Antimicrobial peptides (AMPs) are endogenous polypeptides produced by multicellular organisms in order to protect a host from pathogenic microbes. AMPs are also defined as host defense peptides because of their essential role in constituting the innate immunity system [1,2,3,4]. AMPs are generally comprised of less than 50 amino acids approximately, and characterized by cationic amphipathic properties. In general, when AMPs are folded in membrane mimetic environments, one side of AMPs is positively charged (mainly due to lysine and arginine residues) and the other side contains a considerable proportion of hydrophobic residues [1,2,5,6].

AMPs show broad-spectrum antimicrobial activities against various microorganisms, including Gram-positive and Gram-negative bacteria, fungi, and viruses [1]. Of particular interest, many AMPs are effective against multi-drug resistant (MDR) bacteria and possess low propensity for developing resistance [7,8,9]. Bacterial resistance to antibiotics can be achieved by diverse routes including inhibition of the drug-target interaction, modification of the drug-binding site in target proteins, and efflux of the drug from target cells [10]. Microorganisms can also alter their genetic patterns in response to environmental changes using their own complex systems called sensor-transducer response systems. For instance, bacteria can modify their gene expression in the presence of AMPs [11]. AMPs possess low propensity for developing resistance, probably due to their distinguished mode of action. Most AMPs, with their amphipathic nature, directly act on the membrane of the pathogen. The cationic properties of AMPs are implicated in their selective interaction with the negatively charged surfaces of microbial membranes, resulting in the accumulation of AMPs on the membrane surface. Then, their hydrophobic portions are responsible for the interaction with hydrophobic components of the membrane. From this complex interaction with the membrane, major rearrangements of its structure occur, which may result from the formation of peptide-lipid specific interactions, the peptide translocation across the membrane and interaction with intracellular targets or the most common mechanism, a membranolytic effect [3,11,12,13,14,15,16]. Such a characteristic mechanism of action, distinct from that of conventional antibiotics, enables AMPs to avoid the common resistance mechanisms observed for classic antibiotics. Consequently, AMPs are receiving great attention as promising alternatives to conventional antibiotics to overcome the current drug resistance crisis. In this review, we describe the advantages and limitations of AMPs as novel antibiotic agents and structural information aids for developing new AMPs. Especially, we focus on small peptides (less than twelve amino acids in length) in clinical trials and their structure-activity relationships (SAR).

AMPs can be classified into four groups based on their structures: α-helical peptides, β-sheet peptides, extended peptides, and loop peptides [1,2,3,6]. The α-helical AMPs, including magainin, cecropin, and pexiganan, constitute a representative class of AMPs that are the most well established in structure-activity relationships. This group of peptides is usually unstructured in aqueous solution and forms amphipathic helices in membranes or membrane-mimicking environments. Most α-helical AMPs disrupt bacterial membranes, and several mechanisms of action employed by various AMPs have been proposed. The α-helical amphipathic peptides form barrel-like bundles in the bacterial membranes, and these transmembrane clusters line amphipathic pores (barrel-stave model). Many α-helical AMPs, including some magainins and cecropins, can dirupt bacterial membranes by forming carpet-like clusters of peptides. The peptides adsorb and align in parallel to the surface of bacterial membranes, then the membranes are collapsed into micelle-like structures by high concentrations of peptides (carpet model). The α-helical AMPs, such as some magainins and protegrins, form toroidal pores to disrupt the bacterial membranes (toroidal pore model) [1,2,3,4,5,6,7,9,11].

The β-sheet AMPs, such as α-, β-defensins, and protegrin, are stabilized by disulfide bridges, and form relatively rigid structures. Many of β-sheet AMPs exert their antimicrobial activities by disrupting bacterial membranes. They are perpendicularly inserted or tilted into the lipid bilayer to form toroidal pores, and hydrophilic regions of the peptides are associated with the polar head groups of the membranes [3].

The extended AMPs, which are predominantly rich in specific amino acids such as proline, tryptophan, arginine, and histidine, have no regular secondary structure elements. Indolicidin is a tryptophan/proline-rich extended peptide and Bac5 and Bac7 are proline/arginine-rich peptides [17,18]. Many extended AMPs are not active against the membranes of pathogens, but they can achieve their antimicrobial activities by penetrating across the membranes and interacting with bacterial proteins inside [3]. On the other hand, some extended peptides, such as indolicidin, are membrane active and induce membrane leakage. Indolicidin is a 13-amino acid AMP containing five tryptophan and three proline residues. The peptide adopts a poly-L-II helical structure in the presence of liposomes, and the high content of tryptophan residues is responsible for the interaction with lipid membranes [17]. The loop AMPs, including bactenecin, adopt a loop formation with one disulfide bridge.

Understanding the structure-activity relationships (SAR) of AMPs is essential for the design and development of novel antimicrobial agents with improved properties. In particular, the atomic level structures of AMPs can provide versatile information for all stages of drug development, including the peptide design and modification for pharmaceutical application. Pexiganan (also known as MSI-78), a synthetic variant of magainin 2, is one of the best-investigated AMPs in terms of drug development [19,20]. Pexiganan has reached clinical trials as a novel topical broad-spectrum antibiotic for the treatment of mild-to-moderate diabetic foot ulcer infections [4,20,21]. The three-dimensional structure of pexiganan, determined by Nuclear Magnetic Resonance (NMR) spectroscopy, revealed that the peptide forms a dimeric antiparallel α-helical structure in the presence of membrane mimetics [22,23]. The atomic resolution structure also demonstrated that the side chains of three phenylalanine residues are important for the self-dimerization [23]. In addition to the peptide structure, its orientation in membrane is critical to understand the exact mode of membrane interaction. A solid-state NMR study of pexiganan suggested that the peptide adopts a helical conformation interacting with the membrane surface and then the dimeric peptide is inserted into the membrane [22].

Knowledge on the functional structures of AMPs also enables a rational design of synthetic model peptides. In this respect, minimalistic de novo approaches to design model amphipathic helical peptides are noteworthy. For example, certain 14- or 15-mer peptides (namely LK peptides) composed of two kinds of amino acids (leucines and lysines), have been characterized as possessing strong antimicrobial activity [24,25]. Then, by introducing a single tryptophan residue at a specific position, 9- to 11-mer LKW peptides could exhibit antimicrobial activity. Now, the de novo designed model peptides remain to be further improved by optimizing the amino acid sequence to enhance stability and to reduce potential toxicity. In addition, their activities need to be checked using clinically isolated bacteria. In the LKW peptide design, the tryptophan residue was specifically incorporated in expectation of structural role for conferring activity [26]. Tryptophan is known to stabilize the helical conformation and membrane interaction of peptides. The specific position where the tryptophan should be incorporated could be determined in the helical wheel projection, by aids of structural information [27,28,29,30]. Three-dimensional structures of engineered peptides could provide useful information for the design of shorter and more potent AMPs. Taken together, all these structural studies contribute to a better understanding of the mechanism of action employed by AMPs, and fine structures of natural and synthetic peptides can be used as a scaffold to generate novel AMPs more suitable for pharmaceutical applications.

AMPs are fascinating targets as novel antibiotics because of their broad-spectrum activity, which include drug-resistant bacteria. Since the isolation of magainins from frog skin in 1987 [31], there have been many attempts to develop antibiotics from the natural AMPs. However, despite the efforts over more than two decades, there is no AMP agent currently approved by Food and Drug Administration (FDA) [4,32,33]. Although AMPs have considerable advantages for therapeutic applications, including broad-spectrum activity, rapid onset of activity, and relatively low possibility of resistance emergence, they also have some limitations for drug development. The natural AMPs are labile, depending on the surrounding environments, such as the presence of protease, pH change, and so on [34,35,36]. Other obstacles for the use of peptide antibiotics are the potential toxicity of AMPs for oral application and high cost of peptide production [2]. In general, many AMPs are considered to be less toxic to eukaryotes, but systematical toxicity of AMPs for oral application has not been evaluated. In order to overcome those obstacles, many methods have been proposed. For instances, introduction of unusual amino acids (mainly D-form amino acids) or modification of the terminal regions (acetylation or amidation) improved the stability of peptides by preserving them from proteolytic degradation [36,37]. Also, the use of efficient drug delivery systems, such as liposome encapsulation, can be effective for the improvement of the stability and reduction of potential toxicity [38,39]. Presumably, the practical obstacle may be the cost issue. Production costs are estimated to be roughly $50-$400 per 1 gram of amino acid when running commercial quantities [7]. The most certain solution for the high production cost would be the reduction of the peptide size with retention of the activity. Actually, there have been several successful examples of peptide engineering to reduce the peptide size with improved antimicrobial activity. As an example, the inactive, eleven-residue fragment of gaegurin 5 (GGN5^N11) could recover the antimicrobial activity by a single tryptophanyl substitution at the hydrophobic-hydrophilic interface of the amphipathic helix [29,30].

The serious problems caused by drug resistant bacteria have created an urgent need for the development of alternative therapeutics. In this respect, AMPs are considered as promising antimicrobial agents for producing new generation antibiotics. Additionally, atomic level structures of AMPs are prerequisite information for the generation of improved peptide antibiotic candidates. Although there are several obstacles to be overcome for clinical applications, natural and synthetic AMPs are still attractive sources to the pharmaceutical companies. In order to facilitate commercial development of peptide antibiotics, it is reasonable to focus on small peptides. Successful generation of short antimicrobial peptide molecules includes the A4W-GGN5^N11 [29,30], hLF1-11 [43,44,45,46,47,48,49,50,51], (CKPV)₂ [58,59,60,61,62], P-113 [66,67,71], and LKW [24,25,26,27] peptides. In terms of production cost, the use of those peptides would be advantageous in the pharmaceutical field.