肽类抗生素对细胞选择性作用研究进展

抗菌肽是一种广泛存在于生物界的抵抗病原微生物入侵的重要防御分子。抗菌肽具有广谱抗细菌、真菌、肿瘤细胞等活性，具有自身随着物种适应环境而产生的结构多样性以及具有区别于传统抗生素的杀菌机理的独特性，是一类极有潜力的肽类抗生素。不同抗菌肽对不同类型靶细胞表现出活性上的巨大差异，即是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。探讨抗菌肽作用细胞的机理，特别是选择性作用的机理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。     

Synthetic peptides derived from the β2−β3 loop of Raphanus sativus antifungal protein 2 that mimic the active site

Abstract: Rs‐AFPs are antifungal proteins, isolated from radish (Raphanus sativus) seed or leaves, which consist of 50 or 51 amino acids and belong to the plant defensin family of proteins. Four highly homologous Rs‐AFPs have been isolated (Rs‐AFP1–4). The structure of Rs‐AFP1 consists of three β‐strands and an α‐helix, and is stabilized by four cystine bridges. Small peptides deduced from the native sequence, still having biological activity, are not only important tools to study structure?function relationships, but may also constitute a commercially interesting target. In an earlier study, we showed that the antifungal activity of Rs‐AFP2 is concentrated mainly in the β2?β3 loop. In this study, we synthesized linear 19‐mer peptides, spanning the entire β2?β3 loop, that were found to be almost as potent as Rs‐AFP2. Cysteines, highly conserved in the native protein, are essential for maintaining the secondary structure of the protein. Surprisingly, in the 19‐mer loop peptides, cysteines can be replaced by α‐aminobutyric acid, which even improves the antifungal potency of the peptides. Analogous cyclic 19‐mer peptides, forced to adopt a hairpin structure by the introduction of one or two non‐native disulfide bridges, were also found to possess high antifungal activity. The synthetic 19‐mer peptides, like Rs‐AFP2 itself, cause increased Ca²⁺ influx in pregerminated fungal hyphae.     

Design and synthesis of amphipathic antimicrobial peptides

A large proportion of antimicrobial peptides share a common structural feature that is critical to their antimicrobial activity, i.e. amphipathic α-helices. The amphipathy of a polypeptide chain can be quantitated through the value of the hydrophobic moment. Generally, antimicrobial peptides are characterized by high hydrophobic moment and low hydrophobicity values. Using these criteria we have identified two short segments that possess hydrophobic moment properties associated with known antimicrobial peptides. Using in vitro assays the segment derived from the protein perforin displays no antifungal or antibacterial activity and, while showing no α-helicity in buffer or liposomes, exhibits a modest degree of α-helical structure in the presence of the a-helical inducer, 2,2,2-trifluoroethanol. However, rational modifications result in a derivative which assumes an α-helical conformation in the presence of liposomes, exhibits potent antifungal activity against plant fungal pathogens, has significant antibacterial activity, effects leakage of a fluorescent dye from acidic liposomes and is devoid of hemolytic activity. Results are also presented for a segment derived from the human immunodeficiency virus envelope protein. We suggest that the identification of putative amphipathic structures in proteins may provide a useful starting strategy in the design and synthesis of antimicrobial peptides.     

Antifungal activity of human salivary mucin‐derived peptide,MUC7 12‐mer,in a murine model of oral candidiasis

Abstract: Previous studies have shown that peptides derived from the N‐terminal region of the low molecular mass human salivary mucin, MUC7, possess potent in vitro cidal activity against Candida albicans and other medically important fungi. MUC7 12‐mer (residues 40–51 of the parent MUC7) peptide, having the optimal size and a net charge of +6, was found to be anticandidal in human saliva (clarified and unclarified), and its candidacidal potency was found to be superior to that of histatin 5 12‐mer (Hsn5 12‐mer). We have, therefore, explored the candidacidal potency of MUC7 12‐mer (l and d isomers) and Hsn5 12‐mer peptides in vivo. In vitro killing assay was performed to establish killing activity of the peptides against C. albicans prior to in vivo experiments. A murine model of oral candidiasis that has the characteristics of oral thrush in humans was employed for the in vivo studies, based on a previous protocol. Upon candidal induction, antifungal treatment application using agents emulsified in Pluronic F127 was performed for six consecutive days. Amphotericin B and clotrimazole emulsified in the same delivery system were used as positive control drugs. Candidacidal efficacy was evaluated microbiologically and histopathologically. Results demonstrated a considerable reduction of fungal burden by the MUC7 12‐mer peptides (l and d ), comparable to control drugs, and this effect was statistically significant, unlike the effect seen with Hsn5 12‐mer. Murine oral candidiasis model employed in this study is suitable to test the candidacidal agents employing Pluronic F127. In conclusion, MUC7 12‐mer appears to be a promising candidate as an antifungal agent for oral candidiasis.     

Structure-activity relationship study: short antimicrobial peptides

Many short antimicrobial peptides (< 18mer) have been identified for the development of therapeutic agents. However, Structure-activity relationship (SAR) studies about short antimicrobial peptides have not been extensively performed. To investigate the relationship between activity and structural parameters such as an α-helical structure, a net positive charge and a hydrophobicity, we synthesized and characterized diastereomers, scramble peptides and substituted peptides of the short antimicrobial peptide identified by combinatorial libraries. Circular dichroism (CD) spectra and in vitro activity indicated that an α-helical structure correlated with the antimicrobial activity and a β-sheet structure also satisfied a structural requirement for antimicrobial activity. Most peptides consisting of l -amino acids lost antifungal activity in the presence of heat-inactivated serum, while active diastereomers and a scramble peptide with the β-sheet structure retained antifungal activity in the same condition.     

Cathelicidin peptide SMAP‐29: comprehensive review of its properties and potential as a novel class of antibiotics

Sheep myeloid antimicrobial peptide of 29 amino acids (SMAP‐29) is one of the most potent antimicrobial peptides known, with a broad spectrum of activity against bacteria, fungi, and viruses. It has also been shown to prevent infections in animals. SMAP‐29 is an α‐helical cathelicidin that acts rapidly to permeabilize membranes of susceptible organisms. However, it is also cytotoxic and hemolytic to mammalian cells. In this review, all published data on inhibition constants and hemolytic activities of SMAP‐29 are brought together for the first time, including data for the peptide of 28 residues that lacks the C‐terminal glycine and that also has the new C‐terminal residue amidated. Both peptides have been called SMAP‐29 in the literature. Antimicrobial activity of SMAP‐29 variants (including ovispirins) against Gram‐positive bacteria is also comprehensively tabulated, and anomalies in the nomenclature of the ovispirins are highlighted. The secondary structure, mode of action, and structure–activity relationships of SMAP‐29 are outlined, and the properties that indicate its therapeutic potential are identified; these are its broad‐spectrum antimicrobial activity with high potency, rapid biocidal action, limited development of bacterial resistance, synthesis by recombinant technology, ability to bind lipopolysaccharide, and demonstrated protection against infection in animal models. The limitations of SMAP‐29 are its high cytotoxicity and hemolytic activity, reduced activity under physiological conditions, and limited evidence for synergistic effects in combination with conventional antibiotics. Strategies with the potential to improve the selectivity toward pathogenic microorganisms over mammalian cells have been devised by the authors and are outlined. Drug Dev Res 70: 481–498, 2009. © 2009 Commonwealth of Australia; Published 2009 Wiley‐Liss, Inc.     

Isolation,functional characterization,and biological properties of MCh‐AMP1, a novel antifungal peptide from Matricaria chamomilla L.

The antimicrobial activities of natural products have attracted much attention due to the increasing incidence of pathogens that have become resistant to drugs. Thus, it has been attempted to promisingly manage infectious diseases via a new group of therapeutic agents called antimicrobial peptides. In this study, a novel antifungal peptide, MCh‐AMP1, was purified by reverse phase HPLC and sequenced by de novo sequencing and Edman degradation. The antifungal activity, safety, thermal, and pH stability of MCh‐AMP1 were determined. This peptide demonstrated an antifungal activity against the tested Candida and Aspergillus species with MIC values in the range of 3.33–6.66 μM and 6.66–13.32 μM, respectively. Further, physicochemical properties and molecular modeling of MCh‐AMP1 were evaluated. MCh‐AMP1 demonstrated 3.65% hemolytic activity at the concentration of 13.32 μM on human red blood cells and 10% toxicity after 48 hr at the same concentration on HEK293 cell lines. The antifungal activity of MCh‐AMP1 against Candida albicans was stable at a temperature range of 30–50°C and at the pH level of 7–11. The present study indicates that MCh‐AMP1 may be considered as a new antifungal agent with therapeutic potential against major human pathogenic fungi.     

A comprehensive review on potential applications of metallic nanoparticles as antifungal therapies to combat human fungal diseases

Human pathogenic fungi are responsible for causing a range of infection types including mucosal, skin, and invasive infections. Life-threatening and invasive fungal infections (FIs) are responsible for mortality and morbidity, especially for individuals with compromised immune function. The number of currently available therapeutic agents against invasive FIs is limited compared to that against bacterial infections. In addition, the increased mortality and morbidity caused by FIs are linked to the limited number of available antifungal agents, antifungal resistance, and the increased toxicity of these agents. Currently available antifungal agents have several drawbacks in efficiency, efficacy, toxicity, activity spectrum, and selectivity. It has already been demonstrated with numerous metallic nanoparticles (MNPs) that these nanoparticles can serve as an effective and alternative solution as fungicidal agents. MNPs have great potential owing to their intrinsic antifungal properties and potential to deliver antifungal drugs. For instance, gold nanoparticles (AuNPs) have the capacity to disturb mitochondrial calcium homeostasis induced AuNP-mediated cell death in Candida albicans. In addition, both copper nanoparticles and copper oxide nanoparticles exerted significant suppressive properties against pathogenic fungi. Silver nanoparticles showed strong antifungal properties against numerous pathogenic fungi, such as Stachybotrys chartarum, Mortierella alpina, Chaetomium globosum, A. fumigatus, Cladosporium cladosporioides, Penicillium brevicompactum, Trichophyton rubrum, C. tropicalis, and C. albicans. Iron oxide nanoparticles showed potent antifungal activities against A. niger and P. chrysogenum. It has also been reported that zinc oxide nanoparticles can significantly inhibit fungal growth. These NPs have already exerted potent antifungal properties against a number of pathogenic fungal species including Candida, Aspergillus, Fusarium, and many others. Several strategies are currently used for the research and development of antifungal NPs including chemical modification of NPs and combination with the available drugs. This review has comprehensively presented the current and innovative antifungal approach using MNPs. Moreover, different types of MNPs, their physicochemical characteristics, and production techniques have been summarized in this review.     

The echinocandin micafungin: a review of the pharmacology,spectrum of activity,clinical efficacy and safety

Micafungin is a relatively broad-spectrum antifungal agent available for clinical use in the US and Japan. By inhibiting the production of β-1,3-glucan, an essential fungal cell wall component, micafungin has reduced toxicity to mammalian cells while maintaining potent antifungal activity against many pathogenic fungi including polyene- and azole-resistant isolates. Indeed, micafungin has been shown to be efficacious in the treatment of infections caused by Candida and Aspergillus species in clinical trials without the associated toxicities of amphotericin B formulations and drug interactions that occur with the azoles. In this review, the pharmacology, spectrum of activity, clinical efficacy and safety profile of micafungin are discussed.     

Design and evaluation of small peptides mapping the exposed surface of IL-8

In an effort to determine which regions of IL-8 are involved in interactions with its receptors, eight peptides were designed to correspond to distinct exposed regions of the IL-8 monomer, using the proton NMR-derived structure of the dimer as a basis. The peptides were evaluated singularly, and as equimolar mixtures of two to six peptides, in an IL-8 receptor binding assay and found to have no binding interaction with either α or β IL-8 receptor as single peptides or mixtures of two peptides. In contrast, one of these peptides having the sequence AVLPRSAKEL, which corresponds to the N-terminal 10 amino acid residues of the 77 amino acid form of IL-8, exhibited potent chemotactic activity in human neutrophils. These results indicate that there is no contiguous ligand that can be designed based on the NMR and X-ray determined structure of IL-8 and that there may be multiple receptors responsible for neutrophil activation and chemotaxis.     

1-{2-[(4-取代苯基)甲氧基]-2-(取代苯基)乙基}-1H-氮唑类化合物的合成及抗真菌活性

根据氮唑类抗真菌化合物的构效关系和作用机理,设计合成了29个1-{2-[(4-取代苯基)甲氧基]-2-(取代苯基)乙基}-1H-氮唑类化合物,其中九个为首次报道。初步体外抑菌试验结果表明,大多数目标化合物对八种试验菌株都有不同程度的抗真菌活性。化合物14对白念珠菌的活性与克霉唑及益康唑相当,对其它七种试验菌株的活性明显强于克霉唑及益康唑。化合物4,12对白念珠菌活性差,对其它七种试验菌株的活性也强于克霉唑和益康唑。化合物5,6和23除对白念珠菌外,对其它七种试验菌株,也有较强活性。     

Divergent solid‐phase synthesis and candidacidal activity of MUC7 D1, a 51‐residue histidine‐rich N‐terminal domain of human salivary mucin MUC7

Abstract: Domain 1 of the low‐molecular‐weight human salivary mucin, designated MUC7 D1, spans the 51 N‐terminal amino acid residues. This domain contains a 15‐residue basic histidine‐rich subdomain (R3‐Q17) which has 53% sequence similarity to histatin 5 (Hsn‐5), a salivary molecule known to exert potent in vitro cidal activity against Candida albicans and many other medically important fungi. The MUC7 D1‐15mer and its derivatives have previously been synthesized in our laboratory and their candidacidal activities have been found to be inferior to that of Hsn‐5. We were therefore intrigued to explore the candidacidal potency of the full‐length MUC7 D1 (51‐mer). Linear solid‐phase synthesis of this domain has been accomplished following standard Fmoc chemistry. The problems of partial coupling, owing to the peptide chain length, at several stages of the solid‐phase step‐by‐step synthesis were circumvented either by double‐coupling techniques or efficient coupling procedures. The MUC7 D1 peptide was purified to homogeneity by conventional reverse‐phase HPLC using two columns connected in series. Secondary structure of the purified peptide was assessed by circular dichroism (CD) spectroscopy in phosphate buffer and trifluoroethanol and compared to that of MUC7 D1‐15mer and Hsn‐5. The MUC7 D1 candidacidal activity was assessed against azole‐sensitive and azole‐resistant C. albicans strains and was found, unlike that of the MUC7 D1‐15mer, to be comparable with that of Hsn‐5, indicating that in addition to Hsn‐5, MUC7 D1 could provide an attractive alternative to the classical antifungal agents. The candidacidal potency of MUC7 D1, like that of MUC7 D1‐15mer, and of Hsn‐5, appears to be largely dependent on peptide charge, irrespective of α‐helical structure.     

Synergistic effect between silver nanoparticles and antifungal agents on Candida albicans revealed by dynamic surface-enhanced Raman spectroscopy

Abstract

Identifying the mechanisms of action of new potential antibiotics is a necessary but time-consuming and costly process. We have developed an ultra-rapid, highly sensitive, and reproducible dynamic surface-enhanced Raman spectroscopy (D-SERS) method to discriminate and evaluate the sensitivity of Candida albicans to antifungal agents with different mechanisms by using silver nanoparticles (Ag NPs). Although Ag NPs have been used conventionally for the enhancement of Raman signals, the accompanying influence of Ag NPs on the microbes has not been investigated. Herein, surface charge and concentration of Ag NPs are likely to be the main influencing factors. Then different concentrations of Ag NPs with the same surface charge as C. albicans were prepared to find the optimal conditions for enhancement of Raman signals while minimally affecting tested fungi. Spectral variations were observed with increasing concentrations of Ag NPs, as well as those of antifungal agents, including echinocandin and azole drugs. The results indicated that the combination of sub-lethal Ag NPs and echinocandin drugs revealed potent synergistic effects against fungi. This could be explained by the metabolism of fungi, the result of which has also been verified by transmission electron microscopy (TEM). Lastly, the combination of sub-lethal Ag NPs and echinocandin drugs was used for a mammalian cell toxicity assay to demonstrate whether the optimal combination could cause lower cytotoxicity to mammalian cells. This work opens a window not only for the evaluation of antifungal agents with different mechanisms, but also for the clinical treatment of fungal infections or even new drug development.     

A defensin antimicrobial peptide from the venoms of Nasonia vitripennis

Although many antimicrobial components (i.e. antimicrobial peptides) have been found in many social Hymenoptera venoms, no antimicrobial compound is purified and characterized from parasitic Hymenoptera. From the venoms of the ectoparasitic wasp, Nasonia vitripennis, a defensin-like antimicrobial peptide named defensin-NV was purified and characterized. Defensin-NV is composed of 52 amino acid residues including 6 cysteines forming 3 disulfide bridges. Its amino acid sequence is VTCELLMFGGVVGDSACAANCLSMGKAGGSCNGGLCDCRKTTFKELWDKRFG. By BLAST search, defensin-NV showed significant sequence similarity to other insect defensin antimicrobial peptides. Defensin-NV exerted strong antimicrobial activity against tested microorganisms including Gram-positive bacteria, Gram-negative bacteria and fungi. The cDNA encoding defensin-NV was cloned from the venom reservoir cDNA library of N. vitripennis. The current work firstly purified and characterized an antimicrobial peptide from parasitic Hymenoptera.     

Targeting human secretory phospholipase A2 with designed peptide inhibitors for inflammatory therapy

Phospholipase A2 (PLA2) is potentially an important target for anti-inflammatory therapeutics. Here, we described a systematic scheme that integrated protein docking and peptide redocking, molecular dynamics simulation, and binding affinity analysis to rationally design PLA2 inhibitory peptides based on a solved PLA2 crystal structure. The scheme employed protein docking to sample the interaction modes of PLA2 with its natural inhibitor Clara cell protein, from which a number of peptide fragments, including a pentapeptide LLLGS, were cut off and redocked to serve as the lead entities of PLA2 inhibitory peptides. In addition, a systematic mutation energy map that characterized the binding free energy changes ΔG upon mutations of each position of the putative pentapeptide to 20 amino acids was also profiled, which was subsequently used to guide peptide structure optimization. In order to solidify the computational findings, we performed kinetic and inhibition studies of few designed peptides against human secretory PLA2. Consequently, eight peptides were successfully identified to have potent inhibition potency, in which the LLAYK and AVFRS were found to suppress enzymatic activity significantly (K_i?=?0.75?±?0.06 and 4.2?±?0.3?μM, respectively). A further structure examination revealed that the designed peptides can form intensive nonpolar networks of van der Waals contacts and hydrophobic interactions at their complex interfaces with PLA2, conferring considerable stability and affinity for the formed complex systems.     

Rational Evolution of Antimicrobial Peptides Containing Unnatural Amino Acids to Combat Burn Wound Infections

Antimicrobial peptides have long been raised as a promising strategy to combat bacterial infection in burn wounds. Here, we attempted to rationally design small antimicrobial peptides containing unnatural amino acids by integrating in silico analysis and in vitro assay. Predictive quantitative sequence‐activity models were established and validated rigorously based on a large panel of nonamer antimicrobial peptides with known antibacterial activity. The best quantitative sequence‐activity model predictor was employed to guide genetic evolution of a peptide population. In the evolution procedure, a number of unnatural amino acids with desired physicochemical properties were introduced, resulting in a genetic evolution‐improved population, from which seven peptide candidates with top scores, containing 1–3 unnatural amino acids, and having diverse structures were successfully identified, and their antibacterial potencies against two antibiotic‐resistant bacterial strains isolated from infected burn wounds were measured using in vitro susceptibility test. Consequently, four (WL‐Orn‐LARKIV‐NH₂, ARKRWF‐Dab‐FL‐NH₂, KFI‐Hag‐IWR‐Orn‐R‐NH₂ and YW‐Hag‐R‐Cit‐RF‐Orn‐N‐NH₂) of the seven tested peptides were found to be more potent than reference Bac2A, the smallest naturally occurring broad spectrum antimicrobial peptide. Molecular dynamics simulations revealed that the designed peptides can fold into amphipathic helical structure that allows them to interact directly with microbial membranes.     

两亲α-螺旋抗菌肽的分子设计研究现状与进展

抗菌肽以其广谱、快速、特异性作用及抗菌、抗病毒、抗肿瘤等活性,成为具有重要潜在价值的新型药物.最大限度地发挥其活性和降低毒性,是抗菌肽新药开发的首要问题.用分子设计手段改造抗菌肽已成为解决这一问题的关键.以两亲α-螺旋抗菌肽为对象,对分子设计过程中遵守阳离子性和两亲性的原则及影响活性的物理化学参数,可以采取序列修饰或全新设计的方法,以达到定向改造的目的.对分子设计在医药上的应用和发展前景进行了综述和展望.     

A new nonadride derivative, dihydroepiheveadride, as characteristic antifungal agent against filamentous fungi, isolated from unidentified fungus IFM 52672

In the screening of searching for new antifungal agents, a new nonaride compound, dihydroepiheveadride (1), was isolated from unidentified fungus IFM 52672 as the most potent antifungal principle from this organism. The structure of 1 was established on the basis of spectroscopic and chemical investigation, as well as detailed comparison of the spectroscopic and physico-chemical data of the oxidized derivative (3) from 1 with those of heveadride (2). Compound 1 showed strong antifungal activity against various filamentous fungi including human pathogens Aspergillus fumigatus, Penicillium marneffei and Trichophyton spp. It also showed the growth inhibition activity against certain human pathogenic yeasts such as Trichosporon species, while it had weak or no antifungal activity against Candida spp. and Cryptococcus neoformans, and no antibacterial activity against Bacillus subtilis nor against Escherichia coli. The antifungal potencies of compounds 2 and 3 were found to be weaker than that of 1.     

1-(1H-1,2,4-三唑-1-基)-(2,4-二氟苯基)-3-[N-环丙基-N-(4-取代苄基)]-2-丙醇的合成及抗真菌活性

目的：以氟康唑为先导化合物,设计合成新的三唑醇类化合物,并研究其抗真菌活性。方法：引入4位羧酸酯取代的苄基侧链结构,合成一系列目标化合物,所有化合物结构均经MS、^1H-NMR等谱确证;选择8种真菌为实验菌株,测定其体外抗真菌活性。结果：合成了15个未见文献报道的目标化合物;所有化合物对所选真菌均表现出了一定的抑菌活性,其中化合物（1）,（2）和（3）对除薰烟曲霉菌外的7种菌都表现出了较好的抑菌活性。结论：4位羧酸酯取代的苄基侧链结构的引入对目标化合物的抗菌活性有一定的影响,侧链越短．抑菌活性越好.     

Natural Products as Sources of New Fungicides (I): Synthesis and Antifungal Activity of Acetophenone Derivatives Against Phytopathogenic Fungi

Several series of 45 acetophenone derivatives bearing various alkyl or benzyl substituents were conveniently synthesized and their structures characterized by ¹H and ¹³C NMR spectroscopy, HRMS and single‐crystal X‐ray analysis. Their in vitro antifungal activities against a panel of phytopathogenic fungi were evaluated by mycelial growth rate assay. Of them, 12 derivatives (e.g., 3a–c , 4c and 4e ) exhibited more potent antifungal effects on some phytopathogens than a commercial fungicide hymexazol as positive control. In particular, compound 3b with IC₅₀ values of 10–19 μg/mL was found to be the most active in this series and might be a potential lead structure for further optimization. The preliminary structure–activity relationship (SAR) studies of a series of acetophenones are also discussed.