Introduction to Antimicrobial Peptides
Antibiotics, one of the most significant medical advancements of the 20th century, are widely used to treat infectious diseases, safeguarding human health. However, their overuse and misuse in recent years have led to the emergence of bacterial resistance, giving rise to resistant bacteria and superbugs. According to the 2015 report from the National Bacterial Resistance Monitoring Network, methicillin-resistant Staphylococcus aureus (MRSA) was found in 35.8% of common Gram-positive bacteria, and methicillin-resistant coagulase-negative staphylococci reached 79.4%. Additionally, the resistance rate of Escherichia coli to third-generation cephalosporins remains high at 59%. This highlights the growing issue of bacterial resistance, while the development of new antibiotics struggles to keep pace. Pharmaceutical companies have reduced investment in antibiotic research due to high costs, low economic returns, and regulatory hurdles. As a result, the number of new antibiotics continues to decline.
In response, antimicrobial peptides (AMPs) have emerged as a promising solution. These naturally occurring molecules play a crucial role in immune defense across many organisms, offering broad-spectrum antibacterial activity with low toxicity. AMPs disrupt bacterial membranes, making it difficult for bacteria to develop resistance. Their dual nature—positively charged amino acids and hydrophobic residues—enhances their effectiveness. AMPs also exhibit antiviral, antitumor, immune-regulatory, and wound-healing properties. They are not only potential clinical drugs but can also be applied in agriculture and food preservation. AMPs' ability to self-assemble further broadens their potential in drug delivery systems.
Antimicrobial Peptides
Source of Antimicrobial Peptides
The earliest discovered natural antimicrobial peptide was thionin, found in plants. Through experiments, it was confirmed that this peptide inhibited a variety of plant pathogens. It wasn't until the 1980s that antimicrobial peptides, such as Cecropins extracted from silkworm pupae, were reported. Since then, more antimicrobial peptides with biological activity have been isolated from microorganisms, amphibians, plants, mammals, and even humans. Over 2,900 antimicrobial peptides from different organisms have been discovered. Based on an analysis of the antimicrobial peptide database in 2013, it was found that animals (76%), plants (13%), and bacteria (1.8%) are the primary sources of natural antimicrobial peptides.
Bacterial Antimicrobial Peptides
Bacterial antimicrobial peptides, also known as bacteriocins, are produced through ribosomal synthesis during bacterial metabolism. These peptides can effectively inhibit drug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE), with high selectivity. Bacteriocins can be classified into four categories based on their biochemical properties: Class I bacteriocins, Class II bacteriocins, Class III bacteriocins, and Class IV bacteriocins. Class I bacteriocins are small modified peptides consisting of 19-37 amino acids and are also called lanthionine-containing antibiotics. Their active sites typically contain rare amino acid derivatives such as lanthionine, methyl-lanthionine, dehydrotyrosine, and dehydroalanine. Examples include nisin and mersacidin. Class II bacteriocins are unmodified small peptides with thermal stability, including pediocin PA-1 and leucocin A. In contrast to Class I and II, Class III bacteriocins are heat-sensitive large peptides, usually with a molecular weight greater than 30 kDa, such as zoocin A, lysostaphin, and helveticin J. Class IV bacteriocins are complex macromolecules containing lipids or carbon chain compounds, such as plantaricin S and leucocin S.
Plant Antimicrobial Peptides
Antimicrobial peptides are an important part of the non-specific immune defense system, which is especially crucial for plants that lack a specific immune system. Plant antimicrobial peptides exhibit activity against plant pathogens as well as bacterial pathogens that infect humans. Based on differences in amino acids and secondary structures, common plant antimicrobial peptides can be classified into thionins, plant defensins, cyclic peptide proteins, lipid transfer proteins, rubberin, knottins, impatiensin, arabidopsis thionin, and ecdysteroids.
Animal Antimicrobial Peptides
Animal antimicrobial peptides are immune defense components produced by animal bodies in response to external conditions. These peptides not only exhibit excellent antimicrobial activity but also perform various immune regulatory functions within the body, which is why they are also known as host defense peptides. Animal antimicrobial peptides are found in a wide range of organisms and can be further classified into insect antimicrobial peptides, fish antimicrobial peptides, mammalian antimicrobial peptides, and reptilian antimicrobial peptides.
Structure of Antimicrobial Peptides
The primary structure of antimicrobial peptides (AMPs) is characterized by their amino acid sequence, typically consisting of 10 to 60 residues. These peptides often contain lysine and arginine, which interact with water molecules to impart a positive charge, making them cationic. In addition to hydrophilic amino acids like lysine and arginine, AMPs also contain hydrophobic amino acids. This combination enables them to electrostatically interact with the negatively charged bacterial cell membrane, stabilizing their binding. The hydrophobic regions then insert into the membrane, disrupting its structure. This unique mechanism differentiates AMPs from traditional antibiotics.
The secondary structure of AMPs is diverse, and they can be classified into four main types: α-helix, β-sheet, a combination of both, and extended structures. α-Helix peptides typically lack cysteine and are the most common, accounting for 14.63% of known AMPs. β-sheet AMPs usually contain cysteine, forming disulfide bonds that give the peptide a specific folded shape, such as in plant peptide kalata B1. Some AMPs, like human β-defensin HBD-1, contain both α-helix and β-sheet structures. Others, such as bovine indolicidin, have linear chains, referred to as extended structures. Despite this diversity, around 39.92% of AMP structures remain undefined, highlighting the challenges in fully understanding their structure-activity relationship.
Table.1 Antimicrobial peptides at Creative Peptides.
Mechanism of Action of Antimicrobial Peptides
Bactericidal Mechanism of Antimicrobial Peptide
The mechanism of AMPs with broad-spectrum antibacterial activity is still debated, but their action generally relies on interactions with bacterial cell membranes. AMPs are positively charged and electrostatically bind to the negatively charged lipid headgroups of the membrane, stabilizing their adsorption. The hydrophobic regions of AMPs then interact with the membrane's hydrophobic interior, leading to structural changes in the membrane. Four models explain the specific interactions between AMPs and bacterial membranes: the pore model, barrel-stave model, carpet model, and aggregate model.
In the pore model, the hydrophilic part of the AMP binds to the lipid membrane's polar head, while the hydrophobic part interacts with the nonpolar tails. The peptide aggregates and inserts vertically into the membrane, forming pores that cause leakage of genetic material, leading to cell death. AMPs like magainins and LL-37 follow this mechanism. The barrel-stave model involves the peptide forming a "barrel" structure, with the positive charges facing the hollow center. Gramicidin S follows this model.
The carpet model involves AMPs aligning parallel to the membrane surface, disrupting it like detergents when the concentration threshold is reached, forming micelles. In the aggregate model, AMPs form micelle complexes and cross the membrane without a specific orientation. Additionally, some AMPs translocate across the membrane to target cytoplasmic substances, disrupting cellular processes like nucleic acid and protein synthesis.
Selective Mechanism of Antimicrobial Peptides
Antimicrobial peptides exert antibacterial effects in organisms while maintaining good biocompatibility with normal cells, exhibiting excellent selectivity. This property is mainly based on the differences between bacterial cell membranes and mammalian cell membranes. The outer monolayer of the bacterial cell membrane's phospholipid bilayer is rich in negatively charged phospholipid components, and the electrostatic interaction between antimicrobial peptides and the negatively charged bacterial membrane is the main driving force for their binding. In contrast, the outer layer of the phospholipid bilayer of mammalian cell membranes is mainly composed of neutral lipids with no net charge, with negatively charged phospholipids mostly isolated in the inner monolayer of the bilayer. Therefore, the interaction between antimicrobial peptides and mammalian cell membranes is primarily based on hydrophobic interactions between their hydrophobic parts (repelling water and aggregating). Compared to electrostatic interactions, hydrophobic interactions are weaker, so antimicrobial peptides preferentially interact with bacterial cells. Furthermore, mammalian cell membranes contain abundant cholesterol, which enhances the stability of the plasma membrane and reduces the toxicity of antimicrobial peptides to host cells.
Synthesis of Antimicrobial Peptides
In the early stages of research, natural antimicrobial peptides were directly extracted and isolated from organisms. However, this method was challenging, time-consuming, and costly, with low yields, making it unsuitable for large-scale production. As a result, researchers have focused on the artificial synthesis of AMPs over the past few decades to overcome these limitations.
The synthesis of AMPs typically involves amino acid condensation reactions, often requiring functional group protection and deprotection to ensure directed synthesis. The two primary methods for peptide synthesis are liquid-phase and solid-phase synthesis. Liquid-phase synthesis, where amino acids or short peptides react in solution, is inefficient and challenging due to the need for frequent purification, making it less popular for AMP production.
Solid-phase synthesis, on the other hand, is more commonly used. This method involves attaching the amino acid to a solid resin carrier, followed by deprotection and addition of subsequent amino acids. The process is repeated until the desired peptide sequence is obtained, which is then cleaved from the resin. Solid-phase synthesis offers advantages in terms of convenience, rapidity, and precision, making it ideal for synthesizing AMPs with controlled sequences.
However, this method has limitations, including high costs and long synthesis times, making it unsuitable for large-scale production. To address these issues, researchers developed NCA ring-opening polymerization, which allows for the synthesis of high-molecular-weight peptides at lower costs. While the peptides often have random coil structures, modifications, such as amphiphilic block copolymers, have shown improved antibacterial efficacy. For instance, block antimicrobial peptides synthesized via NCA polymerization demonstrated significantly lower minimum inhibitory concentrations (MICs) against bacteria like E. coli and Staphylococcus aureus compared to random copolymers. Additionally, combining chitosan with NCA polymerization improved biocompatibility while maintaining antimicrobial activity, offering potential for safer clinical applications.
Table.2 Peptide synthesis services at Creative Peptides.
Antimicrobial Peptides Analogs
The discovery of AMPs has provided a molecular framework for the development of novel antimicrobial drugs. Studies show that the D-enantiomer of magainin exhibits similar biological activity as the natural form, suggesting that antimicrobial effects are not reliant on specific protein interactions. Interestingly, no universal amino acid sequence has been identified for AMPs, and while many natural AMPs rely on α-helix structures to generate amphipathic properties, some still exhibit strong antimicrobial activity even when parts of their amino acids are replaced by D-enantiomers, which do not stabilize helical structures. This indicates that stable helical structures are not essential for antimicrobial activity. Researchers emphasize that the key factors for antimicrobial activity are the presence of cationic and hydrophobic amino acids rather than specific sequences or stable secondary structures.
This insight has led to the creation of antimicrobial peptide-like polymers that mimic the essential features of natural AMPs, combining cationic and hydrophobic groups. These polymers share a membrane-disrupting mechanism with AMPs, making it difficult for bacteria to develop resistance. They offer advantages over natural AMPs, including lower production costs, ease of large-scale synthesis, and flexibility for chemical modifications, expanding their potential applications in biomedicine.
Antimicrobial peptide-like polymers fall into two main categories. The first includes peptide-based polymers, which incorporate small molecules or biocompatible components to enhance properties like drug delivery or reduced toxicity. The second category consists of non-peptide-based polycationic polymers that simulate AMP cationic charges and hydrophobicity. These polymers can effectively disrupt bacterial membranes, though their higher toxicity limits biological applications. To mitigate this, researchers have introduced "neutral" components, such as glycine-like or serine-like residues, into ternary copolymers, maintaining antimicrobial activity while reducing toxicity.
Table.3 Catalogue of amino acid products at Creative Peptides.
Antimicrobial Peptide Assembly
In 1993, the discovery of the first self-assembling peptide, EAK16 (n-AEAEAKAEAKEA-K-c), marked a significant milestone in peptide research. Since then, self-assembling peptides have garnered considerable attention due to their broad applications in fields such as functional materials, gene therapy, biosensors, tissue engineering, and clinical medicine. Antimicrobial peptides, as functional peptides, have become crucial in advancing biomedical applications, particularly in combating infections. The morphology of peptide assemblies is influenced by various factors, including the polymer structure, the hydrophilic-hydrophobic component ratio, and the assembly environment. These peptides can form diverse structures, such as vesicles, micelles, and nanolamellar formations.
One study focused on a dendritic polymer grafted with antimicrobial peptides that self-assembled into a nanolamellar structure with a low cationic charge (+6.1 mV). During the bactericidal process, this structure encapsulated bacteria and penetrated their cell membranes, leading to bacterial death even at low cationic concentrations. This "encapsulation-penetration" mechanism achieved high bactericidal efficiency (MICs = 16 μg/mL), offering a promising approach for designing novel antimicrobial agents.
Another study involved the synthesis of a chitosan-based graft copolymer with antimicrobial peptide side chains, which self-assembled into polymer vesicles. These vesicles exhibited superior antimicrobial performance and reduced toxicity compared to pure peptide counterparts, with MICs as low as 8 μg/mL. The copolymer vesicles also demonstrated efficient encapsulation and controlled release of anticancer and antiepileptic drugs, suggesting potential applications in cancer treatment and infection suppression.
Moreover, triblock copolymers composed of polycaprolactone (PCL), polyethylene glycol, and ε-polylysine were developed to self-assemble into antimicrobial peptide vesicles. These vesicles showed excellent biocompatibility, lower biological toxicity, and the ability to load clinical therapeutic drugs. Additionally, peptide-like alternating copolymer vesicles carrying bone repair growth factors were designed to aid in bone repair and combat infections, demonstrating their multifunctional potential in controlled drug release and medical applications.
Summary
Antimicrobial peptides and peptide-like antimicrobial polymers are promising alternatives to antibiotics, offering unique mechanisms that make them powerful weapons against drug-resistant bacteria. With continued research, these peptides have the potential to revolutionize the development of novel antimicrobial drugs, with applications spanning plant and animal disease control, food preservation, cosmetics, and clinical antimicrobial treatments. Their multifunctional assemblies further extend their use in drug delivery, tissue engineering, and biosensing.
However, several challenges remain in the production and application of antimicrobial peptides. The exact bactericidal mechanisms are not yet fully understood, and the toxicity of antimicrobial peptides, particularly their effects on normal cells, needs further investigation. Research on factors like cationic charge density, hydrophobicity, and amphipathicity has provided valuable insights into designing more selective peptides. Additionally, while antimicrobial peptides show high antibacterial activity in vitro, their effectiveness in vivo is often reduced by factors such as salt sensitivity, enzyme degradation, and aggregation in the body. More research on their pharmacodynamics and pharmacokinetics is crucial to optimizing their clinical use.
The high cost of synthesizing antimicrobial peptides is another significant barrier to their clinical application. Efforts are underway to reduce these costs through the development of short peptides. Ultimately, antimicrobial peptides for clinical use should maintain stable activity in complex environments, resist rapid degradation, and exhibit minimal toxicity, making them a powerful tool in combating antibiotic resistance.
Reference
- Lei, Jun, et al., The antimicrobial peptides and their potential clinical applications. American journal of translational research 11.7 (2019): 3919.
