Peptides with antimicrobial properties and their analogs created through modification.

Summary: The observed increase in antibiotic resistance is currently one of the main challenges in modern medicine. Improper use and overuse of available preparations have significantly weakened their effectiveness due to the emergence of an increasing number of resistant microorganisms. Contemporary research aims to develop more effective therapeutic agents targeting pathogenic organisms based on natural defense mechanisms (antimicrobial peptides) and their possible modifications, producing their analogues.

Keywords: antimicrobial peptides; chemical modifications; cyclization; drug conjugates; lipidation

List of abbreviations: AMP – antimicrobial peptides

Concept of drug resistance

Drug resistance refers to the resistance that pathogens and parasites have to the action of drugs. This means that these pathogens can live and reproduce in the presence of a drug that should destroy or inhibit them, but this does not happen. Drug resistance can be divided into two types: innate resistance and acquired resistance. While the first is a typical trait of microbes, acquired resistance results from contact with the drug, through changes in their DNA material, leading to the development of resistance to the drug.

Antimicrobial peptides – characteristics

Antimicrobial peptides (AMP) are a group of compounds composed of 10 to 50 amino acid residues. The net charge, ranging from +2 to +9, results from the presence of L-arginine, L-lysine, or L-histidine residues in the peptide chain. AMP synthesis can occur in two ways. The first is ribosomal translation of mRNA occurring in all organisms, while the second is non-ribosomal peptide synthesis carried out mainly by bacteria. Peptides synthesized via non-ribosomal synthesis, such as polymyxin- and gramicidin S-based antibiotics, are widely used for their antimicrobial action. Increasingly, however, due to their properties stimulating innate immunity, peptides produced by ribosomal synthesis are being applied. Antimicrobial peptides are isolated from various organisms.

Defensins as animal AMPs

Most antimicrobial peptides have been isolated from fish, amphibians, and mammals. The greatest amounts are found in phagocytes, neutrophils, macrophages, and secretions of epithelial cells. Among the compounds with the highest biocidal activity are defensins, due to their ability to modulate the host organism’s immune response. Defensins are amphipathic antimicrobial peptides rich in basic amino acid residues and L-cysteine, found in animal and plant organisms. Their biocidal activity targets a wide range of Gram-positive and Gram-negative bacteria and fungi. There are three classes of defensins: α-, β-, and θ-, which differ in the topology of their disulfide bridges.

The best-known α-defensins are HNP1-4, produced mainly in the placenta, cervix, and intestinal mucosa; HD5 and HD6, found in salivary glands, the walls of the digestive and urinary tracts, and the eye mucosa; and NP5, present in Paneth cells. β-defensins form the most diverse class of AMPs, due to their longest evolutionary development, as they have been detected in the genetic material of all classified vertebrates so far. The most recently discovered are θ-defensins, including peptides RTD1-3. Defensins exhibit a broad spectrum of antimicrobial activity, actively participating in the immune defense of organisms; for example, human α-defensin HD5 effectively eliminates infections caused by Salmonella typhimurium and Staphylococcus aureus, while RTD-1 shows biocidal action against Escherichia coli.

Plant AMPs

Antimicrobial peptides are found in all plant species. A characteristic feature of plant AMPs is the presence of L-cysteine residues and several disulfide bridges, which contribute to maintaining a compact structure, ensuring resistance to proteolytic and chemical degradation. Plant AMPs, including particularly thionins, defensins, and cyclotides, consist of 45 to 47 amino acid residues in the chain. Two subgroups of thionins are distinguished: 8c, which have eight L-cysteine residues in the sequence forming four disulfide bridges, and 6c, which have six such residues and three disulfide bonds accordingly.

Antimicrobial peptides – properties

As an innovative method for treating drug resistance, antimicrobial peptides are increasingly and more successfully used. They show high activity against Gram-negative and Gram-positive bacteria, viruses, and fungi. Additionally, antimicrobial peptides can neutralize bacterial toxins, inhibit pro-inflammatory reactions and biofilm formation processes, and accelerate wound healing.

Mechanism of AMP entry into the cell

AMP entry into bacterial cells can occur through various mechanisms. In most cases, it involves disintegration of microbial cell membranes during lysis, through electrostatic and hydrophobic interactions between positively charged fragments of L-arginine or L-lysine residues and negatively charged areas of bacterial membranes. Three main models describe how antimicrobial peptides penetrate the outer layers of microorganisms: barrel-stave, carpet, and toroidal pore models.

a) The barrel-stave model is based on the interaction of amphipathic α-helical peptides with the bacterial membrane, forming transmembrane channels or pores with hydrophilic fragments directed inward. This causes the AMP to embed vertically into the lipid framework of the membrane, disrupting the transmembrane potential and ion gradient. These effects inhibit ATP synthesis and increase membrane permeability, leading to cell swelling and osmosis;

b) The carpet model involves the peptide binding to the membrane and forming a "carpet" on its surface. Peptide chains arrange themselves on the membrane exterior so that their hydrophilic regions face the hydrophilic parts of phospholipids, and hydrophobic regions face the membrane core. Due to electrostatic interactions, positively charged peptide chain fragments bind to negatively charged phospholipids, limiting membrane permeability by the peptide carpet structure, eventually destroying the membrane and forming micellar structures;

c) The toroidal pore model is based on AMP aggregation on the lipid bilayer surface, causing it to bend inward. Hydrophilic regions of the peptide chain bind to the polar heads of membrane lipids, leading to membrane disintegration and formation of pores larger than those in the barrel-stave model.

Examples of chemical modifications of AMPs

Despite their many advantages, antimicrobial peptides also have several limitations related to their use, which has led to the design of synthetic analogues containing key sequences for antimicrobial action or based on native AMPs. Below are examples of some of these:

1. Cyclization

Four types of cyclization of the peptide chain in natural AMPs are known: between the N- and C-terminal fragments of the chain; between the N- or C-terminus and a functional group located in the side chain of one of the amino acids in the sequence; and within the side chains themselves (Fig. 4). These processes improve peptide stability, resulting in greater resistance to degradation by proteolytic enzymes. AMP analogues formed by cyclization showed properties such as increased antimicrobial activity against Escherichia coli and Bacillus subtilis strains, biocidal action against Gram-positive bacteria (various strains of Staphylococcus aureus, Enterococcus faecalis, Micrococcus luteus, Bacillus subtilis, Bacillus cereus, Corynebacterium bovis) and Gram-negative bacteria (Escherichia coli, Shigella dysenteriae, Salmonella enteritidis, Proteus vulgaris, Proteus mirabilis, Serratia marcescens, Pseudomonas aeruginosa, Klebsiella pneumoniae), and use in treating skin burns, postoperative wound care, and infection prevention.

2. Drug conjugates

Another type of chemical modification of AMPs is covalent bonding with antibiotics, which improves their antimicrobial effect and reduces the therapeutic drug dose, thereby eliminating side effects. AMP analogues formed by drug conjugation showed properties such as increased antimicrobial activity against Escherichia coli and Bacillus subtilis strains, biocidal action against Gram-positive bacteria, no toxicity to epithelial cells and human red blood cells, biocidal action against staphylococcal strains, and use in treating community-acquired pneumonia, acute bacterial sinusitis, and pyelonephritis.

3. Lipidation

One of the important post-translational modifications is lipidation, which, besides regulating peptide and protein functions, also increases their affinity for cell membranes. The use of designed analogues depends on the amount and type of attached fatty acids and the length of carbon chains. Incorporating lipid groups into peptide chains allows, among other things, changes in water solubility of newly synthesized compounds, their self-organization ability, and thermal stability. AMP analogues formed by lipidation showed properties such as increased antimicrobial activity against Gram-positive bacteria (Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Enterococcus faecalis), Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris, Pseudomonas aeruginosa), and fungi (Candida albicans, Candida tropicalis, and Aspergillus brasiliensis).

Summary

One of the significant problems in modern medicine is the frequent use of antibiotics, which results in the formation of new species of microorganisms resistant to them. A way to eliminate this growing problem may be the use of antimicrobial peptides, which are part of the innate immune system. The term AMP most often refers to compounds with a positive charge and amphipathic structure, responsible for modulating their antimicrobial properties against a wide range of bacteria, viruses, and fungi. High production costs and limited bioavailability of natural AMPs have necessitated the search for new model compounds whose action is based on known mechanisms.

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