具有双活性序列的新型抗菌肽的设计及性质

设计合成了具有2个活性序列的线性和环状多肽及具有单个活性序列的短链多肽, 研究了它们的杀菌活性、 细胞毒性及溶血性. 结果表明, 线性肽和环状肽的杀菌活性高于短链肽. 利用计算模拟的方法计算了多肽与细菌细胞膜中一种重要的成分磷脂酰甘油(DMPG)的结合能. 结果表明, 多肽-DMPG的结合能与多肽的杀菌活性具有较高的相关性, 线性和环状多肽与DMPG的结合能大于短链肽. 线性和环状多肽均含有2个活性序列, 可提供多个荷正电氨基酸与荷负电的磷脂结合, 结合能较大, 杀菌活性较强. 采用模拟生物膜对其中几条多肽的作用机理进行了初步研究. 结果表明, 该类多肽有可能使正常哺乳动物细胞的细胞膜产生孔洞; 而对于细菌细胞膜, 多肽并未在膜上产生明显孔洞, 而是引起了细菌细胞膜的聚集.     

Dendropsophin 1, a novel antimicrobial peptide from the skin secretion of the endemic Colombian frog Dendropsophus columbianus

In efforts to find new antimicrobial peptides (AMPs), we studied the skin secretion of the endemic Colombian frog Dendropsophus columbianus belonging to a genus that has not been investigated previously. From HPLC-fractionated secretion, we identified one peptide with slightly antibacterial activity. Its peptide sequence showed no sequence similarity to current annotated peptides. We named this novel peptide dendropsophin 1 (Dc1). Afterward, two analogues were designed (Dc1.1 and Dc1.2) to improve the cationic and amphipathic features. Then, their antiproliferative and cytotoxic properties were evaluated against several pathogens including bacteria, fungi, protozoa and also mammalian cells. Dc1 and its two analogues exhibited moderate antibacterial activities and no hemolytic and cytotoxic effects on mammalian cells. Analogue Dc1.2 showed slightly improved antibacterial properties. Their secondary structures were characterised using CD spectroscopy and Dc1.2 displayed a higher α-helix content and thermal stability compared to Dc1 and Dc1.1 in hydrophobic experimental conditions.     

一种中国林蛙抗菌肽的光谱研究

从中国林蛙皮中纯化得到了一种抗多种临床多重耐药菌的抗菌肽(RTCⅠ), 初步氨基酸组成分析结果表明其不含碱性氨基酸, 此抗菌肽在276.5和356.5 nm波长光的激发下发射出448 nm的荧光, 利用傅里叶变换红外光谱、拉曼光谱、电子吸收光谱及荧光光谱等技术研究了此特定荧光产生的结构依据. 此抗菌肽的主要组成是Tyr, Asn(Asp)和Glu(Gln), 抗菌肽特殊的荧光光谱和电子吸收谱与Tyr的酚羟基和Asn侧链的强氢键有关. 这一特殊的荧光(448 nm)及圆二色谱(259, 263和267 nm)信号为进一步在分子水平上研究此抗菌肽的抗菌机理提供了依据.     

New Found Hope for Antibiotic Discovery: Lipid II Inhibitors

Research into antibacterial agents has recently gathered pace in light of the disturbing crisis of antimicrobial resistance. The development of modern tools offers the opportunity of reviving the fallen era of antibacterial discovery through uncovering novel lead compounds that target vital bacterial cell components, such as lipid II. This paper provides a summary of the role of lipid II as well as an overview and insight into the structural features of macrocyclic peptides that inhibit this bacterial cell wall component. The recent discovery of teixobactin, a new class of lipid II inhibitor has generated substantial research interests. As such, the significant progress that has been achieved towards its development as a promising antibacterial agent is discussed.     

新型抗菌肽的设计、活性研究及与磷脂相互作用的计算模拟

设计合成了多个具有2个活性序列的线性和环状多肽及具有单个活性序列的短链多肽, 研究了它们的杀菌活性, 发现其杀菌活性顺序为长链肽>环状肽>短链肽, 特别是线性的Linear-KT和Linear-KS对多种革兰氏阴性菌和阳性菌均具有较高的杀菌活性. 采用MTT法考察了Linear-KT和Linear-KS对正常细胞的毒性, 其中Linear-KS表现出较低的细胞毒性, 优于阳性对照多粘菌素B. 利用计算模拟的方法计算了多肽与细菌细胞膜中磷脂酰甘油(DMPG)的相互作用. 结果表明, 多肽和DMPG的结合能也表现出长链肽>环状肽>短链肽的规律, 特别是Linear-KT和Linear-KS具有较高的结合能. 长链肽含有2个活性序列, 可提供多个荷正电的氨基酸与荷负电的磷脂结合, 结合能较大, 杀菌活性较强. 同时, 柔性的结构及Linear-KT和Linear-KS中丝氨酸和苏氨酸的β碳上的羟基可与磷脂上的羰基形成多个氢键, 进一步增大了结合能. 计算模拟的方法为抗菌肽的杀菌活性从理论上提供了一定的依据.     

Purification and in vitro Activity of an Antimicrobial Peptide from Skin of Rana Temporaria Chensinensis, David

In this study,an antimicrobial component(RTCI)was purified from the skin of Rana temporaria chensinensis,David.Antimicrobial activities of RTCI against clinical multi-drug resistant bacterial strains,including Escherichia coli,Pseudomonas aeruginosa,Staphylococcus aureaus,Klebsiella oxytoca,Klebsiella pneumoniae,Enterococcus faecalis,and Proteus mirabilis were measured in vitro by means of minimal inhibitory concentration and time-kill studies.The results indicate that RTCI could inhibit the growth of these bacteria at a proper concentration and suggest that RTCI shows a better antimicrobial activity to Gram-negative bacterial strains than to Gram-positive bacterial strains.     

蜂毒肽类似物的合成和生物活性研究

Melittin(GIGAVLKVLTTGLPALISWIKRKRQQ-NH₂)是蜂毒中含26个氨基酸残基的多肽,具有抗菌和溶血等生物活性,是典型的阳离子抗菌肽.本文设计合成了蜂毒肽C端15残基肽片段(GLPALISWIKRKRQQ-NH₂)及其类似物(15残基).研究了Melittin及这些合成肽的抗菌活性、溶血活性、疏水性及二级结构.结果表明,合成的类似物的溶血活性明显降低,抗菌活性基本保留,且与其疏水性相关.类似物中与碱性氨基酸簇(KRKR)距离较远的残基的疏水性对其抗菌活性有较大的贡献.多肽溶血与抗菌机理不同.类似物的抗菌活性和溶血活性与其二级结构(α-螺旋结构)没有明显的相关性.     

Hydrogel Complex Containing the Antimicrobial Peptide HX-12C Accelerates Healing of Infected Wounds

Bacterial infections of the wound surface can be painful for patients, and traditional dressings do not effectively address this problem. In this study, an antimicrobial wound dressing is prepared using a novel antimicrobial peptide, HX-12C. This hydrogel system is based on the natural biomaterials sodium alginate and gelatin, utilizing calcium carbonate as a source of Ca²⁺, and ionic cross-linking is facilitated by lowering the solution pH. The resulting sodium alginate/gelatin HX-12C-loaded hydrogel (CaAGEAM) has good mechanical and adhesion properties, biocompatibility and in vitro degradability. Its extraordinary antibacterial efficacy (>98%) is verified by an antibacterial experiment. More importantly, in vivo experiments further demonstrate its healing-promotion effect, with a 95% wound healing rate by day 9. Tissue staining demonstrates that the hydrogel containing antimicrobial peptides is effective in suppressing inflammation. The dressing promotes wound healing by stimulating the deposition of skin appendages and collagen. The results of this study suggest that composite hydrogels containing antimicrobial peptides are a promising new type of dressing to promote the healing of infected wounds.     

家蝇幼虫抗菌肽MDL-1的构象分析

用红外光谱、圆二色谱和荧光光谱研究家蝇幼虫抗菌肽MDL-1的结构特征及其在不同条件下的构象变化. 红外光谱检测结果显示抗菌肽MDL-1结构中含有螺旋、无规卷曲、折叠构象的吸收特征; 圆二色谱显示抗菌肽MDL-1结构相对比较稳定, 抗菌肽在不同浓度溶液中的构象发生改变; 荧光光谱法研究发现家蝇幼虫抗菌肽MDL-1在280 nm波长的激发光下, 荧光光谱为Tyr残基和Trp残基共同提供, 而且Trp残基不是位于抗菌肽分子的表面, 而是位于分子的内部, 该研究结果为进一步探讨抗菌肽的抗菌机理奠定了基础.     

Photoprotection in tadpoles of the common frog, Rana temporaria

The common frog, Rana temporaria, spawning at altitudes up to 2600 m, is potentially threatened by UVB radiation. In laboratory experiments we found a specifically UVB absorbing substance (UVAS) in the skin of tadpoles with maximum absorption between 290 and 300 nm. The production of UVAS is induced by both visible light and ultraviolet radiation. The concentrations of UVAS found in tadpoles from the field are about as high as those found in tadpoles from laboratory experiments with UVB radiation under simulated mid-summer conditions. The existence of two powerful sun screen factors (UVAS and melanin pigmentation) may explain the high resistance of R. temporaria tadpoles to the intensive UVB radiation at high altitudes.     

Bio-Organometallic Derivatives of Antibacterial Drugs

Overuse and misuse of antibacterial drugs has resulted in bacteria resistance and in an increase in mortality rates due to bacterial infections. Therefore, there is an imperative necessity of new antibacterial drugs. Bio-organometallic derivatives of antibacterial agents offer an opportunity to discover new active antibacterial drugs. These compounds are well-characterized products and, in several examples, their antibacterial activities have been studied. Both inhibition of the antibacterial activity and strong increase in the antibiotic activity of the parent drug have been found. The synthesis of the main classes of bio-organometallic derivatives of these drugs, as well as examples of the use of structure–activity relation (SAR) studies to increase the activity and to understand the mode of action of bio-organometallic antimicrobial peptides (BOAMPs) and platensimicyn bio-organometallic mimics is presented in this article.     

In Vitro and In Vivo Activity of 14-O-[(4,6-Diamino-pyrimidine-2-yl) thioacetyl] Mutilin against Methicillin-Resistant Staphylococcus aureus

Staphylococcus aureus (S. aureus) is a major human pathogen that requires new antibiotics with unique mechanism. A new pleuromutilin derivative, 14-O-[(4,6-Diamino-pyrimidine-2-yl) thioacetyl] mutilin (DPTM), has been synthesized and proved as a potent antibacterial agent using in vitro and in vivo assays. In the present study, DPTM was further in vitro evaluated against methicillin-resistant Staphylococcus aureus (MRSA) isolated from dairy farms and outperformed tiamulin fumarate, a pleuromutilin drug used for veterinary. Moreover, a murine skin wound model caused by MRSA infection was established, and the healing effect of DPTM was investigated. The results showed that DPTM could promote the healing of MRSA skin infection, reduce the bacterial burden of infected skin MRSA and decrease the secretion of IL-6 and TNF-α inflammatory cytokines in plasma. These results provided the basis for further in-depth drug targeted studies of DPTM as a novel antibacterial agent.     

Antibacterial Activity of Pharmaceutical-Grade Rose Bengal: An Application of a Synthetic Dye in Antibacterial Therapies

Rose bengal has been used in the diagnosis of ophthalmic disorders and liver function, and has been studied for the treatment of solid tumor cancers. To date, the antibacterial activity of rose bengal has been sporadically reported; however, these data have been generated with a commercial grade of rose bengal, which contains major uncontrolled impurities generated by the manufacturing process (80–95% dye content). A high-purity form of rose bengal formulation (HP-RBf, >99.5% dye content) kills a battery of Gram-positive bacteria, including drug-resistant strains at low concentrations (0.01–3.13 μg/mL) under fluorescent, LED, and natural light in a few minutes. Significantly, HP-RBf effectively eradicates Gram-positive bacterial biofilms. The frequency that Gram-positive bacteria spontaneously developed resistance to HP-RB is extremely low (less than 1 × 10⁻¹³). Toxicity data obtained through our research programs indicate that HP-RB is feasible as an anti-infective drug for the treatment of skin and soft tissue infections (SSTIs) involving multidrug-resistant (MDR) microbial invasion of the skin, and for eradicating biofilms. This article summarizes the antibacterial activity of pharmaceutical-grade rose bengal, HP-RB, against Gram-positive bacteria, its cytotoxicity against skin cells under illumination conditions, and mechanistic insights into rose bengal’s bactericidal activity under dark conditions.     

Influence of the Hydrophobicity of Galleria Mellonella Antibacterial Peptides on the Parameters of Their Chromatographic Retention

Colloid Journal - The hydrophobicity of antibacterial peptides of Galleria mellonella calculated using the HyperChem and SSRCalc algorithms of the additive chromatographic separation model has been...     

Facile Synthesis of (±)-2,6-Dimethethyl-6-hydroxy-2,7-octadienoic Acid and its Methyl Ester and Amide (Acacialactam)

The monoterpenoid acid 1, which is a component of some traditional medicine such as Gymnocladus chinensis baillon in glycoside form,¹ was recently isolated from Artemisia sieberi in Iran in free form.². Its structure has been established to be (E)-2,6-dimethyl-6-hydroxy-2,7-octadienoic acid (1) by chemical and spectroscopic analysis. Its methyl ester derivative 2,was isolated from the antibacterial extracts of the leaves of Piper aduncum³ as well. Acacialactam, a natural product isolated from the seeds of Acacia concinna DC.(Legunninosae) used in folk medicine of several tropical countries for treating some skin diseases, was assigned structure 4 by Sekine et al⁴ on the basis of its physical and spectral properties.     

Optimized grafting of antimicrobial peptides on stainless steel surface and biofilm resistance tests

Antibacterial peptides, magainin I and nisin were covalently bound to stainless steel surfaces. Several procedures of surface functionalisation processes have been investigated and optimized, each step being characterized by polarization modulation reflection absorption infrared spectroscopy (PM-RAIRS) and X-ray photoemission spectroscopy (XPS). Grafting of antibacterial peptides was successfully achieved by a 3 steps functionalisation process on a chitosan polymeric layer. The antibacterial activity of the anchored magainin and nisin was tested against a gram-positive bacteria, Listeria ivanovii, i.e., the possible survival and attachment of this bacteria, was characterized on modified stainless steel surfaces. The results revealed that the adsorbed peptides reduced the adhesion of bacteria on the functionalised stainless steel surface.     

High-throughput sequence determination of cyclic peptide library members by partial Edman degradation/mass spectrometry

Cyclic peptides provide attractive lead compounds for drug discovery and excellent molecular probes in biomedical research. Large combinatorial libraries of cyclic peptides can now be routinely synthesized by the split-and-pool method and screened against biological targets. However, post-screening sequence determination of hit peptides has been problematic. In this report, a high-throughput method for the sequence determination of cyclic peptide library members has been developed. TentaGel microbeads (90 mum) were spatially segregated into outer and inner layers; cyclic peptides were displayed on the bead surface, whereas the inner core of each bead contained the corresponding linear peptide as the encoding sequence. After screening of the cyclic peptide library against a macromolecular target, the identity of hit peptides was determined by sequencing the linear encoding peptides inside the bead using a partial Edman degradation/mass spectrometry method. On-bead screening of an octapeptide library (theoretical diversity of 160 000) identified cyclic peptides that bind to streptavidin. A 400-member library of tyrocidine A analogues was synthesized on TentaGel macrobeads and solution-phase screening of the library directly against bacterial cells identified a tyrocidine analogue of improved antibacterial activity. Our results demonstrate that the new method for cyclic peptide sequence determination is reliable, operationally simple, rapid, and inexpensive and should greatly expand the utility of cyclic peptides in biomedical research.     

Identification of four cornua by ultra‐performance liquid chromatography with time‐of‐flight mass spectrometry coupled with principal component analysis

An ultra‐high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry method coupled with principal component analysis was developed and applied to the identification of Cornu Antelopis, Cornu Bubali, Cornu Naemorhedi, and Cornu Bovis. The data obtained from the trypsin‐digested samples were subjected to principal component analysis to classify these four cornua. Additionally, marker peptides of the cornua were determined by orthogonal partial least‐squares discriminant analysis, and fragmentation tandem mass spectra of these marker peptides were evaluated. The results from this study indicate that the proposed method is reliable, and it has been successfully applied to the identification of variants of cornua commonly used in traditional Chinese medicine.     

Interplay among folding, sequence, and lipophilicity in the antibacterial and hemolytic activities of alpha/beta-peptides

Host-defense peptides inhibit bacterial growth but manifest relatively little toxicity toward eukaryotic cells. Many host-defense peptides adopt alpha-helical conformations in which cationic side chains and lipophilic side chains are segregated to distinct regions of the molecular surface ("globally amphiphilic helices"). Several efforts have been made to develop unnatural oligomers that mimic the selective antibacterial activity of host-defense peptides; these efforts have focused on the creation of molecules that are globally amphiphilic in the preferred conformation. One such endeavor, from our laboratories, focused on helix-forming alpha/beta-peptides, i.e., oligomers containing a 1:1 pattern of alpha- and beta-amino acid residues in the backbone [Schmitt, M. A.; Weisblum, B.; Gellman, S. H. J. Am. Chem. Soc. 2004, 126, 6848-6849]. We found, unexpectedly, that the most favorable biological activity profile was displayed by a "scrambled" sequence, which was designed not to be able to form a globally amphiphilic helix. Here we report new data, involving an expanded set of alpha/beta-peptides, from experiments designed to elucidate the origins of this surprising result. In addition, we evaluate the susceptibility of alpha/beta-peptides to proteolytic degradation. Our results support the hypothesis that the ability to adopt a globally amphiphilic helical conformation is not a prerequisite for selective antibacterial activity. This conclusion represents a significant advance in our understanding of the relationship among molecular composition, conformation, and biological activity. Our results should therefore influence the design of other unnatural oligomers intended to function as antibacterial agents.     

Identification of a potent antibacterial factor isolated from bronchoalveolar lavage fluid: guanidine,N-[3-[(aminoiminomethyl)amino]propyl]-N-dodecyl-, a potential source of error in the analysis of antibacterial agents

The widespread use of antibiotics in modern society has encouraged the search for new antibacterial compounds. In this laboratory investigations are being made to identify and characterise novel antibacterial peptides. With this in mind, the antibacterial properties of human bronchoalveolar lavage (BAL) fluid from sarcoidosis patients is being investigated. In this communication we report on the identification and characterisation of a highly active non-peptide antibacterial compound isolated from BAL fluid. The structure of this active compound was elucidated by high-resolution accurate mass and tandem mass spectrometry to be guanidine, N-[3-[(aminoiminomethyl)amino]propyl]-N-dodecyl-. This compound does not appear to be endogenous, and its presence in BAL fluid extracts presents a potential source of error in analysis of antibacterial agents. The biological effects of guanidine, N-[3-[(aminoiminomethyl)amino]propyl]-N-dodecyl- have not previously been described in the literature.