Cationic antibacterial peptides have been proclaimed as new drugs against multiresistant bacteria. Their limited success so far is partially due to the size of the peptides, which gives rise to unresolved issues regarding administration, bioavailability, metabolic stability, and immunogenicity. We have systematically investigated the minimum antibacterial motif of cationic antibacterial peptides regarding charge and lipophilicity/bulk and found that the pharmacophore was surprisingly small, opening the opportunity for development of short antibacterial peptides for systemic use.

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LTX 109 is a synthetic antimicrobial peptidomimetic (SAMP) currently in clinical phase II trials for topical treatment of infections of multiresistant bacterial strains. All possible eight stereoisomers of the peptidomimetic have been synthesized and tested for antimicrobial effect, hemolysis, and hydrophobicity, revealing a strong and unusual dependence on the stereochemistry for a molecule proposed to act on a general membrane mechanism. The three-dimensional structures were assessed using nuclear magnetic resonance spectroscopy (NMR) and molecular dynamics (MD) simulations in aqueous solution and in phospholipid bilayers. The solution structures of the most active stereoisomers are perfectly preorganized for insertion into the membrane, whereas the less active isomers need to pay an energy penalty in order to enter the lipid bilayer. This effect is also found to be reinforced by a significantly improved water solubility of the less active isomers due to a guanidyl-π stacking that helps to solvate the hydrophobic surfaces.

A series of synthetic antimicrobial peptidomimetics (SAMPs) have been prepared and found to be highly active against several Gram-negative and Gram-positive bacterial strains. These derivatives comprise the minimal structural requirements for cationic antimicrobial peptides and showed high selectivity for Gram-negative and/or Gram-positive bacteria compared to human red blood cells. We have found that SAMPs share many of the attractive properties of cationic antimicrobial peptides inasmuch that a representative SAMP was found to insert into the bilayers of large unilamellar vesicles, permeabilized both the outer and cytoplasmic membrane of Escherichia coli ML-35p, and displayed an extremely rapid bacterial killing for Staphylococcus aureus. However, while antimicrobial peptides are prone to proteolytic degradation, high in vitro stability in human blood plasma was shown for SAMPs. A combination of high antibacterial activity against methicillin-resistant staphylococci and low toxicity against human erythrocytes makes these molecules promising candidates for novel antibacterial therapeutics.

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.

The inherent instability of peptides toward metabolic degradation is an obstacle on the way toward bringing potential peptide drugs onto the market. Truncation can be one way to increase the proteolytic stability of peptides, and in the present study the susceptibility against trypsin, which is one of the major proteolytic enzymes in the gastrointestinal tract, was investigated for several short and diverse libraries of promising cationic antimicrobial tripeptides. Quite surprisingly, trypsin was able to cleave very small cationic antimicrobial peptides at a substantial rate. Isothermal titration calorimetry studies revealed stoichiometric interactions between selected peptides and trypsin, with dissociation constants ranging from 1 to 20 microM. Introduction of hydrophobic C-terminal amide modifications and likewise bulky synthetic side chains on the central amino acid offered an effective way to increased half-life in our assays. Analysis of the degradation products revealed that the location of cleavage changed when different end-capping strategies were employed to increase the stability and the antimicrobial potency. This suggests that trypsin prefers a bulky hydrophobic element in S1' in addition to a positively charged side chain in S1 and that this binding dictates the mode of cleavage for these substrates. Molecular modeling studies supported this hypothesis, and it is shown that small alterations of the tripeptide result in two very different modes of trypsin binding and degradation. The data presented allows for the design of stable cationic antibacterial peptides and/or peptidomimetics based on several novel design principles.

This review focuses on important structural features affecting the antimicrobial activity of 15-residue derivatives of lactoferricins. Our investigations are based on an alanine-scan of a 15-residue bovine lactoferricin derivative that revealed the absolute necessity of two tryptophan residues for antimicrobial activity. This "tryptophan-effect" was further explored in homologous derivatives of human, caprine, and porcine lactoferricins by the incorporation of one additional tryptophan residue, and by increasing the content of tryptophan in the bovine derivative to five residues. Most of the resulting peptides display a substantial increase in antimicrobial activity. To identify which molecular properties make tryptophan so effective, a series of bovine lactoferricin derivatives were prepared containing non-encoded unnatural aromatic amino acids, which represented various aspects of the physicochemical nature of tryptophan. The results clearly demonstrate that tryptophan is not unique since most of the modified peptides were of higher antimicrobial potency than the native peptide. The size and three-dimensional shape of the inserted "super-tryptophans" are the most important determinants for the high antimicrobial activity of the modified peptides. This review also describes the use of a "soft-modeling" approach in order to identify important structural parameters affecting the antimicrobial activity of modified 15-residue murine lactoferricin derivatives. This QSAR-study revealed that the net charge, charge asymmetry, and micelle affinity of the peptides were the most important structural parameters affecting their antimicrobial activity.