The medicinal chemistry of short lactoferricin-based antibacterial peptides.

This review discusses antibacterial peptides from the perspective of development into clinically useful chemotherapeutic drugs using short lactoferricin based peptides as examples. The review shows how important features for antibacterial activity can be identified and explored using the molecular properties of a range of natural and non-natural amino acids. The results have been further refined quantitatively using a "soft-modelling" approach where important structural parameters that influence the antibacterial activity of 15-residue model peptides were identified. The review describes how this knowledge is utilised to generate pharmacophores for antibacterial efficacy. These pharmacophores turn out to be surprisingly small and relatively consistent between typical Gram-negative and Gram-positive bacteria leading to the discovery of a novel class of short synthetic cationic antimicrobial peptides. These compounds are found to have high antibacterial activity against several bacterial strains that are resistant to commercial antibiotics, and are promising as future clinical candidates for treatment of infections caused by several clinically relevant pathogens.

Enhanced antitumor activity and selectivity of lactoferrin-derived peptides.

A number of peptide analogs derived from the N-terminal alpha-helical region of bovine lactoferrin (LFB 14-31), were designed in order to investigate how deviating numbers and positions of positively charged residues and numbers of aromatic residues affected their activity against prokaryotic, normal and transformed eukaryotic cells. Most of the LFB derivatives were highly active against both Escherichia coli and Staphylococcus aureus. The peptides were more active against the tumor cell lines MethA, HT-29 and MT-1 than normal eukaryotic cells. The peptides that were most active against the tumor cell lines had all cationic residues concentrated in one sector of the helical structure. These peptides were less selective against the tumor cell lines than against normal fibroblasts. Quantitative structure-activity relationship studies showed that certain structural parameters affected toxicity against the tumor cell lines more than against fibroblasts. Peptides encompassing these parameters were slightly less active against tumor cells, but gained significant selectivity.

Bulky aromatic amino acids increase the antibacterial activity of 15-residue bovine lactoferricin derivatives.

A model peptide, FKCRRWQWRMKKLGA, residues 17-31 of bovine lactoferricin, has been subjected to structure-antibacterial activity relationship studies. The two Trp residues are very important for antibacterial activity, and analogue studies have demonstrated the significance of the size, shape and aromatic character of the side chains. In the current study we have replaced Trp residues in the model peptide with bulky aromatic amino acids to elucidate further the importance of size and shape. The counterproductive Cys residue in position 3 was also replaced by these aromatic amino acids. The largest aromatic amino acids employed resulted in the most active peptides. The peptides containing these hydrophobic residues were generally more active against Staphylococcus aureus than against Escherichia coli, indicating that the bacterial specificity as well as the antibacterial efficiency can be altered by employing large hydrophobic aromatic amino acid residues.

Short antibacterial peptides and erythromycin act synergically against Escherichia coli.

Five different peptides (6-18 residues) with chain lengths shorter than the required minimum to span the bacterial cell membrane as monomeric helices were designed in order to elucidate whether variation in chain length exerted differences in their mode of action. To gain a better understanding of the possible mode of action of these peptides, they were studied in combination with clinically used antibiotics with different targets. Antibiotic-peptide combinations were tested against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923. No synergy was observed between the peptides and antibiotics when tested against S. aureus. Synergic interactions between all peptides and erythromycin were observed when tested against E. coli. Synergy was also observed with rifampicin and two peptides against E. coli. There was no clear-cut correlation between the ability to interact synergically or antagonistically and the number of residues. We further investigated the combined action of our peptides and PGLa, to elucidate peptide-peptide interactions. In contrast to previously reported synergy between magainin 2 and PGLa, our peptides did not show any synergy when combined with PGLa. Thus, our results indicate an alternative mode of action of these antibacterial peptides as compared with peptides such as magainin 2.

The role of tryptophan in the antibacterial activity of a 15-residue bovine lactoferricin peptide.

Bovine lactoferricin is a 25-residue antibacterial peptide isolated after gastric cleavage of the iron transporting protein lactoferrin. A 15-residue fragment, FKCRRWQWRMKKLGA of this peptide sustains most of the antibacterial activity. In this truncated sequence, the two Trp residues are found to be essential for antibacterial activity. The anchoring properties of Trp, as have been observed in membrane proteins, are believed to be important for the interaction of Trp containing antibacterial peptides with bacterial cell membranes. We have investigated the molecular properties which make Trp important for the antibacterial activity of the 15-residue peptide by replacing Trp with natural and unnatural aromatic amino acids. This series of peptides was tested for antibacterial activity against Echerichia coli and Staphylococcus aureus. We found that neither the hydrogen bonding ability nor the amphipathicity of the indole system are essential properties for the effect of Trp on the antibacterial activity of the peptides. Replacement of Trp with residues containing aromatic hydrocarbon side chains gave the most active peptides. We propose that aromatic hydrocarbon residues are able to position themselves deeper into the bacterial cell membrane, making the peptide more efficient in disrupting the bacterial cell membrane. From our results the size, shape and aromatic character of Trp seem to be the most important features for the activity of this class of Trp containing antibacterial peptides.

Antibiotic activity of pentadecapeptides modelled from amino acid descriptors.

Pentadecapeptides based on modified murine lactoferricin (LFM) sequences show varying degrees of antibacterial activity against Escherichia coli and Staphylococcus aureus. By means of projections to latent structures (PLS), a good correlation is obtained if the biological activity is modelled as a function of variables describing peptide properties, e.g. alpha-helicity, hydrophobicity/hydrophilicity and charge. Using variables derived from a principal component analysis (PCA) of all naturally occurring amino acids, it is possible to describe the amino acid content of the peptides using three variables per amino acid position. The resulting descriptor matrix is then used to develop quantitative structure-activity relationships (QSAR). It is shown that the theoretically derived descriptors model the activity of the peptides better than the earlier model, and that properties of the peptides other than antibacterial activity can be predicted.

Increased antibacterial activity of 15-residue murine lactoferricin derivatives.

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 alpha-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 microM 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.

Antibacterial activity of 15-residue lactoferricin derivatives.

Lactoferricins are a class of antibacterial peptides isolated after gastric-pepsin digest of the mammalian iron-chelating-protein lactoferrin. For investigation of antibacterial activity, we prepared short synthetic derivatives of bovine, human, caprine, murine and porcine lactoferricins with 15-amino-acid residues of high sequence homology. The peptides corresponded to amino-acid residues 17-31 of the mature bovine lactoferrin. Only the bovine and caprine derivatives displayed measurable antibacterial activity, with the bovine one having a minimal inhibitory concentration of 24 microM and being 10 times more active than the caprine one against Escherichia coli. An alanine-scan of the bovine lactoferricin derivative was performed to identify specific amino acids that were important for the antibacterial activity. We found that neither of the two tryptophan residues (Trp 6 and Trp 8) present in the bovine lactoferricin derivative could be replaced by alanine without a major loss of antibacterial activity. The other lactoferricin derivatives tested contained only one tryptophan residue (Trp 6). Modified human, caprine and porcine lactoferricin derivatives containing two tryptophan residues (Trp 6 and Trp 8) displayed minimal inhibitory concentrations of 74, 174 and 219 microM, respectively, which represented up to a six-fold increase in antibacterial activity. The alanine-scan also revealed that the antibacterial activity was increased when acetamidomethyl-protected cysteine and unprotected glutamine (Cys 3 and Gln 7) were replaced with alanine. Only the bovine lactoferricin derivative and a few of its alanine-modified derivatives displayed measurable activity against Staphylococcus aureus.

TNF 41-62 and TNF 78-96 have distinct effects on LPS-induced tissue factor activity and the production of cytokines in human blood cells.

Biological activities of peptides representing two different regions in the TNF molecule were investigated. We have earlier reported that one of the peptides studied, TNF 36-62, induced chemotaxis in granulocytes and monocytes. TNF 41-62, a shorter analog of TNF 36-62, possessed similar chemotactic effects. Both peptides caused a weak enhancement of LPS -induced IL-6 production and tissue factor activity by monocytes in whole blood. The third peptide studied, TNF 78-96, was selected from a region located on the opposite side of the beta-sheet sandwich structure of the TNF molecule, and includes the loop 84-88 that has been shown to be involved in TNF receptor interaction. TNF 78-96 possessed properties quite different from TNF 36-62 and TNF 41-62. It amplified several fold PMA-induced secretion of elastase, and enhanced significantly PMA-induced secretion of cathepsin G from the neutrophils, activities which were effectively abolished by an anti-human TNF antibody. The TNF 78-96 peptide also inhibited LPS-induced TF activity in monocytes of whole blood, and it abolished the TNF enhancing effect of LPS-induced TF activity in a dose dependent manner. This suggests that the TNF 78-96 peptide may bind to the TNF receptor(s), without potentiating the same signals as native TNF. It may thereby prevent binding of the native TNF and the resultant activation effect of TNF. It also, at high concentrations, inhibited LPS-induced IL-6 production whereas it caused a doubling of LPS-induced IL-8 in monocytes and granulocytes in whole blood. These results clearly show that distinct TNF activities can be induced by peptide sequences taken from different regions of TNF. The TNF 78-96 peptide might be useful in downregulation of LPS-induced monocyte activations in vivo.

Initial binding sites of antimicrobial peptides in Staphylococcus aureus and Escherichia coli.

We examined the initial binding sites of magainin 1, cecropin P1 and lactoferricin B in Staphylococcus aureus and Escherichia coli. All 3 peptides were active against E. coli, whereas only lactoferricin B exerted any activity against S. aureus. Soluble lipoteichoic acid and lipopolysaccharide both interacted with all 3 peptides, whereas soluble teichoic acid interacted with lactoferricin B only. Antibodies against teichoic acid diminished the activity of lactoferricin B, while antibodies against lipoteichoic acid had no influence on the activity of lactoferricin B. Antibodies against lipopolysaccharide diminished the activity of lactoferricin B and magainin 1, but had no effect on the activity of cecropin P1 against E. coli. We conclude that the initial binding sites of lactoferricin B in S. aureus, and of lactoferricin B and magainin 1 in E. coli, are teichoic acid and lipopolysaccharide, respectively. Cecropin P1 seems to interact with a different binding site than those of magainin 1 and lactoferricin B in E. coli.

Interference of the antimicrobial peptide lactoferricin B with the action of various antibiotics against Escherichia coli and Staphylococcus aureus.

The antimicrobial peptide, lactoferricin, can be generated upon gastric pepsin cleavage of lactoferrin. We have examined the interaction of lactoferricin of bovine origin, Lf-cin B, with the antibiotics penicillin G, vancomycin, gentamicin, colistin, D-cycloserine and erythromycin against E. coli ATCC 25922 and Staphylococcus aureus ATCC 25923. We demonstrated synergism between Lf-cin B and erythromycin against E. coli, and partial synergism between Lf-cin B and penicillin G, vancomycin and gentamicin against E. coli. Only penicillin G acted in partial synergism with Lf-cin B against S. aureus. Lf-cin B antagonized vancomycin and gentamicin against S. aureus in low concentration. We conclude that Lf-cin B may facilitate the uptake of antibiotics across the cell envelope.

Antibacterial effects of lactoferricin B.

The antimicrobial peptide, lactoferricin, can be generated upon gastric pepsin cleavage of lactoferrin. We have examined the inhibitory efficacy of lactoferricin of bovine origin (Lf-cin B) on Escherichia coli, Proteus mirabilis and Staphylococcus aureus with or without a cell wall. We found that spheroplasts and protoplasts had a lower MIC than their counterparts with a cell wall. We also compared the efficacies of Lf-cin B (17-31) made of all L-amino acids and all D-amino acids. The peptide made of all D-amino acids was more active than the corresponding L-enantiomer. Furthermore, we examined the influence of Lf-cin B on the motility of E. coli and the influence of temperature on the susceptibility of bacteria exposed to Lf-cin B. Bacteria exposed to sub-MIC of Lf-cin B lost their motility. Bacteria exposed to Lf-cin B at 20 degrees C were more sensitive to Lf-cin B than when exposed at 37 degrees C. These findings indicate that the cell envelope is a limiting step for Lf-cin B to exert its antibiotic effect. We cannot rule out a receptor-mediated first step for Lf-cin B (17-31).

Lactoferricin of bovine origin is more active than lactoferricins of human, murine and caprine origin.

The antimicrobial peptide lactoferricin is generated by gastric pepsin cleavage of lactoferrin. We have examined the antimicrobial activity of lactoferricins derived from lactoferrin of human, murine, caprine and bovine origin with minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) against E. coli ATCC 25922 and S. aureus ATCC 25923. We found that lactoferricin of bovine origin (Lf-cin B) was the most efficacious of the lactoferricins tested. By comparing the linear and cyclic Lf-cin B we found the cyclic peptide to be the most active. Lactoferricin B was moderately active against E. coli ATCC 25922 and S. aureus ATCC 25923, but had no activity against P. mirabilis or Y. enterocolitica. Lf-cin B showed good activity against C. albicans, C. tropicalis and C. neoformans.

Evidence for exclusive role of the p55 tumor necrosis factor (TNF) receptor in mediating the TNF-induced collagenase expression by human dermal fibroblasts.

The aim of this study was to examine the roles of the TNF receptors p55 and p75 in the TNF-enhanced expression of collagenase by human dermal fibroblasts. The agonistic p55 monoclonal antibody Htr9 and TNF induced production of similar amounts of collagenase. Polyclonal or monoclonal agonistic p75 antibodies failed to enhance collagenase production, and the antagonistic p75 antibody 5E12 did not inhibit TNF-enhanced expression of collagenase. This strongly suggests that p55, but not p75, is involved in TNF-induced production of collagenase. Cells continued to produce an elevated level of collagenase after the removal of TNF or Htr9. These data suggest that it may be useful to use specific inhibitors of collagenase rather than to block cytokine action directly in the treatment of diseases with chronic enhanced collagenolytic activity. A peptide of residues 36-62 of TNF previously reported to be chemotactic to leukocytes was also able to enhance the expression of collagenase activity by dermal fibroblasts. Thus, design of peptides with specific TNF effects may offer a novel approach for treatment of fibrotic disorders.

The TNF Receptors p55 and p75 Mediate Chemotaxis of PMN Induced by TNFalpha and a TNFalpha 36-62 Peptide.

The present study was performed to examine whether residues 36-62 of TNFalpha contain the chemotactic domain of TNFalpha, and whether the p55 and p75 TNF receptors are involved in TNFalpha induced chemotaxis. The chemotactic effect of TNFalpha on PMN was inhibited by the mAbs Hrt-7b and Utr-1, against the p55 and p75 TNF receptors, respectively. Both receptors may therefore be required for mediating the chemotactic effect of TNFcz. The synthetic TNFalpha 36-62, similar to TNFalpha, had chemotactic effects on both PMN and monocytes. The chemotactic activity of the TNFalpha 36-62 peptide on PMN, was inhibited by Htr-7b, Utr-1 and soluble p55 receptor, which shows that the peptide possessed the ability to induce chemotaxis through the TNF receptors. In contrast to TNFalpha, the peptide did not show a cytotoxic activity against WEHI 164 flbrosarcoma cells. It is suggested that different domains of the TNFalpha molecule induce distinct biological effects.

Direct demonstration by [13C]NMR spectroscopy that glutamine from astrocytes is a precursor for GABA synthesis in neurons.

Primary cultures of cerebral cortical astrocytes and neurons, as well as neurons growing on top of the astrocytes (sandwich co-cultures), were incubated with 1-[13C]glucose or 2-[13C]acetate and in the presence or absence of the glutamine synthetase inhibitor methionine sulfoximine. [13C]NMR spectroscopy at 125 MHz was performed on perchloric acid extracts of the cells or on media collected from the cultures. In addition, the [13C/12C] ratios of the amino acids glutamine, glutamate and 4-aminobutyrate (GABA) were determined by gas chromatography/mass spectroscopy, showing a larger degree of labeling in GABA than in glutamate and glutamine from glucose. Glutamine and glutamate were predominantly labeled from acetate. A picture of cellular metabolism mainly regarding the tricarboxylic acid cycle and glycolysis was obtained. Due to the fact that acetate is not metabolized by neurons to any significant extent, it could be shown that precursors from astrocytes are incorporated into the GABA pool of neurons grown in co-culture with astrocytes. Spectra of media removed from these cultures revealed that likely precursor candidates for GABA were glutamine and citrate. The importance of glutamine is further substantiated by the finding that inhibition of glutamine synthetase, an enzyme present in astrocytes only, significantly decreased the labeling of GABA in co-cultures incubated with 2-[13C]acetate.

Identification of 3-oxodicarboxylic acids in human urine.

Gas chromatography-mass spectrometry has been used to detect a series of 3-oxodicarboxylic acids in human urine as their corresponding dimethyl ester trimethylsilyl enol ethers. 3-Oxohexanedioic acid, 3-oxooctanedioic acid and 3-oxodecanedioic acid were demonstrated to be normal urine constituents. Increased amounts of these acids were excreted after ingestion of decanedioic acid. These findings support the hypothesis that dicarboxylic acids are degraded by ordinary beta-oxidation.

Absolute configuration of 3-hydroxyadipic acid in human urine.

A method involving derivatization and combined gas chromatography--mass spectrometry has been developed to separate the enantiomers of 3-hydroxyadipic acid. By combining this method with asymmetric synthesis of the same acid, it has been shown that 3-hydroxyadipic acid excreted in urine consists of at least 95% of the L-enantiomer. This finding supports the hypothesis that dicarboxylic acids are degraded by ordinary beta-oxidation, and indicates that adipic acid may be converted into succinic acid.