Progress in research on peptide antibiotics

Progress in the study of peptide antibiotics <br>Sun Chao Wang Hui Sun Bo Peng Xuexian

Abstract Polypeptide antibiotics are a class of biologically active small peptides widely found in the biological world. They generally have antibacterial or fungal effects, and some have the function of antigenic insects, viruses or cancer cells. According to the chemical structure, peptide antibiotics can be divided into five categories: 1 linear polypeptide with helical structure; 2 linear polypeptide rich in certain amino acids; 3 polypeptide containing one disulfide bond; 4 containing two or more two Sulfur-bonded peptide; 5 lantibiotic. Depending on the mechanism of action, peptide antibiotics can be further divided into lytic peptides and non-lytic peptides that cleave cell membranes. Peptide antibiotics have begun to be used in medicine, food and plant disease resistance genetic engineering, and have great potential for development.

The polypeptide antibiotic refers to a polypeptide substance having an antibacterial activity with a relative molecular mass of usually 1 × 104 or less. However, because the distinction between small peptides and proteins is not very strict, different scholars have different views on the molecular weight upper limit of peptide antibiotics. Initially, such antibacterially active polypeptides were referred to as "antibacterial peptides", and Chinese translations were "antibacterial peptides". The original meaning should be "antibacterial peptides". Later, some "antibacterial peptides" were found to have anti-fungal and other microbial functions, which are called "antimicrobial peptides". However, with the deepening of research, people have found that these peptides also have anti-parasitic, viral, cancer cell and other functions, especially with the application of such peptide substances in medicine, many scholars tend to call it "peptide". Antibiotics" - "polypeptide antibiotics". Perhaps because almost all of the peptide antibiotics have anti-bacterial functions, "antibacterial peptides" as a customary name are still widely used in China. Unless otherwise specified, the polypeptide antibiotics herein specifically refer to polypeptide antibiotics encoded by genes and synthesized on ribosomes. Properties of polypeptides synthesized by enzymatic reactions during metabolic processes

The real rise of peptide antibiotic research began in the early 1980s. In 1980, Boman et al. isolated the antibacterial-active peptide cecropins from the American silkworm, and published its amino acid sequence in Nature the following year. Also in 1980, Lefur et al. isolated another peptide antibiotic, defensins, from rabbit macrophages and published its amino acid sequence three years later. In the following years, people have discovered peptide antibiotics from bacteria, fungi, to amphibians, insects, higher plants, mammals, and humans. At present, there are more than 170 kinds of peptide antibiotics derived from insects. For the convenience of research, they were classified according to the structure of the polypeptide antibiotics.

1 Classification of peptide antibiotics

1.1 The linear polypeptide cecropins with helical structure was the first animal peptide antibiotic found. In 1980, it was isolated from Bombyx mori by Boman et al. The polypeptide antibiotics generally contain 37 to 39 amino acid residues, do not contain cysteine, and have a strong basic N-terminal region, which can form a nearly perfect parental helix structure, and a hydrophobic helix can be formed in the C-terminal region. There is a hinge region formed by glycine and proline. The C-terminus of most polypeptides is amidated, and amidation plays an important role in its antibacterial activity. Since then, people have isolated cecropins peptide antibiotics from silkworm, tussah, fruit fly, and flies. In 1989, Lee et al. isolated cecropin P1 from the small intestine of pigs, indicating that cecropins may be widely present in animals. Cecropins have a strong killing effect on Gram-positive and Gram-negative bacteria, but not on fungi and eukaryotic cells. Currently cecropins have been artificially synthesized and commercialized.
Magainins are also a class of peptide antibiotics with a parental helix structure discovered earlier. It was originally isolated from the skin of sputum and later found in mammalian nerve and intestinal tissues. Magainins has a killing effect on Gram-negative bacteria, positive bacteria, fungi and protozoa, but the activity against Gram-negative bacteria is about 10 times lower than that of cecropins.

In addition, some spiral-structured peptides were isolated from the regenerative organs of some animals and various organelles of amphibians, such as dermaseptin derived from the South American frog and bombinnh derived from the tree frog.

1.2 Linear polypeptides rich in certain amino acids apidaenecs are proline-rich peptide antibiotics isolated from honeybees, generally containing 16 to 18 amino acid residues, of which the proline content is as high as 33%, and the arginine content can be Up to 17%. Typical motifs with PRP and PP in their primary structure. Apideecins are highly active against certain Gram-negative bacteria and not on Gram-positive bacteria. The high lethality of apidaecins to certain Gram-negative plant pathogens and Enterobacteriaceae pathogens has a good application prospect in plant antibacterial genetic engineering and food industry.

Drosocin is a proline-rich peptide antibiotic derived from Drosophila. It is structurally similar to apidaecin, but has an O-disaccharide chain attached to its threonine hydroxyl group at position 11 (- N-acetylgalactosamine-galactose).

Coleoptericin and hemiptericin are derived from Coleoptera and Hemiptera, respectively. The primary structure is rich in glycine and the molecular weight is generally large. Oppenheim et al. isolated a group of histidine-rich peptide antibiotics from human parotid and mandibular gland secretions ranging in length from 7 to 38 amino acid residues, known as hisatins. It is active against a variety of microorganisms that cause oral infections. Indolicidin is a peptide antibiotic derived from bovine neutrophils and is named for its five amino acids in 13 amino acids. Its C-terminus is amidated. It has strong bactericidal activity against Escherichia coli and Staphylococcus aureus.

1.3 Polypeptides containing a disulfide bond This is a small number of peptide antibiotics. The first such peptide was found to be bactenecin, derived from bovine neutrophils [5]. It contains 4 arginine in its 12 amino acids and forms a disulfide bond between its 2nd and 11th amino acid residues. Bactenecin is active against Escherichia coli and Staphylococcus aureus.

Some of these peptides also include peptide antibiotics derived from frog skin, usually with a "loop" of 7 amino acids and a long N-terminal "tail" at the C-terminus, such as brevinin-1, brevinin-2.

1.4 Polypeptides containing two or more disulfide bonds A typical representative of such polypeptides is defensins. The α-defensins originally discovered are derived from mammalian tissues and generally contain 29 to 34 amino acid residues, of which 6 are conservative. The cysteine ​​forms three intramolecular disulfide bonds, and in addition, the arginine at positions 6 and 15 and the glycine at position 24 are also conserved. Α-defensins form a 3-layer beta sheet structure that is stabilized by three disulfide bonds and a salt bridge between Arg-6 and Glu-24. Currently, defensins has been synthesized and commercialized. Defensins has a bactericidal effect on a variety of bacteria and certain fungi, and is somewhat toxic to eukaryotic cells. Defensins is more active against Gram-positive bacteria than Gram-negative bacteria. Defensins are less active than cecropins and usually function at low ionic strength.

--defensins are larger than α-defensins and generally contain 38 to 42 amino acid residues. Both contain 3 disulfide bonds and 4-8 arginine.

Insect defensins are similar to α-defensins at the C-terminus, but have only two beta-sheet structures with an alpha-spinning stabilizing effect in between. It mainly acts on Gram-positive bacteria and has no effect on fungi.

Plant defensins generally have 45 to 54 amino acid residues, which can form 4 disulfide bonds, 3 beta sheet structures and an alpha helix structure. Plant defensins generally only work on fungi and have no effect on bacteria. The antibacterial spectrum of different plant defensins on fungi is different.

Thionins are also a class of plant-derived polypeptide antibiotics containing 45 to 47 amino acid residues and 3 or 4 disulfide bonds formed by 6 or 8 cysteines. Its secondary structure can form two anti-parallel alpha helix structures and two anti-parallel beta sheet structures. Thionins inhibit a variety of phytopathogenic bacteria and fungi, but do not work against bacteria of the genus Pseudomonas and Erwinia.
1.5 Lantibiotics Lantibiotics are peptide antibiotics produced by bacteria, encoded by genes, synthesized in ribosomes, and processed by translation to contain specific organic groups. The most widely studied of these is nisin. It is a polypeptide antibiotic derived from lactic acid bacteria. The mature polypeptide consists of 34 amino acids and contains special groups such as lanthionine and methyllanthionine. It mainly acts on Gram-positive bacteria and does not work on Gram-negative bacteria. Has been widely used as a food preservative. The application of nisin and its analogues in medicine is also underway.

2 Biological activity of peptide antibiotics

2.1 Peptide Antibiotics Killing Bacteria Most peptide antibiotics have antibacterial effects. At present, it is believed that many polypeptide antibiotics such as cecropins, magainins, defensins, etc., which form a parental helix structure, form an ion channel on the membrane by acting on the cell membrane of the bacteria, causing leakage of intracellular substances to kill the bacteria. A study of sarcotoxinIA found that when cholesterol is present in liposomes, the cleavage of sarcotoxinIA is diminished. This may partly explain why such polypeptides do not work for eukaryotic cells.

The mechanism of action of apidaecins peptide antibiotics on bacteria is a non-cleavage mechanism, possibly by interacting with chiral molecules on the membrane to kill bacteria.

2.2 Pesticide antibiotics killing fungi Many peptide antibiotics have antibacterial activity in addition to their antibacterial activity. For example, PGQ derived from frogs, dermaseptin and mammalian defensins have a killing effect on some human pathogenic fungi, and rabbit defensinNP-1 also has a role in pathogenic fungi of corn. Some plant-derived polypeptide antibiotics, such as plant defensins, thionins, etc., have a killing effect on a variety of phytopathogenic fungi. Recently, Cavallari et al. found that the 11 amino acid sequence of the N-terminal alpha helix region of the cecropin derivative is related to antifungal activity.

2.3 The role of peptide antibiotics on protozoa Some peptide antibiotics can effectively kill parasites that are parasitic on humans or animals. For example, Shiva-I (an analog of cecropin) can kill Plasmodium; a hybrid peptide of cecropin/melittin can kill Leishmania flagellates.

2.4 The role of peptide antibiotics on viruses It has now been found that peptide antibiotics can act as antivirals in three different mechanisms. The first is to work directly with virions by a polypeptide antibiotic. The effects of α-defensins, modelin-1 on herpesviruses, and the effects of polyphemusins ​​on HIV. The second is to inhibit the proliferation of viruses, such as the effects of mellitin and cecropin A on HIV. The third mechanism works by mimicking the infection process of the virus. For example, the structure of melititn and its analog K7I has similarity to the region in which the tobacco mosaic virus nucleocapsid interacts with mRNA, and exerts an effect on the virus by interfering with the assembly of the virus.

2.5 The role of peptide antibiotics on cancer cells Many studies have shown that tumor cells are more sensitive to peptide antibiotics than normal cells. The reasons for this difference are not fully understood. It is believed to be related to the following factors: 1 due to tumor cells High metabolism causes changes in cell membrane potential; 2 the outer surface of tumor cells contains higher acidic phospholipids; 3 changes in the cytoskeleton or extracellular matrix of tumor cells. It has been found that cecropin and its analogs, magainin2 and its analogs, cecropinA-magainin2, cecropinA-melittin hybrid peptides and their analogues have selective killing effects on tumor cells.

3 Synthesis of peptide antibiotics

3.1 Chemical synthesis

In order to study the mechanism of action of polypeptide antibiotics, many natural polypeptide antibiotics and analogs thereof have been obtained by solid phase synthesis. The most studied of these is a positively charged polypeptide with a parental helix. The conservation of the primary structure of the polypeptide antibiotic can be studied by altering or adding or deleting amino acids at certain positions of the polypeptide. In studying the interaction of such polypeptide antibiotics with bacterial cell membranes, the enantiomers of the native polypeptide were synthesized using D-type amino acids. The natural polypeptide forms a left-handed helix, and its enantiomer forms a right-handed helix. It is found that the enantiomer formed by the D-form amino acid has the same biological activity as the native polypeptide, thereby further demonstrating that the amphiphilic helix has the biological activity of the polypeptide. Important role. In order to find polypeptide antibiotics with higher antibacterial activity or broader antibacterial spectrum, some hybrid peptides are synthesized one after another. For example, a hybrid peptide CN (1 to 13) MN (1 to 13) composed of the first 13 amino acids of cecropin and melititn has strong antibacterial activity without hemolytic activity. In order to save costs, people are also trying to find shorter peptides with antibacterial activity. The chemical synthesis method can conveniently change the primary structure of the polypeptide during the synthesis process, add special amino acids, and modify the end of the polypeptide, but the expensive cost is the biggest obstacle to limit the industrial application of the method.
3.2 Genetic Engineering Synthesis

The use of genetic engineering methods to produce peptide antibiotics is an effective way to reduce production costs. However, the toxicity of peptide antibiotics to prokaryotic cells limits their use in prokaryotic expression systems to some extent. The low expression efficiency of eukaryotic expression systems is also an obstacle to their industrial production. In order to overcome the toxicity of the polypeptide antibiotic to bacterial cells, prokaryotic expression is carried out using a fusion expression or selection of a strain resistant to the polypeptide antibiotic. The earliest Jaynes expressed the Shiva-I gene in E. coli in 1989. In 1993, Piers et al. fused the defensin, HNP-1, and cecropin/melittin hybrid peptide genes to four different carrier proteins and expressed them in E. coli. The yield, cell localization and protein degradation of the expressed products were systematically studied. After chemical cleavage or enzymatic hydrolysis of some of the fusion proteins, small peptides having antibacterial activity are obtained.

In 1993, Maeno et al. secreted a fusion protein expressing apidaecin using a Streptomyces expression system, and obtained an active product after cleavage. In 1992, Reichhart et al. successfully expressed the green flies defensin A with the correct disulfide pairing in yeast, with a yield of up to 2.5 μg per ml of medium. In 1991, Andersons et al. used the polyhedrosis virus expression system to express the fusion protein of cecropinA in insect cell lines. No amidation was observed at the C-terminus, and later expressed by living insects, the yield was increased by 60-fold, and some products were amidated. In China, Xie Wei et al expressed the silkworm polypeptide antibiotic CMIV in E. coli. In 1999, Shen Junqing et al. expressed an analog of cecropin in yeast.

Tab 1 Therapeutic status of peptide antibiotics

Company Peptide Clinical indication Stage of development
Magainin Pharmaceuticals MSI-78(α-helical) Impetigo Abandoned after phase III (1997)
MSI-78 Topical treatment of diabetic foot ulcers PhaseIII (1997)
Applied Microbiology/Astra/Merck Nisin (lantibiotic) Gastric Helicobacter infection/ulcers Early clinical trails (1997); PhaseI (1998)
Applied Microbiology/Nippon/ Shoji Nisin variants Vancomycin-resistant enterococci (parenteral) Preclinical research (1997)
Micrologix Biotech MBI-11CN+ Gram-positive infection Preclinical research (1997)
MBI-20 series(α-helical) Gram-negative infection; enhancers of conventional antibiotics Research and development (1997)
Intrabiotics IB367 (β-sheet) Topical treatment of oral mucositis(muoth ulcerations) Preclinical research (1997); PhaseI (1998)
Xoma Mycoprex (BPI-derived) Systemic candidiasis; enhancer of fluconazole activity Preclinical research (1997); Phase II completed, Phase III initated (1998)

4 Application and prospects of peptide antibiotics

4.1 Application and prospect of peptide antibiotics in the pharmaceutical industry

At present, all the conventional antibiotics have corresponding resistant strains, and the resistance of pathogenic bacteria has become a serious threat to people's health. Finding new types of antibiotics is an effective way to address drug resistance problems. Polypeptide antibiotics are considered to have broad application prospects in the pharmaceutical industry because of their high antibacterial activity, wide spectrum of antibacterial spectrum, wide variety, wide range of options, and difficulty in producing resistance mutations. At present, a variety of peptide antibiotics have been undergoing preclinical feasibility studies, of which magainins has entered the phase III clinical trial phase. See Table 1 for the progress of some peptide antibiotics in medical research.

Most clinical trials are now used for topical treatments, which should be safe and effective because some of the more toxic peptides and lipopeptides, such as gramicidin S, polymyxin B, have been used to make skin ointments. . These polypeptides can also be used where conventional antibiotics and conventional therapies are ineffective. The use of powders to treat lung infections is a promising development. Oral medications may be used to treat intestinal infections, and nisin is conducting clinical trials of anti-helicobacter. At least two companies are developing treatments for parenteral administration. However, if the peptide antibiotic is to be used clinically, the following problems must be solved: the toxicity, stability, immunogenicity, application method, and drug formulation of the polypeptide antibiotic. Drug formulations are a major problem affecting the effectiveness of treatment. In addition, the degradation of polypeptides by proteases in vivo, especially trypsin, and the absorption of peptides by different tissues and organs have yet to be further studied.

4.2 Application and prospects of peptide antibiotics in other aspects

Since some peptide antibiotics are highly resistant to some phytopathogenic bacteria and fungi, some polypeptide antibiotics have been used for plant disease resistance genetic engineering. For example, Jaynes et al. transferred two cecropin analog genes, Shiva-I gene and SB-37 gene into tobacco, and found that Shiva-I transgenic tobacco has certain resistance to bacterial wilt, while SB-37 transgenic tobacco has no Resistance. Huang et al. showed that the cecropin-like polypeptide MB-39 gene was fused with the barley alpha amylase signal peptide gene and then transferred into tobacco, and the resulting plants were more resistant to wildfire disease. In China, Huang Danian used the cecropinB gene to transform rice, and obtained some plants with different resistance to rice stripe disease.

Some progress has been made in the study of transgenic antibiotics for peptide antibiotics. For example, genetic engineering can be used to block the spread of some insect-borne diseases. Possani et al. [21] showed that expression of Shiva-3 in mosquitoes can inhibit malaria. Spread, but there are still some difficulties in the transgenic technology of mosquitoes; Durasula et al. significantly reduced the number of trypanosomes in the body by expressing Cecropin A in the symbiotic bacteria of the long red scorpion. Reed et al. transferred Shiva-Ia into mice, and the resistance of transgenic mice to Brucella was significantly enhanced, which provided a new idea for artificial breeding of new varieties of disease-resistant animals. In addition, the application of peptide antibiotics in food preservation, flower preservation and animal feed additives is also in progress.

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