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。     

Functional analysis of two lebocin-related proteins from Manduca sexta

Insects produce a group of antimicrobial peptides (AMPs) in response to microbial infections. Most AMPs are synthesized as inactive precursors/pro-proteins and require proteolytic processing to generate small active peptides. Here we report identification and functional analysis of two lebocin-related proteins (Leb-B and Leb-C) from the tobacco hornworm, Manduca sexta. The mRNA levels of Leb-B and Leb-C increased significantly in larval fat body and hemocytes after injection of Escherichia coli, Micrococcus luteus and Saccharomyces cerevisiae. Western blotting using rabbit polyclonal antibody to Leb-B showed accumulation of large protein(s) and small peptide(s) in larval hemolymph after microbial injection. This result and the presence of RXXR motifs in the deduced amino acid sequences led to our postulation that Leb-B/C may be inactive precursors that are processed in larval hemolymph to generate short active peptides. To test this hypothesis, we expressed and purified full-length and various fragments of Leb-B and Leb-C as thioredoxin (TRX) fusion proteins. We found that fusion proteins could be cleaved by induced larval plasma, and the cleavage sites were determined by protein sequencing. Antibacterial activity of peptide fragments was also verified using synthetic peptides, and active M. sexta lebocin peptides were located at the N-termini of Leb-B/C, which are different from Bombyx mori lebocins 1-4 that are located close to the C-termini. In addition, we found that synthetic Leb-B(22-48) peptide not only had higher antibacterial activity but also caused agglutination of E. coli cells. Our results provide valuable information for studying processing of lebocin precursors in lepidopteran insects.     

EcDBS1R6: A novel cationic antimicrobial peptide derived from a signal peptide sequence

BackgroundBacterial infections represent a major worldwide health problem the antimicrobial peptides (AMPs) have been considered as potential alternative agents for treating these infections. Here we demonstrated the antimicrobial activity of EcDBS1R6, a peptide derived from a signal peptide sequence of Escherichia coli that we previously turned into an AMP by making changes through the Joker algorithm.MethodsAntimicrobial activity was measured by broth microdilution method. Membrane integrity was measured using fluorescent probes and through scanning electron microscopy imaging. A sliding window of truncated peptides was used to determine the EcDBS1R6 active core. Molecular dynamics in TFE/water environment was used to assess the EcDBS1R6 structure.ResultsSignal peptides are known to naturally interact with membranes; however, the modifications introduced by Joker transformed this peptide into a membrane-active agent capable of killing bacteria. The C-terminus was unable to fold into an α-helix whereas its fragments showed poor or no antimicrobial activity, suggesting that the EcDBS1R6 antibacterial core was located at the helical N-terminus, corresponding to the signal peptide portion of the parent peptide.ConclusionThe strategy of transforming signal peptides into AMPs appears to be promising and could be used to produce novel antimicrobial agents.General significanceThe process of transforming an inactive signal peptide into an antimicrobial peptide could open a new venue for creating new AMPs derived from signal peptides.     

Design and characterization of novel hybrid antimicrobial peptides based on cecropin A, LL-37 and magainin II

Antimicrobial peptides (AMPs) are a naturally occurring component of the innate immune response of many organisms and can have activity against both Gram-negative and Gram-positive bacterial species. In order to optimize and improve the direct antimicrobial effect of AMPs against a broad spectrum of bacterial species, novel synthetic hybrids were rationally designed from cecropin A, LL-37 and magainin II. AMPs were selected based on their α-helical secondary structure and fragments of these were analyzed and combined in silico to determine which hybrid peptides would form the best amphipathic cationic α-helices. Four hybrid peptides were synthesized (CaLL, CaMA, LLaMA and MALL) and evaluated for direct antimicrobial activity against a range of bacterial species (Bacillus anthracis, Burkholderia cepacia, Francisella tularensis LVS and Yersinia pseudotuberculosis) alongside the original 'parent' AMPs. The hybrid peptides showed greater antimicrobial effects than the parent AMPs (in one case a parent is completely ineffective while a hybrid based on it removes all traces of bacteria by 3h), although they also demonstrated higher hemolytic properties. Modifications were then carried out to the most toxic hybrid AMP (CaLL) to further improve the therapeutic index. Modifications made to the hybrid lowered hemolytic activity and also lowered antimicrobial activity by various degrees. Overall, this work highlights the potential for rational design and synthesis of improved AMPs that have the capability to be used therapeutically for treatment of bacterial infections.     

Non hemolytic short peptidomimetics as a new class of potent and broad-spectrum antimicrobial agents

Since the bacterial resistance to antibiotics is increasing rapidly, numerous studies have contributed to the design and synthesis of potent synthetic mimics of antimicrobial peptides (AMPs). In an attempt to find the pharmacophore of short antimicrobial peptidomimetics through systematic tuning of hydrophobic and hydrophilic patterns, we have identified a set of short histidine-derived antimicrobial peptides (SAMPs) with potent and broad-spectrum activity. A combination of high antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA), without hemolytic activity and proteolytic stability makes these molecules promising candidates for novel antimicrobial therapeutics.     

Stepwise identification of potent antimicrobial peptides from human genome

The increasing incidence of hospital acquired infections caused by antibiotic resistant pathogens has led to an increase in morbidity and mortality, finding alternative antibiotics unaffected by resistance mechanisms is fundamentally important for treating this problem. Naturally occurring proteins usually carry short peptide fragments that exhibit noticeable biological activity against a wide variety of microorganisms such as bacteria, fungi and protozoa. Traditional discovery of such antimicrobially active fragments (i.e. antimicrobial peptides, AMPs) from protein repertoire is either random or led by chance. Here, we report the use of a rational protocol that combines in silico prediction and in vitro assay to identify potential AMPs with high activity and low toxicity from the entire human genome. In the procedure, a three-step inference strategy is first proposed to perform genome-wide analysis to infer AMPs in a high-throughput manner. By employing this strategy we are able to screen more than one million peptide candidates generated from various human proteins, from which we identify four highly promising samples, and subsequently their antibacterial activity on five strains as well as cytotoxicity on human myoblasts are tested experimentally. As a consequence, two high-activity, low-toxicity peptides are discovered, which could be used as the structural basis to further develop new antibiotics. In addition, from 1491 known AMPs we also derive a quantitative measure called antibacterial propensity index (API) for 20 naturally occurring amino acids, which shows a significant allometric correlation with the theoretical minimal inhibitory concentration of putative peptides against Gram-positive and Gram-negative bacteria. This study may provide a proof-of-concept paradigm for the genome-wide discovery of novel antimicrobial peptides by using a combination of in silico and in vitro analyses.     

Knowledge-based computational methods for identifying or designing novel,non-homologous antimicrobial peptides

We describe computational approaches for identifying promising lead candidates for the development of peptide antibiotics, in the context of quantitative structure–activity relationships (QSAR) studies for this type of molecule. A first approach deals with predicting the selectivity properties of generated antimicrobial peptide sequences in terms of measured therapeutic indices (TI) for known antimicrobial peptides (AMPs). Based on a training set of anuran AMPs, the concept of sequence moments was used to construct algorithms that could predict TIs for a second test set of natural AMPs and could also predict the effect of point mutations on TI values. This approach was then used to design peptide antibiotics (adepantins) not homologous to known natural or synthetic AMPs. In a second approach, many novel putative AMPs were identified from DNA sequences in EST databases, using the observation that, as a rule, specific subclasses of highly conserved signal peptides are associated exclusively with AMPs. Both anuran and teleost sequences were used to elucidate this observation and its implications. The predicted therapeutic indices of identified sequences could then be used to identify new types of selective putative AMPs for future experimental verification.     

Designing the antimicrobial peptide with centrosymmetric and amphipathic characterizations for improving antimicrobial activity

Antibiotic-resistant bacterial infections are becoming a serious health issue and will cause 10 million deaths per year by 2050. As a result, the development of new antimicrobial agents is urgently needed. Antimicrobial peptides (AMPs) are found in the innate immune systems of various organisms to effectively fend off invading pathogens. In this study, we designed a series of AMPs (THL-2-1 to THL-2-9) with centrosymmetric and amphipathic properties, through substituting different amino acids on the hydrophobic side and at the centrosymmetric position to improve their antimicrobial activity. The results showed that leucine as a residue on the hydrophobic side of the peptide could enhance its antimicrobial activity and that glutamic acid as a centrosymmetric residue could increase the salt resistance of the peptide. Thus, the THL-2-3 peptide (KRLLRELKRLL-NH₂) showed the greatest antimicrobial activity (MIC₉₀ of 16 μM) against Gram-negative bacteria and had the highest salt resistance and cell selectivity among all the designed peptides. In summary, the results of this study provide useful references for the design of AMPs to enhance antimicrobial activity.     

Characterization of two novel antimicrobial peptides from the cuticular extracts of the ant Trichomyrmex criniceps (Mayr), (Hymenoptera: Formicidae)

Antimicrobial peptides (AMPs) from cuticular extracts of worker ants of Trichomyrmex criniceps (Mayr, Hymenoptera: Formicidae) were isolated and evaluated for their antimicrobial activity. Eight peptides ranging in mass from 804.42 to 1541.04 Da were characterized using a combination of analytical and bioinformatics approach. All the eight peptides were novel with no similarity to any of the AMPs archived in the Antimicrobial Peptide Database. Two of the eight novel peptides, the smallest and the largest by mass were named Crinicepsin‐1 and Crinicepsin‐2 and were chemically synthesized by solid phase peptide synthesis. The two synthetic peptides had antibacterial and weak hemolytic activity.     

Antimicrobial properties of membrane-active dodecapeptides derived from MSI-78

Antimicrobial peptides (AMPs) are a class of broad-spectrum antibiotics known by their ability to disrupt bacterial membranes and their low tendency to induce bacterial resistance, arising as excellent candidates to fight bacterial infections. In this study we aimed at designing short 12-mer AMPs, derived from a highly effective and broad spectrum synthetic AMP, MSI-78 (22 residues), by truncating this peptide at the N- and/or C-termini while spanning its entire sequence with 1 amino acid (aa) shifts. These designed peptides were evaluated regarding antimicrobial activity against selected gram-positive Staphylococcus strains and the gram-negative Pseudomonas aeruginosa (P. aeruginosa).The short 12-mer peptide CEM1 (GIGKFLKKAKKF) was identified as an excellent candidate to fight P. aeruginosa infections as it displays antimicrobial activity against this strain and selectivity, with negligible toxicity to mammalian cells even at high concentrations. However, in general most of the short 12-mer peptides tested showed a reduction in antimicrobial activity, an effect that was more pronounced for gram-positive Staphylococcus strains. Interestingly, CEM1 and a highly similar peptide differing by only one aa-shift (CEM2: IGKFLKKAKKFG), showed a remarkably contrasting AMP activity. These two peptides were chosen for a more detailed study regarding their mechanism of action, using several biophysical assays and simple membrane models that mimic the mammalian and bacterial lipid composition.We confirmed the correlation between peptide helicity and antimicrobial activity and propose a mechanism of action based on the disruption of the bacterial membrane permeability barrier.     

Role of cysteine residues and disulfide bonds in the activity of a legume root nodule-specific, cysteine-rich peptide

The root nodules of certain legumes including Medicago truncatula produce >300 different nodule-specific cysteine-rich (NCR) peptides. Medicago NCR antimicrobial peptides (AMPs) mediate the differentiation of the bacterium, Sinorhizobium meliloti into a nitrogen-fixing bacteroid within the legume root nodules. In vitro, NCR AMPs such as NCR247 induced bacteroid features and exhibited antimicrobial activity against S. meliloti. The bacterial BacA protein is critical to prevent S. meliloti from being hypersensitive toward NCR AMPs. NCR AMPs are cationic and have conserved cysteine residues, which form disulfide (S-S) bridges. However, the natural configuration of NCR AMP S-S bridges and the role of these in the activity of the peptide are unknown. In this study, we found that either cysteine replacements or S-S bond modifications influenced the activity of NCR247 against S. meliloti. Specifically, either substitution of cysteines for serines, changing the S-S bridges from cysteines 1-2, 3-4 to 1-3, 2-4 or oxidation of NCR247 lowered its activity against S. meliloti. We also determined that BacA specifically protected S. meliloti against oxidized NCR247. Due to the large number of different NCRs synthesized by legume root nodules and the importance of bacterial BacA proteins for prolonged host infections, these findings have important implications for analyzing the function of these novel peptides and the protective role of BacA in the bacterial response toward these peptides.     

The novel antimicrobial peptide PXL150 in the local treatment of skin and soft tissue infections

Dramatic increase in bacterial resistance towards conventional antibiotics emphasises the importance to identify novel, more potent antimicrobial therapies. Antimicrobial peptides (AMPs) have emerged as a promising new group to be evaluated in therapeutic intervention of infectious diseases. Here we describe a novel AMP, PXL150, which demonstrates in vitro a broad spectrum microbicidal action against both Gram-positive and Gram-negative bacteria, including resistant strains. The potent microbicidal activity and broad antibacterial spectrum of PXL150 were not associated with any hemolytic activity. Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) failed to develop resistance towards PXL150 during continued selection pressure. PXL150 caused a rapid depolarisation of cytoplasmic membrane of S. aureus, and dissipating membrane potential is likely one mechanism for PXL150 to kill its target bacteria. Studies in human cell lines indicated that PXL150 has anti-inflammatory properties, which might be of additional benefit. PXL150 demonstrated pronounced anti-infectious effect in an in vivo model of full thickness wounds infected with MRSA in rats and in an ex vivo model of pig skin infected with S. aureus. Subcutaneous or topical application of the peptide in rats did not lead to any adverse reactions. In conclusion, PXL150 may constitute a new therapeutic alternative for local treatment of infections, and further studies are warranted to evaluate the applicability of this AMP in clinical settings.     

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.     

Molecular cloning and functional characterization of novel antimicrobial peptides from the skin of brown frog, Rana zhenhaiensis

Rana zhenhaiensis, a species of brown frog, is widely distributed in central and south China. In the present study, a total of 14 cDNA sequences encoding eight novel antimicrobial peptides (AMPs) were cloned from the synthesized cDNAs of R. zhenhaiensis skin. The eight novel AMPs belong to four families: brevinin-1 (four peptides), brevinin-2 (one peptide), ranatuerin-2 (one peptide) and chensinin-1 (two peptides), five AMPs from the four families (brevinin-1ZHa, brevinin-1ZHb, brevinin-2ZHa, ranatuerin-2ZHa and chensinin-1ZHa) were chemically synthesized, their antimicrobial and hemolytic activities were examined. The results indicated that the five AMPs possess different antimicrobial and hemolytic activities. Of these, brevinin-2ZHa exhibited the strongest and most broad-spectrum antimicrobial activity. Furthermore, scanning electron microscopy (SEM) experiment was carried out to investigate the potential antimicrobial mechanism of chensinin-1ZHa. The result indicated that chensinin-1ZHa may exert its function through disruption of the bacterial membrane.     

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.     

Truncated antimicrobial peptides from marine organisms retain anticancer activity and antibacterial activity against multidrug-resistant Staphylococcus aureus

Antimicrobial peptides (AMPs) were recently determined to be potential candidates for treating drug-resistant bacterial infections. The aim of this study was to develop shorter AMP fragments that combine maximal bactericidal effect with minimal synthesis cost. We first synthesized a series of truncated forms of AMPs (anti-lipopolysaccharide factor from shrimp, epinecidin from grouper, and pardaxin from Pardachirus marmoratus). The minimum inhibitory concentrations (MICs) of modified AMPs against ten bacterial species were determined. We also examined the synergy between peptide and non-peptide antibiotics. In addition, we measured the inhibitory rate of cancer cells treated with AMPs by MTS assay. We found that two modified antibacterial peptides (epinecidin-8 and pardaxin-6) had a broad range of action against both gram-positive and gram-negative bacteria. Furthermore, epinecidin and pardaxin were demonstrated to have high antibacterial and anticancer activities, and both AMPs resulted in a significant synergistic improvement in the potencies of streptomycin and kanamycin against methicillin-resistant Staphylococcus aureus. Neither AMP induced significant hemolysis at their MICs. In addition, both AMPs inhibited human epithelial carcinoma (HeLa) and fibrosarcoma (HT-1080) cell growth. The functions of these truncated AMPs were similar to those of their full-length equivalents. In conclusion, we have successfully identified shorter, inexpensive fragments with maximal bactericidal activity. This study also provides an excellent basis for the investigation of potential synergies between peptide and non-peptide antibiotics, for a broad range of antimicrobial and anticancer activities.     

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.     

Antimicrobial properties of analgesic kyotorphin peptides unraveled through atomic force microscopy

Antimicrobial peptides (AMPs) are promising candidates as alternatives to conventional antibiotics for the treatment of resistant pathogens. In the last decades, new AMPs have been found from the cleavage of intact proteins with no antibacterial activity themselves. Bovine hemoglobin hydrolysis, for instance, results in AMPs and the minimal antimicrobial peptide sequence was defined as Tyr-Arg plus a positively charged amino acid residue. The Tyr-Arg dipeptide alone, known as kyotorphin (KTP), is an endogenous analgesic neuropeptide but has no antimicrobial activity itself. In previous studies new KTP derivatives combining C-terminal amidation and Ibuprofen (Ib) - KTP-NH(2), IbKTP, IbKTP-NH(2) - were designed in order to improve KTP brain targeting. Those modifications succeeded in enhancing peptide-cell membrane affinity towards fluid anionic lipids and higher analgesic activity after systemic injection resulted therefrom. Here, we investigated if this affinity for anionic lipid membranes also translates into antimicrobial activity because bacteria have anionic membranes. Atomic force microscopy revealed that KTP derivatives perturbed Staphylococcus aureus membrane structure by inducing membrane blebbing, disruption and lysis. In addition, these peptides bind to red blood cells but are non-hemolytic. From the KTP derivatives tested, amidated KTP proves to be the most active antibacterial agent. The combination of analgesia and antibacterial activities with absence of toxicity is highly appealing from the clinical point of view and broadens the therapeutic potential and application of kyotorphin peptides.     

Bi-functional peptides with both trypsin-inhibitory and antimicrobial activities are frequent defensive molecules in Ranidae amphibian skins

Amphibian skins act as the first line against noxious aggression by microorganisms, parasites, and predators. Anti-microorganism activity is an important task of amphibian skins. A large amount of gene-encoded antimicrobial peptides (AMPs) has been identified from amphibian skins. Only a few of small protease inhibitors have been found in amphibian skins. From skin secretions of 5 species (Odorrana livida, Hylarana nigrovittata, Limnonectes kuhlii, Odorrana grahami, and Amolops loloensis) of Ranidae frogs, 16 small serine protease inhibitor peptides have been purified and characterized. They have lengths of 17-20 amino acid residues (aa). All of them are encoded by precursors with length of 65-70 aa. These small peptides show strong trypsin-inhibitory abilities. Some of them can exert antimicrobial activities. They share the conserved GCWTKSXXPKPC fragment in their primary structures, suggesting they belong to the same families of peptide. Signal peptides of precursors encoding these serine protease inhibitors share obvious sequence similarity with those of precursors encoding AMPs from Ranidae frogs. The current results suggest that these small serine protease inhibitors are the common defensive compounds in frog skin of Ranidae as amphibian skin AMPs.