Hundreds of peptide antibiotics have been described in the past half-century (20,28,35). These fall into two classes, nonribosomally synthesized peptides, such as the gramicidins, polymyxins, bacitracins, glycopeptides, etc., and ribosomally synthesized (natural) peptides. The former are often drastically modified and are largely produced by bacteria, whereas the latter are produced by all species of life (including bacteria) as a major component of the natural host defense molecules of these species. The former have been well described to date (28, 35) and will be briefly summarized here with emphasis on their clinical importance, similarities in function to the natural peptides, and future prospects. The latter represent a new opportunity for the medicinal chemist and will be described in more detail with emphasis on the role in natural host defenses (as nature's antibiotics) and the clinical potential of peptides derived from these natural peptides.

An 11-amino-acid amphipathic synthetic peptide homologous to a helical region on helix 1 of human lactoferrin HLP-2 exhibited bactericidal activity against Escherichia coli serotype O111, whereas an analogue synthesized with Pro substituted for Met, HLP-6, had greatly reduced antimicrobial activity. The bactericidal activity of HLP-2 was 10-fold greater than that of HLP-6 in both buffer and growth medium by time-kill assays. These assays also showed a pronounced lag phase that was both concentration and time dependent and that was far greater for HLP-2 than for HLP-6. Both peptides, however, were shown to be equally efficient in destabilizing the outer membrane when the hydrophobic probe 1-N-phenylnaphthylamine was used and to have the same lipopolysaccharide (LPS) binding affinity, as shown by polymyxin B displacement. Circular dichroism (CD) spectroscopy was used to study the structure and the organization of the peptides in solution and upon interaction with E. coli LPS. In the presence of LPS, HLP-2 and HLP-6 were found to bind and adopt a ␤-strand conformation rather than an ␣-helix, as shown by nonimmobilized ligand interaction assay-CD spectroscopy. Furthermore, this assay was used to show that there is a time-dependent association of peptide that results in an ordered formation of peptide aggregates. The rate of interpeptide association was far greater in HLP-2 LPS than in HLP-6 LPS, which was consistent with the lag phase observed on the killing curves. These results allow us to propose a mechanism by which HLP-2 folds and self-assembles at the outer membrane surface before exerting its activity.

Human lactoferrin contains a 46 residue sequence named lactoferricin H thought to be responsible for its antimicrobial properties. Synthetic peptides HLTI, correspomling to the loop region of human lactoferricin (FQWQR-NMRKVRGPPVS) and HLT2, corresponding to its charged portion (FQWQRNMRKVR), exerted significant antibacterial effects against £. co//serotype O111 strains NCTC 8007 and ML35. The corresponding sequences In native human lactoferrin were shown to adopt a charged helix and hydrophoble tall within the N-lobe remote from the iron binding site. Sequence similarities between lactoferricin and dermaseptin and magainins suggest that laetoferricin may act as an amphipathie alpha helix.

Lactoferricin includes an 11-amino-acid amphipathic alpha-helical region which is exhibited on the outer surface of the amino-terminal lobe of lactoferrin. Synthetic peptides homologous to this region exhibited potent antibacterial activity against a selected range of both gram-negative and gram-positive bacteria. An analog synthesized with methionine substituted for proline at position 26, which is predicted to disrupt the helical region, abolished antibacterial activity against Escherichia coli and considerably reduced antibacterial activity against Staphylococcus aureus and anAcinetobacter strain. The mode of action of human lactoferrin peptide (HLP) 2 against E. coli serotype O111 (NCTC 8007) was established by using flow cytometry, surface plasmon resonance, and transmission electron microscopy. Flow cytometry was used to monitor membrane potential, membrane integrity, and metabolic processes by using the fluorescent probes bis-1,3-(dibutylbarbituric acid)-trimethine oxonol, propidium iodide, and carbonyl cyanide m-chlorophenylhydrazone, respectively. HLP 2 was found to act at the cell membrane, causing complete loss of membrane potential after 10 min and of membrane integrity within 30 min, with irreversible damage to the cell as shown by rapid loss of viability. The number of particles, measured by light scatter on the flow cytometer, dropped significantly, showing that bacterial lysis resulted. The peptide was shown to bind toE. coli O111 lipopolysaccharide by using surface plasmon resonance. Transmission electron microscopy revealed bacterial distortion, with the outer membrane becoming detached from the inner cytoplasmic membrane. We conclude that HLP 2 causes membrane disruption of the outer membrane, resulting in lysis, and that structural considerations are important for antibacterial activity.