Peptides with antimicrobial properties and their analogues created through modification.

Abstract: The observed increase in antibiotic resistance is currently one of the main problems in modern medicine. Inappropriate use and abuse of available preparations has significantly weakened their effectiveness due to the emergence of an increasing number of resistant microorganisms. Research is currently being conducted to develop more effective therapeutic agents that act on pathogenic organisms according to natural defence mechanisms (antimicrobial peptides) and their possible modifications, obtaining their analogues.

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

List of abbreviations: AMP – antimicrobial peptides

The concept of drug resistance

Drug resistance is a term referring to the resistance of pathogens and parasites to the effects of drugs. This means that these pathogens have the ability to 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 and acquired. While the former is a characteristic typically found in microbes, acquired resistance is the result of contact with a drug, through a change in their DNA, which causes resistance to the drug.

Peptides with antimicrobial properties – characteristics

Antimicrobial peptides (AMPs) are a group of compounds consisting 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 proceed in two ways. The first is through ribosomal mRNA translation, which occurs in all organisms, while the second is through non-ribosomal peptide synthesis, which is mainly carried out by bacteria. Peptides synthesised by non-ribosomal synthesis, which include polymyxin- and gramicidin S-based antibiotics, are widely used for their antimicrobial activity. However, due to their innate immune-stimulating properties, peptides produced by ribosomal synthesis are increasingly being used. Antimicrobial peptides are isolated from various organisms. (Fig. 1)

Figure 1. Organisms from which antimicrobial peptides (AMPs) were isolated

Defensins as animal AMPs

Grupa peptydów przeciwdrobnoustrojowych w większości została wyizolowana z ryb, płazów i ssaków. Największą ilość zaobserwowano w fagocytach, neutrofilach, makrofagach i wydzielinach komórek nabłonkowych. Do związków z największą aktywności biobójczą należą defensyny, ze względu na ich właściwości pozwalające na modulowanie odpowiedzi immunologicznej organizmu gospodarza. Defensyny są amfipatycznymi peptydami przeciwdrobnoustrojowymi będącymi związkami bogatymi w reszty aminokwasów zasadowych oraz L-cysteiny, występującymi w organizmach zwierzęcych i roślinnych. Ich aktywność biobójcza skierowana jest względem szerokiej gamy bakterii Gram-dodatnich, Gram-ujemnych i grzybów. Wyróżniamy trzy klasy defensyn: α-, β- i θ-, które różnią się między sobą topologią mostków disulfidowych (Rys.2)

Figure 2. Three basic classes of defensins found in the environment

The most well-known α-defensins are: HNP1-4, produced mainly in the placenta, cervix and intestinal mucosa; HD5 and HD6, found in the salivary glands, gastrointestinal tract, urinary tract and ocular mucosa; and NP5, present in Paneth cells. β-defensins are the most diverse class of AMPs, due to their longest evolution, as they have been detected in the genetic material of all vertebrates classified to date. The most recently discovered are θ-defensins, which include the RTD1-3 peptides. Defensins exhibit a broad spectrum of antimicrobial activity, actively participating in the immune defence of organisms. For example, human α-defensin HD5 effectively eliminates infections caused by Salmonella typhimurium and Staphylococcus aureus, while RTD-1 exhibits biocidal activity against Escherichia coli.

Plant peptides AMP 

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 proteolytic and chemical resistance. Plant AMPs, which include thionins, defensins, and cycloids, are composed of 45 to 47 amino acid residues in a chain. There are two subgroups of thionins, namely 8c, which have eight L-cysteine residues in the sequence and form four disulfide bridges, and 6c, which have six such residues and three -S-S bonds, respectively.

Antimicrobial peptides – properties

Antimicrobial peptides are increasingly being used as an innovative method of treating drug resistance, with growing success. They exhibit high activity against Gram-negative and Gram-positive bacteria, viruses and fungi. In addition, peptides with antimicrobial properties have the ability to neutralise bacterial toxins, inhibit pro-inflammatory reactions and biofilm formation processes, and accelerate wound healing.

The mechanism of AMP penetration into the cell

AMP penetration into bacterial cells can occur through various mechanisms. In the vast majority of cases, the cell membranes of microorganisms disintegrate in the process of lysis due to electrostatic and hydrophobic interactions between positively charged fragments of L-arginine or L-lysine residues and negatively charged areas of bacterial membranes. There are three main models of antimicrobial peptide penetration through the outer membranes of microorganisms: barrel, carpet and toroidal (Fig. 3).

Figure 3. Diagram of mechanisms of cell membrane disintegration by AMP: a) barrel-stave model b) carpet model c) toroidal model

a) The barrel clamp model is based on the interaction of amphipathic peptides with an α-helical structure with the bacterial membrane, with the formation of transmembrane channels or pores with hydrophilic fragments directed towards their interior. This causes AMP to embed itself vertically into the lipid skeleton of the membrane and disrupt the transmembrane potential and ion gradient. As a result of these phenomena, ATP synthesis is inhibited and membrane permeability increases, leading to cell swelling and osmosis; b) The carpet model involves binding the peptide to the membrane and forming a ‘carpet’ on its surface. The peptide chains are arranged on the outside of the membrane in such a way that their hydrophilic regions face the hydrophilic fragments of phospholipids, and their hydrophobic regions face the core of the membrane. As a result of electrostatic interactions, the positively charged fragments of the AMP peptide chain bind to negatively charged phospholipids, limiting the permeability of the membrane through the peptide carpet structure, and then the membrane is destroyed, ultimately forming micellar structures; c) The toroidal pore model is based on the fact that AMPs aggregate on the surface of the lipid bilayer, causing it to bend inwards. The hydrophilic regions of the peptide chain bind to the polar heads of the membrane lipids, leading to the disintegration of the membrane and the formation of pores larger than in the barrel-staple model.

Examples of chemical modifications of AMP

Despite their numerous advantages, antimicrobial peptides also have many limitations related to their use, which has led to the design of synthetic analogues containing a sequence that is key to antimicrobial activity or based on native AMPs. Below are some examples of these:

 1. Cyclisation

There are four known types of peptide chain cyclisation in natural AMPs: between the N- and C-terminal fragments of the chain, the N- or C-terminus of the peptide chain and a functional group located in the side chain of one of the amino acids present in the sequence, and within the side chains themselves (Fig. 4). These processes result in improved peptide stability, which translates into greater resistance to degradation by proteolytic enzymes. AMP analogues resulting from cyclisation modification exhibited properties such as increased antimicrobial activity against Escherichia coli and Bacillus subtilis strains, biocidal activity 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) or the use of this AMP analogue in skin burns, postoperative wound care and infection prevention.

Figure 4. An example of AMP modification, namely cyclisation

2. Conjugates with drugs

Another type of chemical modification of AMP is covalent binding with antibiotics, which improves their antimicrobial activity and reduces the therapeutic dose of the drug, thus eliminating the occurrence of adverse effects. AMP analogues resulting from modification, such as drug conjugates, exhibited properties such as: increased antimicrobial activity against Escherichia coli and Bacillus subtilis strains, biocidal activity against Gram-positive bacteria, no toxicity to epithelial cells and human erythrocytes, biocidal activity against staphylococcal strains, and the use of the analogue in the treatment of community-acquired pneumonia, acute bacterial sinusitis and pyelonephritis.

 3. Lipidacja

One of the most important post-translational modifications is lipidation, which, in addition to regulating the function of peptides and proteins, also increases their affinity for cell membranes. The use of designed analogues is determined by the amount and type of attached fatty acids and the length of carbon chains. The incorporation of lipid groups into peptide chains allows, among other things, for changes in the water solubility of newly synthesised compounds, their ability to self-organise and their thermal stability. AMP analogues resulting from lipidation modification exhibited 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 of modern medicine is the frequent use of antibiotics, which results in the development of new, resistant species of microorganisms. One way to eliminate this growing problem may be the use of antimicrobial peptides, which are part of the body's innate immune system. The term AMP is most often used to describe compounds with a positive charge and an amphipathic structure, which is responsible for modulating their antimicrobial properties against a wide range of bacteria, viruses and fungi. The high production costs and limited bioavailability of natural AMPs have necessitated the search for new model compounds whose action is based on previously known mechanisms.

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