FK-13, core sequence of LL-37, is protected from bacterial proteolysis by actin. FK-13 showed activity against HIV-1, EC?? 3.4 μM. As removal of the N-terminal phenylalanine leads to inactivation, FKRIVQRIKDFLR corresponds to the minimal anti-HIV region of human LL-37.

人源性抗菌肽LL-37具有很強(qiáng)的抗菌和抗炎活性，廣譜抗菌效果號(hào)，但由于其分子量比較大，并且在生物體內(nèi)的細(xì)胞選擇性低，加之生產(chǎn)成本高，因此限制了LL-37在治療方面的應(yīng)用。因此開發(fā)出新的LL-37衍生的短α-螺旋抗菌肽，這類序列較短的抗菌肽具有更好的細(xì)胞選擇性，并且與LL-37相比，其抗炎活性沒(méi)有明顯的損失。LL-37 FK-13就是LL-37衍生的短α-螺旋抗菌肽中的一種，由13個(gè)氨基酸組成，具有很高的細(xì)胞選擇性和抗炎活性。研究表明LL-37 FK-13可以通過(guò)滲透細(xì)胞膜和破壞膜的完整性來(lái)殺死微生物細(xì)胞。

抗菌肽介紹一

AMPs是由相對(duì)較小的分子組成的異質(zhì)基團(tuán)，通常含有不到100個(gè)氨基酸。 它們最初是在20世紀(jì)60年代由Zeya和Spitznagel 在多形核白細(xì)胞溶酶體中描述的。 迄今為止，已在數(shù)據(jù)庫(kù)（如數(shù)據(jù)庫(kù)）中 確定和登記了2600多個(gè)AMP。  它們是由幾乎所有的生物群產(chǎn)生的，包括細(xì)菌、真菌、植物和動(dòng)物。 許多脊椎動(dòng)物AMPs是由上皮表面分泌的，如 哺乳動(dòng)物的氣管、舌、腸粘膜或兩棲動(dòng)物的皮膚。 有些在中性粒細(xì)胞、單核 細(xì)胞和巨噬細(xì)胞中表達(dá)。 AMPs參與動(dòng)物和植物的免疫防御系統(tǒng)。 構(gòu)成表達(dá)或誘導(dǎo)它們?cè)诘钟⑸锶肭终?的第一道防線中起著關(guān)鍵作用。

結(jié)構(gòu)/分類 AMPs可以根據(jù)其氨基酸組成和結(jié)構(gòu)進(jìn)行分類。 可以區(qū)分兩大類AMP。

第一類由線性分子組成，它們要么傾向于采用α螺旋結(jié)構(gòu)，要么富含精氨酸、甘氨 酸、組氨酸、脯氨酸和色氨酸等某些氨 基酸。

第二類由含半胱氨酸的肽組成， 可分為單一或多個(gè)二硫結(jié)構(gòu)。 在許多情 況下，抗菌活性需要存在二硫橋。 大多數(shù)AMPs是陽(yáng)離子肽，但也有陰離子肽，如真皮素，一種富含天冬氨酸 的人肽和兩棲動(dòng)物的最大蛋白H5皮膚。 其他非陽(yáng)離子AMPs包括神經(jīng)肽前體分子的片段，如原啡肽A， 芳香二肽主要從二翅目幼蟲中分離出來(lái)，或從節(jié)肢動(dòng)物或茴香物種的氧結(jié)合 蛋白中提取的肽。

專肽生物可定制合成各類序列的抗菌肽，可標(biāo)記FITC/FAM/TAMRA等常見(jiàn)熒光素。

Definition

Antimicrobial peptides (AMPs) are as widespread as bacterial inactivator molecules in the innate immune systems of insects, fungi, plants, and mammals. These peptides are also known as host defense peptides (HDPs) as they have other immuno-modulatory functions besides the direct antimicrobial actions and are even capable of killing cancerous cells 1,2. 

Classification

Three broad categories of HDPs have been identified: 1) the linear peptides with helical structures, 2) the cysteine stabilized peptides with beta-sheet, and 3) a group of linear peptides rich in proline and arginine that primarily have been identified in non-mammalian species. 

Structural characteristics

In mammals, cathelicidins and defensins are the two principal AMP families. Cathelicidins are peptides with a conserved proregion and a variable C-terminal antimicrobial domain. Defensins are the best-characterized AMPs, they have six invariant cysteines, forming three intramolecular cystine-disulfide bonds. 

Mode of action

The mode of action of AMPs elucidated to date include inhibition of cell wall formation, formation of pores in the cell membrane resulting in the disruption of membrane potential with eventual lysis of the cell. These peptides also inhibit nuclease activity of both RNase and DNase. 

Functions

They have a broad ability to kill microbes. AMPs form an important means of host defense in eukaryotes. Large AMPs (>100 amino acids), are often lytic, nutrient-binding proteins or specifically target microbial macromolecules. Small AMPs act by disrupting the structure of microbial cell membranes. It plays an active role in wound repair and regulation of the adaptive immune system. They have multiple roles as mediators of inflammation with impact on epithelial and inflammatory cells, influencing diverse processes such as cell proliferation, wound healing, cytokine release, chemotaxis and  immune induction 3. 

References 

1.     Gottlieb CT, Thomsen LE, Ingmer H, Mygind PH, Kristensen HH, Gram L(2008). Antimicrobial peptides effectively kill a broad spectrum of Listeria monocytogenes and Staphylococcus aureus strains independently of origin, sub-type, or virulence factor expression. BMC Microbiol., 8:205.

2.     Yeaman MR and Yount NY (2003). Mechanisms of Antimicrobial Peptide Action and Resistance.  Pharmocological Reviews, 55(1).

3.     Hanna Galkowska H and Olszewski WL (2003). Antimicrobial peptides – their role in immunity and therapeutic potential. Centr Eur J Immunol., 28 (3):138–141.

抗菌肽介紹二

Ribosomally synthesized antimicrobial peptides (AMPs) constitute a structurally diverse group of molecules found virtually in all organisms. Most antimicrobial peptides contain less than 100 amino acid residues, have a net positive charge, and are membrane active. They are major players in the innate immune defense but can also have roles in processes as chemokine induction, chemotaxis, inflammation, and wound healing. In addition to their antimicrobial effects, many of them show antiviral and antineoplastic activities.

INTRODUCTION
AMPs are a heterogeneous group of relatively small molecules usually containing less than a hundred amino acids. They were first described in the 1960’s by Zeya and Spitznagel in polymorphonuclear leukocyte lysosomes.

To date, more than 2600 AMPs have been identified and registered in databases. They are produced by nearly all groups of organisms, including bacteria, fungi, plants, and animals. Many vertebrate AMPs are secreted by epithelial surfaces such as the tracheal, lingual, or intestinal mucosa of mammals or the skin of amphibia. Some are expressed in neutrophils, monocytes, and macrophages.

AMPs are involved in both animal and plant immune defense systems. Constitutively expressed or induced they play a key role in the first line of defense against microbial intruders.

STRUCTURE/CLASSIFICATION
AMPs can be classified on the basis of their amino acid composition and structure. Two major groups of AMPs can be distinguished. The first group consists of linear molecules which either tend to adopt α-helical structure or are enriched in certain amino acids such as arginine, glycine, histidine, proline, and tryptophan. The second group consists of cysteine-containing peptides which can be divided into single or multiple disulfide structures. In many cases, the presence of disulfide bridges is required for antimicrobial activity.

Most AMPs are cationic peptides, but there are also anionic peptides such as dermcidin, an aspartic acid-rich peptide from human and maximin H5 from amphibian skin. Other non-cationic AMPs include fragments from neuropeptide precursor molecules such as proenkephalin A, aromatic dipeptides primarily isolated from dipteran larvae, or peptides derived from oxygen-binding proteins from arthropod or annelid species.

MODE OF ACTION
Most AMPs act by provoking an increase in plasma membrane permeability. They preferentially target microbial versus mammalian cells. Selectivity is influenced by several factors such as differences in membrane composition: membranes of many bacterial pathogens contain negatively charged lipid moieties such as phosphatidylglycerol (PG), cardiolipin, and phosphatidylserine (PS), whereas mammalian membranes, commonly enriched in phosphatidylethanolamine (PE), phosphatidylcholine (PC) and sphingomyelin, are generally neutral in net charge.

The presence of sterols such as cholesterol and ergesterol within the membrane may be a further means by which AMPs can distinguish between mammalian or fungal cells and prokaryotes. A first step in the mechanism of membrane permeabilization is the electrostatic interaction between the positively charged AMP with the negatively charged membrane surface of the microorganism. Subsequent disruption of the membrane by creation of  pores within the microbial membrane ultimately results in cell death of the organism due to leakage of ions, metabolites, cessation of membrane-coupled respiration, and biosynthesis.

Several models for pore formation such as the Barrel-Stave, the Toroidal or Wormhole Model, and the Carpet Model have been proposed (Fig. 1).

FIG. 1. MODE OF ACTION A BARREL-STAVE MODEL B TOROIDAL PORE OR WORMHOLE MODEL C CARPET MODEL

THE BARREL-STAVE MODEL
The Barrel-Stave model describes a mechanism in which AMPs form a barrellike pore within the bacterial membrane with the individual AMPs or AMP complexes being the staves. Arranged in this manner, the hydrophobic regions of the AMPs point outwards towards the acyl chains of the membrane whereas the hydrophilic areas form the pore.

THE TOROIDAL PORE OR WORMHOLE MODEL
The pores described by this model differ from those of the Barrel-Stave model. Primarily, the outer and inner leaflet of the membrane are not intercalated in the transmembrane channel.

THE CARPET MODEL
A different mechanism is proposed in the Carpet model where AMPs first cover the outer surface of the membrane and then disrupt the membrane like detergents by forming micelle-like units. Certain AMPs penetrate the bacterial membrane without channel formation. They act on intracellular targets by e.g. inhibiting nucleic acid and/or protein synthesis.

RESISTANCE
Resistance to AMPs can either be constitutive or inducible. Inherited resistance mechanisms include altered surface charge, active efflux, production of peptidases or trapping proteins, and modification of host cellular processes. For instance, Staphylococcus aureus manages to reduce the overall cell surface charge by esterification of the cell wall component teichoic acid with D-alanine and thereby increases its resistance against human AMPs. Another example for changing the surface net charge is the production of cationic lysine-substituted phosphatidylglycerol (L-PG) found in certain Staphylococcus aureus strains. In Gram-negative bacteria, addition of 4-aminoarabinose (Ara4N) to the phosphate group of the lipid A backbone or increased acylation of lipopolysaccharides (LPS) are important mechanisms of AMP resistance. Exposure to AMPs may also induce stress responses by which microorganisms try to survive. Inducible regulatory mechanisms have been described in a variety of organisms. For instance, the PhoP/PhoQ regulon in Salmonella has been demonstrated to regulate transcriptional activation of surface and secretory proteins, enzymes that modify lipopolysaccharide, lipid and protein constituents of the outer membrane and proteases that likely degrade certain AMPs.

EXAMPLES OF ANTIMICROBIAL PEPTIDES

ADAPTED FROM K.A. BROGDEN, NAT. REV. MICROBIOL. 3, 238-250 (2005)

IMPORTANT FAMILIES OF AMPS
BOMBININS
Bombinins constitute a family of AMPs produced in fire-bellied toads (Bombina species) active against Gram-negative and Gram-positive bacteria and fungi. Bombinins, bombinin-like peptides (BLPs), and Bombinin H molecules are found in the species Bombina bombina, Bombina variegata, and Bombina orientalis, whereas the homologous maximins and maximin H peptides are derived from the giant fire-bellied toad Bombina maxima. Bombinin H peptides contain either 17 or 20 amino acid residues and are more hydrophobic than bombinins, some of them contain D-alloisoleucine at position 2. They exhibit lower antibacterial activity than bombinins but, in contrast to them, they possess haemolytic activity.

CATHELICIDINS
Members of this family are amphipathic, cationic peptides with a broad-spectrum antimicrobial activity. Cathelicidins typically act by disrupting the integrity of bacterial membranes. They are characterized by an evolutionary conserved N-terminal cathelin- like domain of approximately 99-114 amino acid residues linked to a C-terminal antimicrobial domain of 12-100 residues that can be released upon proteolytic processing. Members of this family include linear peptides amongst them a number of proline-rich AMPs that show different types of proline repeat motifs (Bac5, Bac7, PR-39, prophenins) and the tryptophan-rich indolicidin characterized by three regularly spaced proline residues. The protegrins (PG-1 to PG-5) contain two disulfide bridges and an amidated C-terminus. Cathelicidins have been found in every mammalian species examined. In human, LL-37 (Product 4042456) is the only member of the cathelicidin family. The peptide consists of 37 amino acids and contains two leucine residues at the N-terminus. It is proteolytically cleaved from the 18 kDa precursor protein human cathelicidin antimicrobial protein CAP-18. LL-37 is primarily produced by phagocytic leucocytes and epithelial cells, and is involved in various processes such as direct killing of microorganisms, binding and neutralizing LPS, chemotaxis and chemokine induction, regulation of inflammatory responses, and wound healing. Its production is influenced by several factors such as microbial products, host cytokines, vitamin D3, and availability of oxygen. LL-37 orthologues in mouse and rat are CRAMP (mouse) (Product 4056438) and CRAMP (rat), respectively.

CECROPINS
Cecropins were first isolated from the giant silk moth Hyalophora cecropia. They can form amphipathic, α-helical structures and are structurally related to other cecropins as bactericidin, lepidopteran, and sarcotoxin. Cecropin P1 (Product 4039862), found in pig intestine, also belongs to this family. Most cecropins show broad-spectrum antibacterial activity. Cecropin A (Product 4030488) and B (Product 4030477) have also been demonstrated to possess tumoricidal activity against mammalian leukemia, lymphoma, and carcinoma cell lines.

CERATOTOXINS
This family consists of cationic α-helical amphipathic peptides expressed in the female reproductive accessory glands of the Mediterranean fruit fly Ceratitis capitata. The production of the peptides is enhanced by mating. Ceratotoxin A and ceratotoxin B are 29 amino acid peptides differing in two amino acids. Ceratotoxin C and D consist of 32 and 36 amino acids, respectively. The peptides of this family are active against Gram-negative as well as Grampositive bacteria and are supposed to act via the Barrel-Stave model. Ceratotoxin A has been shown to be mainly antibacterial for Gram-negative organisms.

DEFENSINS
Defensins are small cysteine-rich cationic peptides containing three or four disulfide bridges. They have been isolated from molluscs, acari, arachnids, insects, mammals, and plants. They are further divided into families on the basis of the spatial distribution of their cysteine residues. Three families, the α-, β- and θ-defensins, can be distinguished in mammals. α- and β-defensins are characterized by antiparallel β-sheet structures stabilized by three disulfide bonds. The θ-defensins are found in rhesus monkey and some other non-human primates but not in human, chimpanzee and gorilla. They consist of two nine amino acid peptides derived from different precursor proteins joined by head-to-tail cyclization. Invertebrate and plant defensins contain three or four disulfide  bridges, respectively. Insect and mammalian defensins are mainly active against bacteria while most plant defensins possess antifungal activity.

DERMASEPTINS
The peptides of the dermaseptin family are closely related and consist of 28-34 amino acids. They were originally isolated from skin extracts of the South American arboreal frog Phyllomedusa sauvagei and contain a conserved tryptophan residue at position 3. Dermaseptins exhibit broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria.

HISTATINS
Histatins are histidine-rich and mostly cationic peptides found in the saliva of humans and some higher primates. They are active against a broad-spectrum of bacteria and fungi. The antifungal activity of the human salivary peptide histatin-5 has been extensively studied and is supposed to be due to inhibition of mitochondrial respiration and the formation of reactive oxygen species. Histatin-5 has also been shown to inhibit both host-derived and bacterial proteolytc enzymes involved in peridontal diseases. Histatin-8, a peptide from human parotid secretion, has been shown to inhibit hemagglutination activity of Porphyromonas gingivalis 381, a Gram-negative bacterium involved in certain forms of periodontal disease. The peptide may function as a binding domain for the hemagglutinins of Porphyromonas gingivalis during agglutination.

MAGAININS
Magainins constitute a family of linear amphipathic cationic AMPs discovered in the skin of Xenopus laevis. The two closely related members of this family, magainin I (Product 4012844) and magainin II (Product 4013706) differ merely in two positions and are 23 amino acids in length. Magainins exhibit broad-spectrum antimicrobial activity against Gram-negative and Gram-positive bacteria, fungi and protozoa and are also cytotoxic for many murine and human cancer cell lines.

CONCLUSIONS
The structures of AMPs represent a unique source for the targeted exploration of new applications in the therapy of microbial and viral infection, cancer, and sepsis. Modern synthetic methods will allow the relatively cheap and accurate production of lead compounds and peptide candidates. The achievements in peptide library generation, analytical methods as mass spectrometry, and screening and formulation technologies may contribute to solve intrinsic problems associated with the use of AMPs as therapeutic agents such as susceptibility to proteases and host toxicity. Bachem has considerable expertise and long-standing experience in peptide synthesis. With our capacity to upscale the production of simple and modified peptides, we are the partner of choice for the pharmaceutical industries.