The therapeutic applications of antimicrobial peptides (AMPs): a patent review

Antimicrobial peptides (AMPs) are small molecules with a broad spectrum of antibiotic activities against bacteria, yeasts, fungi, and viruses and cytotoxic activity on cancer cells, in addition to anti-inflammatory and immunomodulatory activities. Therefore, AMPs have garnered interest as novel therapeutic agents. Because of the rapid increase in drug-resistant pathogenic microorganisms, AMPs from synthetic and natural sources have been developed using alternative antimicrobial strategies. This article presents a broad analysis of patents referring to the therapeutic applications of AMPs since 2009. The review focuses on the universal trends in the effective design, mechanism, and biological evolution of AMPs.     

Bacterial resistance to antimicrobial peptides

Antimicrobial peptides (AMPs) or host defense peptides (HDPs) are vital components of human innate defense system targeting human‐related bacteria. Many bacteria have various mechanisms interfering with AMP activity, causing resistance to AMPs. Since AMPs are considered as potential novel antimicrobial drugs, understanding the mechanisms of bacterial resistance to direct killing of AMPs is of great significance. In this review, a comparative overview of bacterial strategies for resistance to direct killing of various AMPs is presented. Such strategies include bacterial cell envelope modification, AMP degradation, sequestration, expelling, and capsule.     

LAMP: A Database Linking Antimicrobial Peptides

The frequent emergence of drug-resistant bacteria has created an urgent demand for new antimicrobial agents. Traditional methods of novel antibiotic development are almost obsolete. Antimicrobial peptides (AMPs) are now regarded as a potential solution to revive the traditional methods of antibiotic development, although, until now, many AMPs have failed in clinical trials. A comprehensive database of AMPs with information about their antimicrobial activity and cytotoxicity will help promote the process of finding novel AMPs with improved antimicrobial activity and reduced cytotoxicity and eventually accelerate the speed of translating the discovery of new AMPs into clinical or preclinical trials. LAMP, a database linking AMPs, serves as a tool to aid the discovery and design of AMPs as new antimicrobial agents. The current version of LAMP has 5,547 entries, comprising 3,904 natural AMPs and 1,643 synthetic peptides. The database can be queried using either simply keywords or combinatorial conditions searches. Equipped with the detailed antimicrobial activity and cytotoxicity data, the cross-linking and top similar AMPs functions implemented in LAMP will help enhance our current understanding of AMPs and this may speed up the development of new AMPs for medical applications. LAMP is freely available at: http://biotechlab.fudan.edu.cn/database/lamp.     

Display of Multimeric Antimicrobial Peptides on the Escherichia coli Cell Surface and Its Application as Whole-Cell Antibiotics

Concerns over the increasing emergence of antibiotic-resistant pathogenic microorganisms due to the overuse of antibiotics and the lack of effective antibiotics for livestock have prompted efforts to develop alternatives to conventional antibiotics. Antimicrobial peptides (AMPs) with a broad-spectrum activity and rapid killing, along with little opportunity for the development of resistance, represent one of the promising novel alternatives. Their high production cost and cytotoxicity, however, limit the use of AMPs as effective antibiotic agents to livestock. To overcome these problems, we developed potent antimicrobial Escherichia coli displaying multimeric AMPs on the cell surface so that the AMP multimers can be converted into active AMP monomers by the pepsin in the stomach of livestock. Buf IIIb, a strong AMP without cytotoxicity, was expressed on the surface of E. coli as Lpp-OmpA-fused tandem multimers with a pepsin substrate residue, leucine, at the C-terminus of each monomer. The AMP multimers were successfully converted into active AMPs upon pepsin cleavage, and the liberated Buf IIIb-L monomers inhibited the growth of two major oral infectious pathogens of livestock, Salmonella enteritidis and Listeria monocytogenes. Live antimicrobial microorganisms developed in this study may represent the most effective means of providing potent AMPs to livestock, and have a great impact on controlling over pathogenic microorganisms in the livestock production.     

Antimicrobial peptides with cell-penetrating peptide properties and vice versa

Antimicrobial peptides (AMPs) are a group of peptides that are active against a diverse spectrum of microorganisms. Due to their mode of action, AMPs are a promising class of molecules that could overcome the problems of increasing resistance of bacteria to conventional antibiotics. Furthermore, AMPs are strongly membrane-active and some are able to translocate into cells without the necessity for permanent membrane permeabilization. This feature has brought them into focus for use as transport vectors in the context of drug delivery. Since the plasma membrane restricts transport of bioactive substances into cells, great research interest lies in the development of innovative ways to overcome this barrier and to increase bioavailability. In this context, peptide-based transport systems, such as cell-penetrating peptides (CPPs), have come into focus, and their efficiency has been demonstrated in many different applications. However, more recently, also some AMPs have been used as efficient vectors for intracellular translocation of various active molecules. This review summarizes recent efforts in this interesting field of drug delivery. Moreover, some examples of the application of CPPs as efficient antimicrobial substances will be discussed.     

综述人工合成型抗菌肽及其药学应用研究进展

天然抗菌肽（antimicrobial peptides, AMPs）是一类小分子阳离子多肽,具备多种杀菌机制,呈现出高效、广谱的杀菌特性,在抑制耐药性细菌、制备新型抗菌素等方面具有重要的研究价值。以天然抗菌肽为蓝本,设计和开发的人工合成型抗菌肽可以有效克服天然抗菌肽对蛋白酶敏感、细胞毒性较大、生产成本高等缺陷,作为抗感染的潜在药物具有更广阔的应用前景。综述了目前主要的抗菌肽人工改造技术,包括化学修饰法、蛋白质工程技术、计算机分子模拟技术和从头设计最小化抗菌肽方法的研究进展,并对人工合成抗菌肽作为抗菌药物的应用现状进行了简介。     

Antimicrobial peptides: Possible anti-infective agents

Multidrug-resistant bacterial, fungal, viral, and parasitic infections are major health threats. The Infectious Diseases Society of America has expressed concern on the decrease of pharmaceutical companies working on antibiotic research and development. However, small companies, along with academic research institutes, are stepping forward to develop novel therapeutic methods to overcome the present healthcare situation. Among the leading alternatives to current drugs are antimicrobial peptides (AMPs), which are abundantly distributed in nature. AMPs exhibit broad-spectrum activity against a wide variety of bacteria, fungi, viruses, and parasites, and even cancerous cells. They also show potential immunomodulatory properties, and are highly responsive to infectious agents and innate immuno-stimulatory molecules. In recent years, many AMPs have undergone or are undergoing clinical development, and a few are commercially available for topical and other applications. In this review, we outline selected anion and cationic AMPs which are at various stages of development, from preliminary analysis to clinical drug development. Moreover, we also consider current production methods and delivery tools for AMPs, which must be improved for the effective use of these agents.     

The expanding scope of antimicrobial peptide structures and their modes of action

Antimicrobial peptides (AMPs) are an integral part of the innate immune system that protect a host from invading pathogenic bacteria. To help overcome the problem of antimicrobial resistance, cationic AMPs are currently being considered as potential alternatives for antibiotics. Although extremely variable in length, amino acid composition and secondary structure, all peptides can adopt a distinct membrane-bound amphipathic conformation. Recent studies demonstrate that they achieve their antimicrobial activity by disrupting various key cellular processes. Some peptides can even use multiple mechanisms. Moreover, several intact proteins or protein fragments are now being shown to have inherent antimicrobial activity. A better understanding of the structure-activity relationships of AMPs is required to facilitate the rational design of novel antimicrobial agents.     

Evaluation of antibacterial activity induced by Staphylococcus aureus and Ent A in the hemolymph of Spodoptera littoralis

The problem of antibiotic resistance considers one of the most dangerous challenges facing the medical field. So, it is necessary to find substitutions to conventional antibiotics. Antimicrobial peptides (AMPs) are a bio-functional derivative that have been observed as one of the important solutions to such upcoming crisis. Owing to their role as the first line of defense against bacteria, fungi, and viruses. This study was conducted to induce the immune response of Spodoptera littoralis larvae by inoculation of sub lethal doses of Staphylococcus aureus and its enterotoxin. Since Staphylococcal enterotoxin A (SEA) considers the major causative agents of Staphylococcal food poisoning, our study oriented to purify and characterize this toxin to provoke its role in yielding AMPs with broad spectrum antimicrobial activity. A great fluctuation was recorded in the biochemical properties of immunized hemolymph not only in the total protein content but also protein banding pattern. Protein bands of ∼22 kDa (attacin-like) and ∼15 kDa (lysozyme-like) were found to be common between the AMPs induced as a result of both treatments. While protein bands of molecular weight ∼70 kDa (phenoloxidase-like) and ∼14 kDa (gloverin-like) were found specific for SEA treatment. Chromatographic analysis using HPLC for the induced AMPs showed different types of amino acids appeared with differences in their quantities and velocities. These peptides exhibited noticeable antimicrobial activity against certain Gram-positive and Gram-negative bacteria. In conclusion, the antimicrobial potential of the antimicrobial peptides (AMP) induced in the larval hemolymph of S. littoralis will be a promising molecule for the development of new therapeutic alternatives.     

Identification and characterization of antimicrobial peptides from skin of Amolops ricketti (Anura: Ranidae)

As one of large amphibian group, there are a total of 45 species of Amolops in the world. However, the antimicrobial peptides (AMPs) existing in this genus has not been extensively studied. In this study, cDNAs encoding five novel AMP precursors were cloned by screening the skin-derived cDNA library of Amolops ricketti, a frog species that exists in southern and western parts of China. Protein sequence analysis led to the identification of five deduced peptides, three belonging to the brevinin-1 family and two belonging to the brevinin-2 family of amphibian AMPs. Thus, they were named as brevinin-1RTa (FLPLLAGVVANFLPQIICKIARKC), brevinin-1RTb (FLGSLLGLVGKVVPTLFCKISKKC), brevinin-1RTc (FLGSLLGLVGKIVPTLICKISKKC), brevinin-2RTa (GLMSTLKDFGKTAAKEIAQSLLSTASCKLAKTC), and brevinin-2RTb (GILDTLKEFGKTAAKGIAQSLLSTASCKLAKTC), respectively. The purification of brevinin-1RTa, brevinin-1RTb, and brevinin-2RTb was carried out by RP-HPLC, and confirmed by the LC-MS/MS-based proteomics approach. All of the peptides displayed different antimicrobial potency against a variety of microorganisms. In addition, brevinin-2RTa and brevinin-2RTb were found to have relatively low hemolytic activity (>400μg/ml) against mammalian red blood cells in vitro, which could potentially be as candidates for developing novel anti-infection agents.     

Sense the moment: A highly sensitive antimicrobial activity predictor based on hydrophobic moment

BackgroundComputer-aided identification and design tools are indispensable for developing antimicrobial agents for controlling antibiotic-resistant bacteria. Antimicrobial peptides (AMPs) have aroused intense interest, since they have a broad spectrum of activity, and therefore, several systems for predicting antimicrobial peptides have been developed, using scalar physicochemical properties; however, regardless of the machine learning algorithm, these systems often fail in discriminating AMPs from their shuffled versions, leading to the need for new training methods to overcome this bias. Aiming to solve this bias, here we present “Sense the Moment”, a prediction system capable of discriminating AMPs and shuffled versions.MethodsThe system was trained using 776 entries: 388 from known AMPs and another 388 based on shuffled versions of known AMPs. Each entry contained the geometric average of three hydrophobic moments measured with different scales.ResultsThe model showed good accuracy (>80%) and excellent sensitivity (>90%) for AMP prediction, exceeding deep-learning-based methods.ConclusionOur results demonstrate the system's applicability, aiding in identifying and discarding non-AMPs, since the number of false negatives is lower than false positives.General significanceThe application of this model in virtual screening protocols for identifying and/or creating antimicrobial agents could aid in the identification of potential drugs to control pathogenic microorganisms and in solving the antibiotic resistance crisis.AvailabilityThe system was implemented as a web application, available at <http://portoreports.com/stm/>.     

病原微生物对抗菌肽抗性机制的研究进展

抗菌肽(antimicrobial peptides, AMPs)是生物先天免疫系统的重要组成部分,可帮助宿主有效应对病原细菌、真菌和病毒等微生物的胁迫,被认为是医疗、食品加工和农业领域最具前途和潜力的抗生素替代物。病原微生物在与抗菌肽的互作中进化出了多种有针对性的抗性机制,本文从病原微生物对AMPs的感应与基因调控、细胞壁/膜成份的修饰、分泌蛋白酶降解及利用外排泵排出等四个方面综述了国内外的研究进展,并对AMPs类制品的研究前景进行了讨论与展望。     

Multivalent Antimicrobial Peptides as Therapeutics: Design Principles and Structural Diversities

This review highlights the design principles, progress and advantages attributed to the structural diversity associated with both natural and synthetic multivalent antimicrobial peptides (AMPs). Natural homo- or hetero-dimers of AMPs linked by intermolecular disulfide bonds existed in the animal kingdom, but the multivalency strategy has been adopted to create synthetic branched or polymeric AMPs that do not exist in nature. The multivalent strategy for the design of multivalent AMPs provides advantages to overcome the challenges faced in clinical applications of AMPs, such as: stability, efficiency, toxicity, maintenance of activity in high salt concentrations and under physiological conditions, and importantly overcoming bacterial resistance which is currently a leading health problem in the world. The multivalency strategy is valuable for moving multivalent AMPs toward clinical applications.     

The role of natural antimicrobial peptides during infection and chronic inflammation

Natural antimicrobial peptides (AMPs), a family of small polypeptides that are produced by constitutive or inducible expression in organisms, are integral components of the host innate immune system. In addition to their broad-spectrum antibacterial activity, natural AMPs also have many biological activities against fungi, viruses and parasites. Natural AMPs exert multiple immunomodulatory roles that may predominate under physiological conditions where they lose their microbicidal properties in serum and tissue environments. Increased drug resistance among microorganisms is occurring far more quickly than the discovery of new antibiotics. Natural AMPs have shown promise as ‘next generation antibiotics’ due to their broad-spectrum curative effects, low toxicity, the fact that they are not residual in animals, and the low rates of resistance exhibited by many pathogens. Many types of synthetic AMPs are currently being tested in clinical trials for the prevention and treatment of various diseases such as chemotherapy-associated infections, diabetic foot ulcers, catheter-related infections, and other conditions. Here, we provide an overview of the types and functions of natural AMPs and their role in combating microorganisms and different infectious and inflammatory diseases.     

Antimicrobial peptides (AMPs): peptide structure and mode of action

Antimicrobial peptides (AMPs) have been isolated and characterized from tissues and organisms representing virtually every kingdom and phylum. Their amino acid composition, amphipathicity, cationic charge, and size allow them to attach to and insert into membrane bilayers to form pores by 'barrel-stave', 'carpet' or 'toroidal-pore' mechanisms. Although these models are helpful for defining mechanisms of AMP activity, their relevance to resolving how peptides damage and kill microorganisms still needs to be clarified. Moreover, many AMPs employ sophisticated and dynamic mechanisms of action to carry out their likely roles in antimicrobial host defense. Recently, it has been speculated that transmembrane pore formation is not the only mechanism of microbial killing by AMPs. In fact, several observations suggest that translocated AMPs can alter cytoplasmic membrane septum formation, reduce cell-wall, nucleic acid, and protein synthesis, and inhibit enzymatic activity. In this review, we present the structures of several AMPs as well as models of how AMPs induce pore formation. AMPs have received special attention as a possible alternative way to combat antibiotic-resistant bacterial strains. It may be possible to design synthetic AMPs with enhanced activity for microbial cells, especially those with antibiotic resistance, as well as synergistic effects with conventional antibiotic agents that lack cytotoxic or hemolytic activity.     

Extremely abundant antimicrobial peptides existed in the skins of nine kinds of Chinese odorous frogs

Peptide agents are regarded as hopeful candidates to solve life-threatening resistance of pathogenic microorganisms to classic antibiotics due to their unique action mechanisms. Peptidomic and genomic investigation of natural antimicrobial peptides (AMPs) from amphibian skin secretions can provide a large amount of structure-functional information to design peptide antibiotics with therapeutic potential. In the present study, we identified a large number of AMPs from the skins of nine kinds of Chinese odorous frogs. Eighty AMPs were purified from three different odorous frogs and confirmed by peptidomic analysis. Our results indicated that post-translational modification of AMPs rarely happened in odorous frogs. cDNAs encoding precursors of 728 AMPs, including all the precursors of the confirmed 80 native peptides, were cloned from the constructed AMP cDNA libraries of nine Chinese odorous frogs. On the basis of the sequence similarity of deduced mature peptides, these 728 AMPs were grouped into 97 different families in which 71 novel families were identified. Out of these 728 AMPs, 662 AMPs were novel and 28 AMPs were reported previously in other frog species. Our results revealed that identical AMPs were widely distributed in odorous frogs; 49 presently identified AMPs could find their identical molecules in different amphibian species. Purified peptides showed strong antimicrobial activities against 4 tested microbe strains. Twenty-three deduced peptides were synthesized and their bioactivities, including antimicrobial, antioxidant, hemolytic, immunomodulatory and insulin-releasing activities, were evaluated. Our findings demonstrate the extreme diversity of AMPs in amphibian skins and provide plenty of templates to develop novel peptide antibiotics.     

Molecular mechanisms of antitumor effect of natural antimicrobial peptides

In spite of all the advances in cancer treatment made in recent years, one of the main problems in this field that remains extremely urgent is the development of drug resistance to the chemotherapeutic agents currently in use due to clonal microevolution of tumor tissue. Numerous publications devoted to the study of cationic antimicrobial peptides (AMPs) as molecular factors of the innate immune system suggest that these compounds possess significant therapeutic potential and can be considered as candidates for the role of not only antimicrobial, but also next-generation anticancer drugs. AMPs are characterized by a variety of mechanisms of cytotoxic action that can lead to either necrosis or apoptosis of the target cells. These effects are based on the selective interaction with the membranes of tumor cells, which have a number of similarities, in structural and physiological aspects, with the microbial membranes. AMPs were found to be able to inhibit tumor growth by interrupting the formation of its vascular network. The antitumor effect of AMPs may also be enhanced by the modulation of host immune system, as previously observed for their antimicrobial effects. The described properties of AMPs give hope for the development of new drugs that will be able to overcome the resistance of tumor cells.     

Studies on anticancer activities of antimicrobial peptides

In spite of great advances in cancer therapy, there is considerable current interest in developing anticancer agents with a new mode of action because of the development of resistance by cancer cells towards current anticancer drugs. A growing number of studies have shown that some of the cationic antimicrobial peptides (AMPs), which are toxic to bacteria but not to normal mammalian cells, exhibit a broad spectrum of cytotoxic activity against cancer cells. Such studies have considerably enhanced the significance of AMPs, both synthetic and from natural sources, which have been of importance both for an increased understanding of the immune system and for their potential as clinical antibiotics. The electrostatic attraction between the negatively charged components of bacterial and cancer cells and the positively charged AMPs is believed to play a major role in the strong binding and selective disruption of bacterial and cancer cell membranes, respectively. However, it is unclear why some host defense peptides are able to kill cancer cells when others do not. In addition, it is not clear whether the molecular mechanism(s) underlying the antibacterial and anticancer activities of AMPs are the same or different. In this article, we review various studies on different AMPs that exhibit cytotoxic activity against cancer cells. The suitability of cancer cell-targeting AMPs as cancer therapeutics is also discussed.     

Studies on anticancer activities of antimicrobial peptides

In spite of great advances in cancer therapy, there is considerable current interest in developing anticancer agents with a new mode of action because of the development of resistance by cancer cells towards current anticancer drugs. A growing number of studies have shown that some of the cationic antimicrobial peptides (AMPs), which are toxic to bacteria but not to normal mammalian cells, exhibit a broad spectrum of cytotoxic activity against cancer cells. Such studies have considerably enhanced the significance of AMPs, both synthetic and from natural sources, which have been of importance both for an increased understanding of the immune system and for their potential as clinical antibiotics. The electrostatic attraction between the negatively charged components of bacterial and cancer cells and the positively charged AMPs is believed to play a major role in the strong binding and selective disruption of bacterial and cancer cell membranes, respectively. However, it is unclear why some host defense peptides are able to kill cancer cells when others do not. In addition, it is not clear whether the molecular mechanism(s) underlying the antibacterial and anticancer activities of AMPs are the same or different. In this article, we review various studies on different AMPs that exhibit cytotoxic activity against cancer cells. The suitability of cancer cell-targeting AMPs as cancer therapeutics is also discussed.