicity in sodium dodecyl sulfate micelles and in 1,1,1,3,3,3‐hexafluoro‐2‐propanol, were the most important structural parameters affecting antibacterial activity. The most active derivative, LFM R1,9 W8 Y13, displayed a minimal inhibitory concentration of 10 and 12 µm against E. coli and S. aureus, respectively. This represented more than 50‐fold and 40‐fold increases in antibacterial activity, respectively, compared with LFM W8.