Current Pharmaceutical Design - Volume 8, Issue 9, 2002

The increasing development of bacterial resistance to traditional antibiotics has reached alarming levels, thus necessitating the strong need to develop new antimicrobial agents. These new antimicrobials should possess both novel modes of action as well as different cellular targets compared with the existing antibiotics. Lysozyme, muramidase, and aprotinin, a protease inhibitor, both exhibit antimicrobial activities against different microorganisms, were chosen as model proteins to develop more potent bactericidal agents with broader antimicrobial specificity. The antibacterial specificity of lysozyme is basically directed against certain Gram-positive bacteria and to a lesser extent against Gram-negative ones, thus its potential use as antimicrobial agent in food and drug systems is hampered. Several strategies were attempted to convert lysozyme to be active in killing Gram-negative bacteria which would be an important contribution for modern biotechnology and medicine. Three strategies were adopted in which membrane-binding hydrophobic domains were introduced to the catalytic function of lysozyme, to enable it to damage the bacterial membrane functions. These successful strategies were based on either equipping the enzyme with a hydrophobic carrier to enable it to penetrate and disrupt the bacterial membrane, or coupling lysozyme with a safe phenolic aldehyde having lethal activity toward bacterial membrane. In a different approach, proteolytically tailored lysozyme and aprotinin have been designed on the basis of modifying the derived peptides to confer the most favorable bactericidal potency and cellular specificity. The results obtained from these strategies show that proteins can be tailored and modelled to achieve particular functions. These approaches introduced, for the first time, a new conceptual utilization of lysozyme and aprotinin, and thus heralded a great opportunity for potential use in drug systems as new antimicrobial agent.

Neutrophils contain several cationic antimicrobial proteins or peptides (CAPs) that exert antibiotic-like action against bacteria. These host-derived antibiotics kill susceptible bacteria by oxygen-independent mechanisms. Considerable interest in their activity has been generated in recent years due not only to their likely important role in innate host defense against infection, but also their possible use as therapeutic agents in treating infections caused by antibiotic-resistant pathogens. We have studied the antibacterial properties of human lysosomal cathepsin G (cat G). This highly cationic serine protease contains at least three antibacterial regions that by themselves can exert antibacterial action against Gram-negative bacteria, such as Pseudomonas aeruginosa. Only one of these peptides, defined by residues 117-136 of full-length cat G, has bactericidal action against Gram-positive pathogens, such as Staphylococcus aureus. Due to the broad-spectrum antibacterial action of this peptide, we have sought to define the amino acids within its primary sequence required for this activity and have developed variants with improved activity. This review emphasizes the importance of both cationicity and hydrophobicity as necessary characteristics for the antibacterial action of CAPs. It also proposes the strategy that naturally occurring large human CAPs can be dissected to smaller CAPs and then modified to enhance their activity in vitro. This approach could prove beneficial to those interested in developing antimicrobial peptides as therapeutic agents.

Cationic antimicrobial peptides are observed throughout nature. In mammals they are observed both at epithelial surfaces and within the granules of phagocytic cells. They are an important component of innate defences, since in addition to their ability to kill microorganisms, they are able to modulate inflammatory responses. With respect to their ability to kill bacteria, it is very difficult to isolate resistant mutants. However there are a few known mechanisms of intrinsic resistance, including PhoPQ-dependent and other alterations in lipopolysaccharide structure that influence self promoted uptake, and protease-mediated resistance.

Antimicrobial peptides are a large group of gene-encoded, net positively charged polypeptides, produced by living organisms of all types including human and plants. They are mobilized shortly after infection as part of the innate immunity of these species and act rapidly to neutralize a broad range of microbes. Nowadays, thousands of native and de-novo designed antimicrobial peptides are available. They vary considerably in length, composition, charge and secondary structure. Despite these variations most antimicrobial peptides use a similar target, which is the bacterial phospholipid membrane. Many of them use a common general mechanism, the ”carpet“mechanism, in which they accumulate on the bacterial membrane up to a threshold concentration, and then effect membrane permeation / disintegration. However, the structure of the permeation pathway may vary for different peptides and may include channel aggregates, toroidal pores or channels. Target specificity is determined by the negatively charged bacterial membrane, the net positive charge of the peptide, its hydrophobicity, oligomeric state in solution and in the membrane, and the stability of its secondary structure. A novel group of non hemolytic antimicrobial peptides were derived from diastereomers (containing D- and L-amino acids) of lytic peptides based on parameters required by the carpet mechanism. Because these disastereomers exhibit several advantages over their all-L amino acid counterparts, they have a potential to be developed for therapeutic use both in vitro and in vivo.

Antimicrobial peptides are important components of innate immunity in species across the evolutionary scale. Unlike therapeutically used antibiotics, this class of peptides exert their activity by permeabilizing bacterial membranes. Despite the seemingly common mechanism of action, there is considerable variation in their primary structures, length and number of positive charges. Host-defense antimicrobial peptides have been the subject of extensive biophysical studies with a view at delineate structural requirements for activity. In this article, the structures of host defence antibacterial peptides and the structural requirements for activity are reviewed.

Gene-encoded, ribosomally synthesised antimicrobial peptides (AMPs) are an ancient and pervasive component of the innate defence mechanisms used by multicellular organisms to control the natural flora and combat pathogens. Bacteria also produce such AMPs to maintain ecological niches free of rival strains. Several hundred different peptides have been characterised to date, and they show a marked degree of variability in both sequence and structure, having evolved to act against distinct microbial targets in different physiological contexts. Many of these peptides appear to function via a selective, but not receptor-mediated, permeabilisation of microbial membranes, while others interact with specific membrane associated or intracellular targets. This review presents a broad survey of different AMP structural classes, emphasising both their molecular diversity and underlying similarities. The mode of action of these peptides and potential for biomedical and other application is also briefly discussed.

Pro-rich antimicrobial peptides are a group of linear peptides of innate immunity isolated from mammals and invertebrates, and characterised by a high content of proline residues (up to 50%). Members of this group are predominantly active against Gram-negative bacterial species which they kill by a non-lytic mechanism, at variance with the majority of the known antimicrobial peptides. Evidence is accumulating that the Pro-rich peptides enter the cells without membrane lysis and, once in the cytoplasm, bind to, and inhibit the activity of specific molecular targets essential to bacterial growth, thereby causing cell death. This mode of action makes these peptides suitable for drug development efforts. In addition to antibacterial action, PR-39, one of the better characterised Pro-rich peptides from mammals, exerts other potentially exploitable biological activities, such as induction of syndecan expression in mesenchymal cells and inhibition of the NADPH oxidase activity of neutrophils, suggesting a role of this peptide in wound repair and inflammation. PR-39 also exerts a protective effect in various animal models of ischemia-reperfusion injury, preventing the post-ischemic oxidant production, and is a potent inducer of angiogenesis both in vitro and in vivo. Although the physiological relevance of all these effects has not yet been established, the above observations underscore the therapeutic potential of this peptide in a number of complex processes such as inflammation, wound repair, ischemia-reperfusion injury, and angiogenesis.

Cathelicidin peptides are a numerous group of mammalian cationic antimicrobial peptides. Despite a common evolutionary origin of their genes, peptides display a remarkable variety of sizes, sequences and structures. Their spectra of antimicrobial activity are varied and cover a range of organisms that includes bacteria, fungi and enveloped viruses. In addition, they bind to and neutralize the effects of endotoxin. These features make this family of peptides good candidates in view of a therapeutic use. The most promising ones are currently under evaluation as leads for the development of novel anti-infectives, and synthetic variants are in an advanced stage of development for specific clinical applications. This review focuses on recent studies on the structure and in vitro and in vivo biological activities of these peptides.

Recent increase of antibiotic-resistant pathogens demands exploration of novel antimicrobial molecules with unexploited mechanisms. Several hundred host defense peptides have been isolated from natural sources and their functions characterized. As host defense peptides have several advantages over classic antibiotics for resistant pathogens, there are many efforts to develop host defense peptides as therapeutic agents. In this review, focusing on the development of short antimicrobial peptides (≤18-mer), several examples are introduced that identify the active fragment from cyclic host peptides, or novel antimicrobial peptides derived from combinatorial libraries. Moreover, structure-activity relationships of short antimicrobial peptides are discussed, and several methods for improving bioavailability as well as specificity of the peptides, such as D-amino acid replacements, unnatural amino acid replacements, and backbone modifications, are discussed.

Lantibiotics are antibiotic peptides distinguished by the presence of the rare thioether amino acids lanthionine and / or methyllanthionine. They are produced by Grampositive bacteria as gene-encoded precursor peptides and undergo post-translational modification to generate the mature peptide. The structural gene for the prepeptide and the genes involved in biosynthesis, processing, export as well as regulation and producer strain self-protection are organized in clusters. Based on their structural and functional features lantibiotics are currently divided into two major groups. The flexible amphiphilic type-A lantibiotics act primarily by pore formation in the bacterial membrane, a mechanism which was recently shown, e.g. for nisin and epidermin, to involve the interaction with specific docking molecules such as the membrane precursor lipid II. The rather rigid and globular type-B lantibiotics inhibit enzyme functions through interaction with the respective substrates: mersacidin and actagardine inhibit the cell wall biosynthesis by complexing lipid II, whereas the cinnamycin-like peptides inhibit phospholipases by binding phosphoethanolamine. Lantibiotics have attracted much attention in recent years and undergone extensive characterization. New insights into the mode of action and structure-function relationships as well as the biochemistry and the genetics will be outlined in this review.