阳离子抗菌肽的研究进展

阳离子抗菌肽是一类具抗微生物活性的小分子多肽，按结构特征可分为α－螺旋肽和β－折叠肽，不同来源或同一抗菌肽的不同结构形式生物活性差别较大，部分抗菌肽之间或抗菌肽与抗生素之间存在协同作用，特别是最近发现一些抗菌肽具有抗内毒素活性。阳离子抗菌肽的结构特征是其发挥作用的重要基础，α－螺旋肽通过其两亲性的α－螺旋上的正电茶与细菌细胞膜磷脂分子的负电荷之间的静电吸引而结合在细菌膜上，并借助于疏水段分子中连接结构的柔性插入到细胞膜中，最终通过膜内分子间的相互位移使抗菌肽分子相互聚集在一起形成离子通道，使细菌失去膜势，不能保持正常的渗透压而死亡。一般认为β－折叠肽也是和细胞膜结合后，结构发生变化而发挥作用。抗菌肽和细胞膜结合及形成离子通道受多种因素的影响，如阳离子抗菌肽的结构、浓度，环境 pH、温度，介质的离子强度。阳离子抗菌肽杀菌力强，抗菌谱广，不良反应少，因此在食品、农业，特别是在医药领域都有很好的应用前景，极可能发展成为一类全新的抗生素。     

膜活性多肽(MAPs)的抗菌活性及其作用机制的研究进展

膜活性多肽(MAPs)是一类具有较好抗菌活性的抗菌肽。作为先天宿主防御分子,广泛的分布于细菌、植物、无脊椎动物、脊椎动物中。膜活性多肽具有抗菌肽的结构特征,肽链通常较短,带正电荷,具有两亲性的α-螺旋或β-折叠结构,通过破坏膜的通透性杀死细菌、真菌和部分肿瘤细胞。膜活性多肽在细胞膜或细胞内部存在特定的分子靶点,并因其独特的作用机制而成为一种新型的肽类抗生素。本文主要对膜活性多肽的抗菌活性及其作用机制的研究现状和发展情况做一综述。     

正电性多肽copoly(Lys/Tyr)与磷脂膜的相互作用研究

目的 研究正电性多肽copoly(Lys/Tyr)(CPLT)在模拟生物膜上的透过性。方法 ①配制由卵磷脂(EPC)、2-油酰基磷脂酰乙醇胺(DOPE)和大豆磷脂(SBPL)组成的二分子磷脂膜。②Zeta电位测定法(zeta potential method，ZP)测定由上述磷脂组成的脂质体在加入CPLT后的膜表面Zeta电位。③园二色谱法(circular dichroism spectrcvscopy，CD)检测CPLT分子与磷脂膜作用时的构象情况。④电生理学方法(electrophy siology tech—nique，ET)测量CPLT分子在磷脂膜上引起的跨膜电流。⑤荧光光度分析法(fluorescence spectroscopy，FS)检测CPLT分子与磷脂膜的作用过程中的荧光强度变化。⑥共焦点激光扫描显微分析法(confocal laser scanning microscopy，CLSM)研究CPLT在磷脂膜上透过及其相对透过效率。结果①随着CPLT的加入和其浓度的增加，磷脂膜Zeta电位逐渐增加并趋向饱和。②CPLT分子在水相取β-sheet构象，当与磷脂膜结合后，其β-sheet构象的波峰发生红移，但构象基本不变。③CPLT分子能在一定浓度和一定外加电压条件下，透过二分子磷脂膜引起膜电流。④CPLT与磷脂膜的作用可分为三步：第一步，CPLT分子吸附于磷脂膜上；第二步，CPLT分子通过磷脂膜的疏水区；第三步，CPLT分子从二分子膜的内膜解吸，进入膜内水相。⑤CPLT分子在磷脂膜上的透过效率主要决定于磷脂膜的组成，在磷脂膜中存在带负电性的磷脂时可降低CPLT分子在磷脂膜上的透过效率。⑥在最初的CPLT分子与磷脂膜的相互吸附过程中，CPLT分子与磷脂膜间的静电作用力起主要作用。结论copoly(Lys／Tyr)分子能透过二分子磷脂膜，其透过效率主要决定于磷脂膜的组成。在最初的肽一膜吸附过程中，当存在静电作用力时，静电力起主要作用，疏水效应次之。     

抗菌肽抗菌机制及与抗生素协同作用的研究进展

抗菌肽(antimicrobial peptides, AMPs)是一种新型抗生素，对多种细菌，多重耐药细菌均具有抗菌活性。然而，其副作用也是制约抗菌肽研发的主要障碍。研究者使用模式细胞膜，揭示抗菌肽与细胞膜之间的作用方式，开展抗菌肽开发和筛选研究。另外，研究者还将抗菌肽与常规抗生素联合使用，可以协同提高抗菌效果。研究初步揭示了AMPs和常规抗生素之间协同作用的机制。本综述探讨了模式细胞膜在AMPs发现筛选中的应用，及AMPs和常规抗生素之间联合用药的研发现状。     

红霉素与细胞被膜的关系和临床应用

目的探讨红霉素与病菌细胞被膜之间的关系和临床应用疗效观察。方法综合分析致病菌细胞被膜与红霉素抗菌治疗46例感染患者,及疗效观察。结果46例经红霉素抗炎治疗的患者有效位42例占91．3％,无效仅有4例占8．7％。结论大环内酯类抗生素可透过细菌细胞膜,抑制细菌蛋白质合成,从而起到杀菌作用。同时,更利于其他抗生素发挥抗菌作用。     

硝酸咪康唑的临床应用

硝酸咪康唑(miconazole Nitrafe)是咪唑类抗真菌药,有很强的抗微生物作用,较宽的抗菌谱和抗菌活性。其机理是通过抑制过氧化物酶,干扰细菌细胞膜的渗透性而发     

嗪诺酮类抗菌药

新的4-喹诺酮类抗菌药物的主要作用机理是抑制细菌DNA转曲酶活性。包括氧氟沙星、环丙沙星、依诺沙星和诺氟沙星。这类药物比萘啶酮酸的活性高1000倍,且有更广的抗菌谱。耐药菌的产生率亦比较低。4-喹诺酮类药物是通过破坏细菌DNA结构而阻止细菌细胞分裂来杀菌的。环丙沙星和氧氟沙星除能抑制DNA转曲酶活性外,似乎还能破坏细菌的细胞膜,导致细胞内含物流失。这两种药物较其它的喹诺酮类药物活性高,且有更广的抗菌谱。人体细胞内含有与细菌转曲酶类似的酶。但据报道,喹诺酮类药物对人体内的这     

抗菌药物的作用机制及细菌耐药性机制的研究进展

抗菌药物为人类的健康生存和发展作出了巨大的贡献。而细菌耐药性问题近年来已经发展到了非常严重的地步。深入了解药物的作用机制及其相关的耐药机制对研制新的有效抗菌药物是非常必需的。近年来对临床常用的抗菌药物β－内酰胺类、氨基糖苷类、喹诺酮类的作用机制和耐药机制进行了研究。其耐药机制涉及多个方面，主要有：酶对药物进行水解、酰化、磷酸化及核苷化；改变修饰药物的靶位；通过改变细胞膜的通透性或增加药物外排而降低细胞体内药物的浓度以及细菌固有的一些特性，如铜绿假单胞菌的生物被膜等，每类药物各有其侧重点。     

细菌分选酶A抑制剂抗革兰阳性超级细菌感染的作用及其应用前景

过去几十年中，全球抗菌药物耐药形势愈发严峻，多重耐药菌所致感染性疾病的发病率及死亡率逐年上升。对抗菌药物耐药机制的研究发现，抗毒力疗法可能成为对抗超级细菌感染的新选择。细菌分选酶A(SrtA)是一种细菌细胞膜酶，可将关键毒力因子固定在革兰阳性菌细胞壁表面，在革兰阳性超级细菌的致病机制中起重要作用。SrtA通过识别、硫酯化和转肽化将微生物表面成分识别黏附基质分子锚定在细菌肽聚糖上，因此抑制SrtA可使细菌无法附着在特定的组织和（或）器官上，从而无法攻击宿主细胞，且无法逃避宿主的免疫反应。此外，SrtA并非细菌生长和生存所必需，在细胞膜上很容易获得，是开发抗毒力疗法药物的理想靶点。本文对合成小分子、多肽和天然产物等SrtA抑制剂的研究进展进行归纳，同时对SrtA抑制剂治疗革兰阳性超级细菌感染的应用潜力进行了分析。     

中药抗菌和逆转耐药作用机制研究进展

目的:为进一步深入研究中药抗菌和逆转耐药作用机制提供参考和研究思路。方法:采用文献综述的方法,阐述国内、外近十年来中药抗菌和逆转耐药作用机制研究进展。结果:中药抗菌可以通过抑制菌体内能量的生成、改变细胞膜和离子通道的通透性、抑制菌体内酶的活性、增强中性粒细胞的吞噬功能、减少细菌内毒素的释放、代谢产物发挥抗菌作用、抑制蛋白质的合成、抑制细菌生物被膜的形成等机制发挥抗菌作用,中药可以通过消除R质粒、抑制细菌主动外排泵、抑制β-内酰胺酶、抑制耐药基因的表达等途径逆转细菌耐药性。结论:在中医药理论指导下结合现代研究思路深入探讨中药抗菌和逆转耐药作用机制尤为重要。     

Adaptive translocation: the role of hydrogen bonding and membrane potential in the uptake of guanidinium-rich transporters into cells

A mechanistic hypothesis is presented for how water-soluble guanidinium-rich transporters attached to small cargoes (MW ca. <3000) can migrate across the non-polar lipid membrane of a cell and enter the cytosol. Positively charged and water-soluble, arginine oligomers can associate with negatively charged, bidentate hydrogen bond acceptor groups of endogenous membrane constituents, leading to the formation of membrane-soluble ion pair complexes. The resultant less polar, ion pair complexes partition into the lipid bilayer and migrate in a direction, and with a rate, influenced by the membrane potential. The complex dissociates on the inner leaf of the membrane and the transporter conjugate enters the cytosol. This mechanism could also be involved in the translocation of guanidinium-rich molecules that are endocytosed due to their size or the conditions of the assay, across the endosomal membrane.     

An atomic and molecular view of the depth dependence of the free energies of solute transfer from water into lipid bilayers

Molecular interactions and orientations responsible for differences in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayer partitioning of three structurally related drug-like molecules (4-ethylphenol, phenethylamine, and tyramine) were investigated. This work is based on previously reported molecular dynamics (MD) simulations that determined their transverse free energy profiles across the bilayer. Previously, the location where the transfer free energy of the three solutes is highest, which defines the barrier domain for permeability, was found to be the bilayer center, while the interfacial region was found to be the preferred binding region. Contributions of the amino (NH2) and hydroxyl (OH) functional groups to the transfer free energies from water to the interfacial region were found to be very small both experimentally and by MD simulation, suggesting that the interfacial binding of these solutes is hydrophobically driven and occurs with minimal loss of hydrogen-bonding interactions of the polar functional groups which can occur with either water or phospholipid head groups. Therefore, interfacial binding is relatively insensitive to the number or type of polar functional groups on the solute. In contrast, the relative solute free energy in the barrier domain is highly sensitive to the number of polar functional groups on the molecule. The number and types of hydrogen bonds formed between the three solutes and polar phospholipid atoms or with water molecules were determined as a function of solute position in the bilayer. Minima were observed in the number of hydrogen bonds formed by each solute at the center of the bilayer, coinciding with a decrease in the number of water molecules in DOPC as a function of distance away from the interfacial region. In all regions, hydrogen bonds with water molecules account for the majority of hydrogen-bonding interactions observed for each solute. Significant orientational preferences for the solutes are evident in certain regions of the bilayer (e.g., within the ordered chain region and near the interfacial region 20-25 ? from the bilayer center). The preferred orientations are those that preserve favorable molecular interactions for each solute, which vary with the solute structure.     

Study of Orientation and Penetration of LAH4 into Lipid Bilayer Membranes: pH and Composition Dependence

LAH4 is an antimicrobial peptide that is believed to possess both antibiotic and DNA delivery capabilities. It is one of a number of membrane‐active peptides that show increased affinity toward anionic lipids. Herein, we have performed molecular dynamics simulations to compare LAH4 effects on anionic palmitoyl–oleoyl–phosphatidylglycerol bilayer, which approximate a prokaryotic membrane environment and zwitterionic palmitoyl–oleoyl–phosphatidylcholine bilayer, which approximate a eukaryotic membrane environment. One particular interest in this work is to study how different kinds of lipid bilayers respond to the attraction of LAH4. Remarkably, our data have shown that the depth of peptide penetration strongly depends on membrane composition and pH. At acidic pH, LAH4 has exhibited a high tendency to interact strongly with and be adsorbed on anionic membrane. We have also shown that electrostatic interactions between His11 and the phosphor atoms of bilayers should have a significant impact on the penetration of LAH4. These results provide insights into the interactions of LAH4 and lipid bilayers at the atomic level, which is useful to understand cell selectivity and mechanism of the peptide action.     

Molecular Dynamics Simulation of Chemokine Receptors in Lipid Bilayer: A Case Study on C–C Chemokine Receptor Type 2

Chemokine receptors belong to the membrane proteins that are included in many physiological phenomena. However, the mechanism of their action is unknown at the atomistic level in different aspects. In this study, a computational pipeline is exploited to investigate the molecular basis of how the structure of C–C chemokine receptor type 2, a prototypical chemokine receptor, is affected by lipid bilayer and an antagonist (INCB3344). This study includes homology modeling, molecular dynamics simulation in lipid bilayer, and docking. A detailed mechanism of INCB3344 has been described. Tyr 49, Trp 98, Tyr 120, His 121, and Glu 291 are proved to play important roles in binding. Integrating results obtained in this study and experimental data help us to suggest a two‐step ligand‐binding mechanism. The N‐terminus of protein first sticks out from the extracellular domain suitable for the contact with the antagonist. Binding of ligand to this segment leads to the geometrical changes to facilitate the ligand interactions with extracellular loop 2 of C–C chemokine receptor type 2. Finally, the interactions occurring between extracellular loop 2 and ligand induce conformational changes in C–C chemokine receptor type 2 structure. These changes bring the ligand closer to the binding pocket, allowing the interaction between INCB3344 and the residues of active site.     

Capsaicin regulates voltage-dependent sodium channels by altering lipid bilayer elasticity

At submicromolar concentrations, capsaicin specifically activates the TRPV1 receptor involved in nociception. At micro- to millimolar concentrations, commonly used in clinical and in vitro studies, capsaicin also modulates the function of a large number of seemingly unrelated membrane proteins, many of which are similarly modulated by the capsaicin antagonist capsazepine. The mechanism(s) underlying this widespread regulation of protein function are not understood. We investigated whether capsaicin could regulate membrane protein function by changing the elasticity of the host lipid bilayer. This was done by studying capsaicin's effects on lipid bilayer stiffness, measured using gramicidin A (gA) channels as molecular force-transducers, and on voltage-dependent sodium channels (VDSC) known to be regulated by bilayer elasticity. Capsaicin and capsazepine (10-100 microM) increase gA channel appearance rate and lifetime without measurably altering bilayer thickness or channel conductance, meaning that the changes in bilayer elasticity are sufficient to alter the conformation of an embedded protein. Capsaicin and capsazepine promote VDSC inactivation, similar to other amphiphiles that decrease bilayer stiffness, producing use-dependent current inhibition. For capsaicin, the quantitative relation between the decrease in bilayer stiffness and the hyperpolarizing shift in inactivation conforms to that previously found for other amphiphiles. Capsaicin's effects on gA channels and VDSC are similar to those of Triton X-100, although these amphiphiles promote opposite lipid monolayer curvature. We conclude that capsaicin can regulate VDSC function by altering bilayer elasticity. This mechanism may underlie the promiscuous regulation of membrane protein function by capsaicin and capsazepine-and by amphiphilic drugs generally.     

The mode of action of chlorpromazine on mitochondrial membrane permeability to K+

The effects of chlorpromazine on parameters related to potassium transport in isolated rat liver intact mitochondria were investigated. The results showed that chlorpromazine induced the passive permeability of mitochondrial membrane to potassium. A competition between spermine and chlorpromazine suggested the same binding sites within the inner mitochondrial membrane. Partial neutralization of mitochondrial surface potential by chlorpromazine indicated that this drug binds to negatively charged superficial sites of the membrane. A mechanism of action of chlorpromazine on potassium transport across the mitochondrial membrane involving alterations of surface potential and disarrangement of lipid bilayer is postulated.     

Molecular Dynamics Simulation to Uncover the Mechanisms of Protein Instability During Freezing

Freezing is a common process applied in the pharmaceutical industry to store and transport biotherapeutics. Herewith, multi-scale molecular dynamics simulations of Lactate dehydrogenase (LDH) protein in phosphate buffer with/without ice formation performed to uncover the still poorly understood mechanisms and molecular details of protein destabilization upon freezing. Both fast and slow ice growing conditions were simulated at 243 K from one or two-side of the simulation box, respectively. The rate of ice formation at all-atom simulations was crucial to LDH stability, as faster freezing rates resulted in enhanced structural stability maintained by a higher number of intramolecular hydrogen bonds, less flexible protein's residues, lower solvent accessibility and greater structural compactness. Further, protein aggregation investigated by coarse-grained simulations was verified to be initiated by extended protein structures and retained by electrostatic interactions of the salt bridges between charged residues and hydrogen bonds between polar residues of the protein. Lastly, the study of free energy of dissociation through steered molecular dynamics simulation revealed LDH was destabilized by the solvation of the hydrophobic core and the loss of hydrophobic interactions. For the first time, experimentally validated molecular simulations revealed the detailed mechanisms of LDH destabilization upon ice formation and cryoconcentration of solutes.     

Ab initio investigation of interactions between models of membrane-active compounds and polar groups of membranes: complexes involving amine, ether, amide, phosphate, and carboxylate.

Ab initio (MINI-1) molecular orbital calculations were performed on model systems to investigate the hydrogen bonds and proton transfer between antiarrhythmics and polar groups of the cell membrane. Methylamine cation, dimethyl ether, and N-methylacetamide served as models of associative sites for the antiarrhythmics mexiletine and tocainide. Formate and phosphate anions, the methylamine cation, and formamide were chosen as models for the membrane polar groups. Protonated methylamine forms a very strong complex with the formate and phosphate anions. However, the formate COO- group is a better proton acceptor than the phosphate PO4- group. The effect of specific hydration on the proton potential functions was investigated in the HCOO- ... +HNH2CH3 and H2PO4- ... +HNH2CH3 systems. The proton potential functions, calculated at the equilibrium distances RO ... N, with a single minimum were found. The ab initio calculations at the longer RO ... N = 0.275 nm distance indicate double-minimum potentials. The increasing hydration stabilizes a second minimum corresponding to the charged O- ... +HN structures. The complexes involving the amide and ether groups of tocainide and mexiletine and the protonated primary amine group of the membrane are considerably weaker. The weakest hydrogen bonds are formed by the amine group of the drug (in its neutral and ionized state) with the peptide group.     

Attachment of rod-like (BAR) proteins and membrane shape

Previous studies have shown that cellular function depends on rod-like membrane proteins, among them Bin/Amphiphysin/Rvs (BAR) proteins may curve the membrane leading to physiologically important membrane invaginations and membrane protrusions. The membrane shaping induced by BAR proteins has a major role in various biological processes such as cell motility and cell growth. Different models of binding of BAR domains to the lipid bilayer are described. The binding includes hydrophobic insertion loops and electrostatic interactions between basic amino acids at the concave region of the BAR domain and negatively charged lipids. To shed light on the elusive binding dynamics, a novel experiment is proposed to expand the technique of single-molecule AFM for the traction of binding energy of a single BAR domain.     

Ion channel formation and membrane-linked pathologies of misfolded hydrophobic proteins: the role of dangerous unchaperoned molecules

1. Protein-membrane interaction includes the interaction of proteins with intrinsic receptors and ion transport pathways and with membrane lipids. Several hypothetical interaction models have been reported for peptide-induced membrane destabilization, including hydrophobic clustering, electrostatic interaction, electrostatic followed by hydrophobic interaction, wedge x type incorporation and hydrophobic mismatch. 2. The present review focuses on the hypothesis of protein interaction with lipid membranes of those unchaperoned positively charged and misfolded proteins that have hydrophobic regions. We advance the hypothesis that protein misfolding that leads to the exposure of hydrophobic regions of proteins renders them potentially cytotoxic. Such proteins include prion, amyloid beta protein (AbetaP), amylin, calcitonin, serum amyloid and C-type natriuretic peptides. These proteins have the ability to interact with lipid membranes, thereby inducing membrane damage and cell malfunction. 3. We propose that the most significant mechanism of membrane damage induced by hydrophobic misfolded proteins is mediated via the formation of ion channels. The hydrophobicity based toxicity of several proteins linked to neurodegenerative pathologies is similar to those observed for antibacterial toxins and viral proteins. 4. It is hypothesized that the membrane damage induced by amyloids, antibacterial toxins and viral proteins represents a common mechanism for cell malfunction, which underlies the associated pathologies and cytotoxicity of such proteins.