The pharmacology of radiolabeled cationic antimicrobial peptides

Cationic antimicrobial peptides are good candidates for new diagnostics and antimicrobial agents. They can rapidly kill a broad range of microbes and have additional activities that have impact on the quality and effectiveness of innate responses and inflammation. Furthermore, the challenge of bacterial resistance to conventional antibiotics and the unique mode of action of antimicrobial peptides have made such peptides promising candidates for the development of a new class of antibiotics. This review focuses on antimicrobial peptides as a topic for molecular imaging, infection detection, treatment monitoring and additionally, displaying microbicidal activities. A scintigraphic approach to studying the pharmacokinetics of antimicrobial peptides in laboratory animals has been developed. The peptides were labeled with technetium-99m and, after intravenous injection into laboratory animals, scintigraphy allowed real-time, whole body imaging and quantitative biodistribution studies of delivery of the peptides to the various body compartments. Antimicrobial peptides rapidly accumulated at sites of infection but not at sites of sterile inflammation, indicating that radiolabeled cationic antimicrobial peptides could be used for the detection of infected sites. As the number of viable micro-organisms determines the rate of accumulation of these peptides, radiolabeled antimicrobial peptides enabled to determine the efficacy of antibacterial therapy in animals to be monitored as well to quantify the delivery of antimicrobial peptides to the site of infection. The scintigraphic approach provides to be a reliable method for investigating the pharmacokinetics of small cationic antimicrobial peptides in animals and offers perspective for diagnosis of infections, monitoring antimicrobial therapy, and most important, alternative antimicrobial treatment infections with multi-drug resistant micro-organisms in humans.     

Novel therapies based on cationic antimicrobial peptides

Cationic antimicrobial peptides serve as critical defense molecules protecting the host from invading bacteria, viruses and fungi. These antimicrobial peptides are widely distributed in nature and in vertebrates they have been localized to numerous tissues and cells. Cationic antimicrobial peptides can be expressed constitutively or under certain circumstances they can be induced in response to infection, inflammatory mediators, and cytokines. Although, their original and primary function was believed to be antimicrobial, it is now becoming clear that these antimicrobial peptides have a wide repertoire of functions with interesting ramifications on the immune system that are not solely antimicrobial. An area of active research is the determination of the mechanism(s) of action of antimicrobial peptides which have yet to be clarified. However, current consensus is that the mechanism is sufficiently different from conventional antibiotics that the development of resistance could be remote. Their broad spectrum activity, low potential to induce resistance and diverse functions make antimicrobial peptides an attractive family of compounds that have potential to be developed as therapeutics for treating certain infections.     

常见抗生素与新型抗菌药物在临床上的研究应用进展

摘要：抗生素的研发与使用有效杀灭和抑制了众多病原菌，挽救了无数人的生命，但由于抗生素的不合理使用，导致细菌耐 药不断出现，耐药菌的感染已严重地威胁人类的生命健康。为此，本文主要对近年来临床上常用的抗生素如β-内酰胺类、喹诺酮 类、氨基糖苷类药物以及新型抗菌药物如抗菌肽和纳米颗粒的应用研究进行总结分析与探讨，为临床上治疗疾病提供一些参考。     

Antimicrobial compounds of low molecular mass are constitutively present in insects: characterisation of beta-alanyl-tyrosine

The number of bacterial and fungal strains that have developed resistance against the classical antibiotics continues to grow. The intensified search for new antibiotic lead compounds has resulted in the discovery of numerous endogenous peptides with antimicrobial properties in plants, bacteria and animals. Their possible applications as anti-infective agents are often limited by their size, in reference to production costs and susceptibility to proteases. In this article, we report recent isolations of antimicrobial compounds from insects, with molecular masses less than 1 kDa. Experimental approaches are discussed and the first data on the antimicrobial properties of beta-alanyl-tyrosine (252 Da), one of such low molecular mass compounds isolated from the fleshfly Neobellieria bullata, are presented. We also offer evidence for the constitutive presence of antimicrobial compounds in insects of different orders, in addition to the previously identified inducible antimicrobial peptides.     

Designing antimicrobial peptides: form follows function

Multidrug-resistant bacteria are a severe threat to public health. Conventional antibiotics are becoming increasingly ineffective as a result of resistance, and it is imperative to find new antibacterial strategies. Natural antimicrobials, known as host defence peptides or antimicrobial peptides, defend host organisms against microbes but most have modest direct antibiotic activity. Enhanced variants have been developed using straightforward design and optimization strategies and are being tested clinically. Here, we describe advanced computer-assisted design strategies that address the difficult problem of relating primary sequence to peptide structure, and are delivering more potent, cost-effective, broad-spectrum peptides as potential next-generation antibiotics.     

Alignment-based design and synthesis of new antimicrobial Aurein-derived peptides with improved activity against Gram-negative bacteria and evaluation of their toxicity on human cells

Considering the worldwide increasing prevalence of resistance to traditional antibiotics, it is necessary to find new antibiotics to deal with this issue. Recently, antimicrobial peptides (AMPs) have been proposed as new antimicrobial agents. Aureins are a family of AMPs that are isolated from Green and Golden Bell Frogs. These peptides have a favorable antibacterial activity against Gram-positive bacteria. We designed two peptides derived from natural Aurein enjoying alignment-based design method. After synthesis of the peptides, their secondary structure was checked by circular dichroism. Consequently, the antibacterial effects of these peptides were investigated by determining the minimum inhibitory concentration (MIC) and bactericidal concentration. Eventually, the toxicity of these peptides was determined by MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assay on normal human skin cells (Hu02 cell line). Natural Aurein1.2 was used as a natural control to compare the properties in all stages. The results indicated that these new peptides had medium-upward antimicrobial activity against Escherichia coli, Pseudomonas aeruginosa, and Bacillus subtilis (MIC of 8–64 μg/mL) and weak bactericidal activity against Staphylococcus aureus (MIC of 128–256 μg/mL). Also, MTT assays results showed that AureinN2 is less toxic than AureinN1 and Aurein1.2. Toxicity of AureinN2 for Hu02 cell lines was between 20 and 40% at the concentration of 8–500 μg/mL. In this study, we were able to improve antimicrobial activity of two synthetic derivatives of the Aurein family against Gram-negative bacteria by using machine-learning algorithm and other in silico methods.     

Clinical development of cationic antimicrobial peptides: from natural to novel antibiotics

Over the past decade, levels of bacterial resistance to antibiotics have risen dramatically and "superbugs" resistant to most or all available agents have appeared in the clinic. Thus there is a growing need to discover and introduce new drugs. One potential source of novel antibiotics is the cationic antimicrobial peptides, which have been isolated from most living entities as components of their non-specific defenses against infectious organisms. Based on these natural templates, scores of structurally diverse antimicrobial cationic peptides have been designed, manufactured both chemically and biologically, and tested for activity against specific pathogens. A few of these peptide antibiotics have entered clinical trials to date, with mixed success. However, their diverse portfolio of structures, activity spectra, biological activities, and modes of action, provide substantial potential.     

Omiganan pentahydrochloride in the front line of clinical applications of antimicrobial peptides

Ribosomally synthesized antimicrobial peptides have very wide killing spectra and bacterial resistance to these peptides seems to be a rare phenomenon. Indolicidin is a ribosomally synthesized antimicrobial peptide that served as a template to omiganan, which is in development for the prevention of catheter-related bloodstream infections; clinical trials also proved its efficiency against acne vulgaris. Omiganan is the most advanced molecule in the front line of clinical applications of antimicrobial peptides. The mode and site of action of omiganan are not yet settled although its interaction with membranes is known to play a fundamental role. The biochemical and biophysical foundations for the action of indolicidin and its analogues are reviewed in this paper, as well as the clinical application of omiganan. The in vitro efficiency tests and the outcome of clinical trials are addressed. Altogether, despite the very specific use of omiganan as a topical antibiotic, it has the potential of being a pioneer of a new generation of antibiotics that carry the promise of ending the multi-resistance problem.     

Current state of a dual behaviour of antimicrobial peptides—Therapeutic agents and promising delivery vectors

Micro‐organism resistance is an important challenge in modern medicine due to the global uncontrolled use of antibiotics. Natural and synthetic antimicrobial peptides (AMPs) symbolize a new family of antibiotics, which have stimulated research and clinical interest as new therapeutic options for infections. They represent one of the most promising antimicrobial substances, due to their broad spectrum of biological activity, against bacteria, fungi, protozoa, viruses, yeast and even tumour cells. Besides, being antimicrobial, AMPs have been shown to bind and neutralize bacterial endotoxins, as well as possess immunomodulatory, anti‐inflammatory, wound‐healing, angiogenic and antitumour properties. In contrast to conventional antibiotics, which have very defined and specific molecular targets, host cationic peptides show varying, complex and very rapid mechanisms of actions that make it difficult to form an effective antimicrobial defence. Importantly, AMPs display their antimicrobial activity at micromolar concentrations or less. To do this, many peptide‐based drugs are commercially available for the treatment of numerous diseases, such as hepatitis C, myeloma, skin infections and diabetes. Herein, we present an overview of the general mechanism of AMPs action, along with recent developments regarding carriers of AMPs and their potential applications in medical fields.     

Mechanisms of antimicrobial peptide action and resistance

Antimicrobial peptides have been isolated and characterized from tissues and organisms representing virtually every kingdom and phylum, ranging from prokaryotes to humans. Yet, recurrent structural and functional themes in mechanisms of action and resistance are observed among peptides of widely diverse source and composition. Biochemical distinctions among the peptides themselves, target versus host cells, and the microenvironments in which these counterparts convene, likely provide for varying degrees of selective toxicity among diverse antimicrobial peptide types. Moreover, many antimicrobial peptides employ sophisticated and dynamic mechanisms of action to effect rapid and potent activities consistent with their likely roles in antimicrobial host defense. In balance, successful microbial pathogens have evolved multifaceted and effective countermeasures to avoid exposure to and subvert mechanisms of antimicrobial peptides. A clearer recognition of these opposing themes will significantly advance our understanding of how antimicrobial peptides function in defense against infection. Furthermore, this understanding may provide new models and strategies for developing novel antimicrobial agents, that may also augment immunity, restore potency or amplify the mechanisms of conventional antibiotics, and minimize antimicrobial resistance mechanisms among pathogens. From these perspectives, the intention of this review is to illustrate the contemporary structural and functional themes among mechanisms of antimicrobial peptide action and resistance.     

Targeted anti bacterial therapy

The increasing development of bacterial resistance to traditional antibiotics has reached alarming levels, thus necessitating a strong need to develop new antimicrobial agents. These new antimicrobials should possess novel modes of action and/or different cellular targets compared with the existing antibiotics. As a result, new classes of compounds designed to avoid defined resistance mechanisms are undergoing pre clinical and clinical evaluation. Microbial and phage genomic sequencing are now being used to find previously unidentified genes and their corresponding proteins. In both traditional and newly developed antibiotics, the target selectivity lies in the drug itself, in its ability to affect a mechanism that is unique to prokaryotes. As a result, a vast number of potent agents that, due to low selectivity, in addition to the pathogen also affect the eukaryote host have been excluded from use as therapeutics. Such compounds could be re-considered for clinical use if applied as part of a targeted delivery platform where the drug selectivity is replaced by target-selectivity borne by the targeting moiety. With a large number of antibodies and antibody-drug conjugates already approved or near approval as cancer therapeutics, targeted therapy is becoming increasingly attractive and additional potential targeting moieties that are non-antibody based, such as peptides, non-antibody ligand-binding proteins and even carbohydrates are receiving increasing attention. Still, targeted therapy is mostly focused on cancer, with targeted anti bacterial therapies being suggested only very recently. This review will focus in the various methods of antimicrobial targeting, by systemic and local application of targeted antimicrobial substances.     

肽类抗生素选择性作用机制研究进展与评价

肽类抗生素(peptide antibiotics)是一类广泛存在于生物体内具有抵抗外界微生物侵害、消除体内突变细胞的小分子多肽,即抗菌肽。肽类抗生素不仅具有高效广谱的抗微生物作用,还参与免疫应答、伤口愈合、细胞因子释放、白细胞趋化等反应过程,是一类广谱、安全、不易产生耐药菌的抗生素。不同抗菌肽对不同类型靶细胞表现出活性上的巨大差异,即抗菌肽作用的选择性,这种作用的选择性受到多种因素的影响。探讨抗菌肽作用于细胞的机制,特别是选择性作用的机制将有助于设计出活性更高的新型肽类抗生素。本文主要从细胞表面结构特异性、抗菌肽自身结构特点阐述近年来抗菌肽活性与选择性作用研究的新进展。     

组合化学方法合成1，3，5-三嗪类化合物及其抗菌活性研究

基于天然或合成抗菌肽具有疏水性及带正电荷的特性,本研究以1,3,5-三嗪作为母体,利用组合化学方法合成了一系列三嗪衍生物。抗菌试验证明这些衍生物具有很强的抗菌活性和低溶血性,并能在低于最低抑菌浓度的情况下有效抑制细菌生物膜的生长。     

Perspectives on synthetic pharmacotherapy for the treatment of nosocomial pneumonia

ABSTRACT

Introduction: Nosocomial pneumonia is the second most common infection in hospital settings, resulting in substantial increases in morbidity, mortality, and length of hospital stay. The rapid increase in resistance of nosocomial pathogens to many antibiotics and the high dissemination of resistance genes highlight the need for innovative approaches to combat difficult-to-treat nosocomial respiratory infections.

Areas covered: This review summarizes the synthetic antimicrobials that are currently in development for the treatment of nosocomial pneumonia, focusing on antibiotics in the final phases of clinical development and on the strategies employed by novel synthetic antimicrobial peptides.

Expert opinion: Several novel synthetic antimicrobials are currently in the pipeline, and it appears that new antimicrobial peptides or mimetics will soon be made available, expanding the opportunities to treat nosocomial pneumonia. However, the approval process for use in the treatment of nosocomial pneumonia is arduous. Given that significant investments by pharmaceutical companies have ended in failure to obtain the approval of regulatory agencies, novel platforms for antimicrobial discovery are needed. The identification of new and fully synthetic chemical structures with activity against nosocomial pathogens needs to be followed by preclinical studies in large animals and by pharmacokinetic and pharmacodynamic studies in specific critically ill populations to assess lung penetration.     

Overcoming multidrug resistance in gram-negative bacteria

The incidence of Gram-negative pathogens resistant to multiple antibiotics and multiple classes of antibiotics is increasing and the resultant deficit in effective therapeutic agents emphasizes the urgent need for novel agents and novel therapeutic approaches to the treatment of Gram-negative infectious disease. While developing versions of existing agents able to overcome resistance, or targeting resistance itself are strategies being considered to deal with multidrug resistance, genomic approaches will ultimately provide a multitude of novel targets for the development of new classes of agents likely to be unaffected by existing resistance mechanisms. The use of 'natural' antibacterials such as cationic antimicrobial peptides and bacteriophage as therapeutic agents is also being pursued. With an increased understanding of the infection process, immunomodulation and vaccinology are increasingly useful approaches to infectious disease management in the face of increasing antimicrobial resistance. Finally, there is a need to rigorously implement appropriate prescribing and infection control practices, to minimize the risk of resistance development and spread. Clearly, the antibiotic era has not heralded the defeat of infectious disease and prudent use of novel therapies is imperative if we are to avoid entering the post-antimicrobial era.     

Novel antimicrobial peptide CPF‐C1 analogs with superior stabilities and activities against multidrug‐resistant bacteria

As numerous clinical isolates are resistant to most conventional antibiotics, infections caused by multidrug‐resistant bacteria are associated with a higher death rate. Antimicrobial peptides show great potential as new antibiotics. However, a major obstacle to the development of these peptides as useful drugs is their low stability. To overcome the problem of the natural antimicrobial peptide CPF‐C1, we designed and synthesized a series of analogs. Our results indicated that by introducing lysine, which could increase the number of positive charges, and by introducing tryptophan, which could increase the hydrophobicity, we could improve the antimicrobial activity of the peptides against multidrug‐resistant strains. The introduction of d ‐amino acids significantly improved stability. Certain analogs demonstrated antibiofilm activities. In mechanistic studies, the analogs eradicated bacteria not just by interrupting the bacterial membranes, but also by linking to DNA, which was not impacted by known mechanisms of resistance. In a mouse model, certain analogs were able to significantly reduce the bacterial load. Among the analogs, CPF‐9 was notable due to its greater antimicrobial potency in vitro and in vivo and its superior stability, lower hemolytic activity, and higher antibiofilm activity. This analog is a potential antibiotic candidate for treating infections induced by multidrug‐resistant bacteria.     

Strategies for new antimicrobial proteins and peptides: lysozyme and aprotinin as model molecules

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.     

Design and characterization of short hybrid antimicrobial peptides from pEM‐2, mastoparan‐VT1, and mastoparan‐B

Antimicrobial peptides are considered to be excellent templates for designing novel antibiotics because of their broad‐spectrum antimicrobial activity and their low prognostic to induce antibiotic resistance. In this study, for the first time, a series of short hybrid antimicrobial peptides combined by different fragments of venom‐derived alpha‐helical antimicrobial peptides pEM‐2, mastoparan‐VT1, and mastoparan‐B were designed with the intent to improve the therapeutic index of the parental peptides. Short hybrid antimicrobial peptides PV, derived from pEM‐2 and mastoparan‐VT1, was found to possess the highest antibacterial, hemolytic, and cytotoxic activity. Short hybrid antimicrobial peptides PV3, derived from pEM‐2 and three fragments of mastoparan‐VT1, showed more than threefold improvement in therapeutic index compared with parental peptides pEM‐2 and mastoparan‐VT1. PV had the highest antimicrobial activity in stability studies. Except BVP, designed based on all three parental peptides, the other short hybrid antimicrobial peptides at their minimal inhibitory concentration and 2× minimal inhibitory concentration required less than 120 and 60 min to reduce >3log10 the initial inoculum, respectively. All peptides had membrane‐disrupting activity in a time‐dependent manner. Collectively, this study highlights the potential for rational design of improved short hybrid antimicrobial peptides such as PV3 that was an ideal candidate for further assessment with the ultimate purpose of development of effective antimicrobial agents.     

Rational design of α-helical antimicrobial peptides to target Gram-negative pathogens, Acinetobacter baumannii and Pseudomonas aeruginosa: utilization of charge, 'specificity determinants,' total hydrophobicity, hydrophobe type and location as design parameters to improve the therapeutic ratio

The rapidly growing problem of increased resistance to classical antibiotics makes the development of new classes of antimicrobial agents with lower rates of resistance urgent. Amphipathic cationic α‐helical antimicrobial peptides have been proposed as a potential new class of antimicrobial agents. The goal of this study was to take a broad‐spectrum, 26‐residue, antimicrobial peptide in the all‐D conformation, peptide D1 (K13) with excellent biologic properties and address the question of whether a rational design approach could be used to enhance the biologic properties if the focus was on Gram‐negative pathogens only. To test this hypothesis, we used 11 and 6 diverse strains of Acinetobacter baumannii and Pseudomonas aeruginosa, respectively. We optimized the number and location of positively charged residues on the polar face, the number, location, and type of hydrophobe on the non‐polar face and varied the number of ‘specificity determinants’ in the center of the non‐polar face from 1 to 2 to develop four new antimicrobial peptides. We demonstrated not only improvements in antimicrobial activity, but also dramatic reductions in hemolytic activity and unprecedented improvements in therapeutic indices. Compared to our original starting peptide D1 (V13), peptide D16 had a 746‐fold improvement in hemolytic activity (i.e. decrease), maintained antimicrobial activity, and improved the therapeutic indices by 1305‐fold and 895‐fold against A. baumannii and P. aeruginosa, respectively. The resulting therapeutic indices for D16 were 3355 and 895 for A. baumannii and P. aeruginosa, respectively. D16 is an ideal candidate for commercialization as a clinical therapeutic to treat Gram‐negative bacterial infections.     

Anti-infective treatment of bacterial urinary tract infections

Bacterial urinary tract infections (UTI) are frequently found in the outpatient as well as in the nosocomial setting. The bacterial UTI can be stratified into uncomplicated and complicated UTI. Antibiotic resistance is continuously increasing in uncomplicated as well as complicated UTI. In uncomplicated UTI efforts are made to use antibiotic substances exclusively for this indication. In complicated UTI as broad spectrum antibiotics are increasingly used, the higher the antimicrobial resistance rates are reported. There are two predominant aims in the antimicrobial treatment of both uncomplicated and complicated UTI: 1.) rapid and effective response to therapy, prevention of complications and prevention of recurrence in the individual patient treated, and 2.) prevention of emergence of resistance to anti-infective agents in the microbial environment. The use of antibiotics has to keep up with the continuous change in antimicrobial resistance and the tailored needs in the individual patient. Antibiotic substances therefore need to become evaluated for each indication and continuously followed for clinical usage. The knowledge of structure-activity relationships of antimicrobial substances and bacterial resistance mechanisms to antibiotics help to use antibiotics better in daily routine and design new derivatives and substances. The aim of this review is to describe the chemistry and structure-activity relationships of current antibiotics and promising substances in development for the treatment of UTI.