Despite the great strides in healthcare during the last century, some challenges still remained unanswered. The development of multi-drug resistant bacteria, the alarming growth of fungal infections, the emerging/re-emerging of viral diseases are yet a worldwide threat. Since the discovery of natural antimicrobial peptides able to broadly hit several pathogens, peptide-based therapeutics have been under the lenses of the researchers. This review aims to focus on synthetic peptides and elucidate their multifaceted mechanisms of action as antiviral, antibacterial and antifungal agents. Antimicrobial peptides generally affect highly preserved structures, e.g., the phospholipid membrane via pore formation or other constitutive targets like peptidoglycans in Gram-negative and Gram-positive bacteria, and glucan in the fungal cell wall. Additionally, some peptides are particularly active on biofilm destabilizing the microbial communities. They can also act intracellularly, e.g., on protein biosynthesis or DNA replication. Their intracellular properties are extended upon viral infection since peptides can influence several steps along the virus life cycle starting from viral receptor-cell interaction to the budding. Besides their mode of action, improvements in manufacturing to increase their half-life and performances are also taken into consideration together with advantages and impairments in the clinical usage. Thus far, the progress of new synthetic peptide-based approaches is making them a promising tool to counteract emerging infections.
All antimicrobial peptides (AMPs) share common features, such as a sequence composed of less than 100 amino acids (aa), [1] with the majority having between 10 and 60 aa [2]. Even if some anionic AMPs, rich in glutamic and aspartic acids, are negatively charged [3], almost all antimicrobial peptides have a net positive charge for the presence of a high number of lysine, arginine and histidine (protonated in acidic conditions) [4]. Finally, another common feature is represented by the hydrophobicity conferred by hydrophobic aa that often overcomes 50% of the total amino acid sequence [5]. The high lipophilicity is useful especially for the penetration in the biological membranes but considering the net charge, overall, AMPs are amphipathic molecules. The classifications are based on their structure or the presence/absence of recognizable motifs. AMPs could be α-helix, β-sheet, linearly extended, both α-helix and β-sheet, cyclic and with complex structure or, seen from a different perspective, tryptophan- and arginine-rich, histidine-rich, proline-rich and glycine-rich [6][7].
In the last decades, the increasing resistance to antibiotic treatments, i.e., Methicillin, Vancomycin-resistant Staphilococus aureus and the rise of species with intrinsic multi-drug resistance, such as Candida auris, highlights the need for the development of new agents [8][9][10]. Studies on the AMPs synthetic analogs provided a new tool to understand the different and unique modes of actions against diverse microorganisms.
Many factors can influence membrane perturbation and disruption by AMPs, i.e., amino acids sequence, the lipid composition of the membrane, peptide concentration as well as differences in membrane composition between eukaryotic and bacterial cells allow the AMPs to distinguish a microbial target from the host. Bacterial membranes are negatively charged due to the presence of anionic phospholipids groups, e.g., phosphatidylglycerol, phosphatidylserine, while eukaryotic cells possess groups with a neutral charge, e.g., phosphatidylcholine and phosphatidylethanolamine [39]. Moreover, the presence of cholesterol, a common feature in eukaryotic cells, is able to interact with AMPs either neutralizing or reducing their activity or stabilizing the phospholipid bilayer [40].
In Gram-positive bacteria, AMPs have to cross first the cell wall composed of crosslinked peptidoglycan with lipoteichoic acid prior to reaching the membrane whereas in Gram-negative they face a coat of lipopolysaccharide (LPS) followed by a phospholipidic outer membrane and a less cross-linked peptidoglycan layer [41]. Electrostatic interactions between the cationic peptide and the negatively charged components, e.g., lipopolysaccharide in Gram-negative and teichoic acid in Gram-positive, are the first steps to contribute to bacterial membrane affinity [42]. However, while AMPs seem to traverse the peptidoglycan layer with ease and access to the cytoplasmic membrane of the Gram-positive, they need to disrupt or perturb both outer and cytoplasmic membrane in Gram-negatives. Impedance in crossing or permeabilization results in loss of antimicrobial activity (Figure 1a (A,B)) [43]. Figure 1. AMPs broad-spectrum antimicrobial activity. (a) Primarily, AMPs’action is based on their action on cytoplasmic membranes, i.e., perturbation or disruption. However, in presence of Gram-negative bacteria (A) AMPs have to firstly cross the outer phospholipidic membrane and secondly traverse the peptidoglycan layer before reaching the inner membrane. In Gram-positive bacteria (B) they navigate through the thick cell wall of peptidoglycan and in fungi (C), they encounter mannitol proteins, glucans and chitin prior to access to the cytoplasmic membrane. Once reached the phospholipidic bilayer, they induce perturbation via pore formation following either (D) (i) carpet-like, (ii) barrel-stave or (iii) or toroidal pore model depending on the peptide composition. (b) Besides pore formation, some AMPs bind some components and receptors on the extracellular side of the membrane, i.e., Toll-like receptors; others manage to enter the cytosol through direct penetration in vesicles or channels thus destabilizing the permeability and activating the inflammatory cytokines cascade. Intracellularly, they could also interfere with DNA or RNA leading to degradation and cell death. They may also affect mitochondrial activity or protein synthesis by targeting ribosome subunits or protein folding. In the case of bacterial cell wall, they can prevent elongation of peptidoglycan chains or hinder teichoic and teichuronic binding acids to amidases. Cell wall components inhibition will promote cell autolysis. In the extracellular space, AMPs can sequestrate LPS reducing the impact of endotoxins on the host’s immune response. In fungal cells, AMPs can intervene on glucan synthesis thus blocking the building pieces of their wall. Further inhibitory action on biofilm matrix impairs the quorum sensing and improves the susceptibility of the single pathogens in both bacterial and fungal communities. In order to explain the perturbation of the phospholipidic membranes operated by the AMPs, three main models have been proposed: carpet-like, barrel-stave and toroidal pore (Figure 1a (D)). Generally, when the ratio of peptide/lipids is low, AMPs interact with the phospholipidic layer of the membrane in a parallel manner, defined as carpet-like model, and interaction among the peptides or penetration in the hydrophobic core of the bilayer are not taking place [44]. Membrane integrity is disrupted and micelles are formed as in a detergent-like process [45]. With increasing AMPs ratio, they move to a perpendicular orientation until reaching such a concentration that they can cross the membrane forming pores (1:50–1:500 and more) [46][47]. A minimum length of ~22 amino acid for α-helix peptides is required to span the phospholipid layer, while β-sheet structures necessitate a minimum of 8 [48]. In the barrel-stave, interaction among peptides is a prerequisite as they mimic a transmembrane pore, whereas, in the case of the toroidal model, peptides are loosely arranged [49][50]. Despite the perturbation of the membrane seems to vary depending on the peptides, actually, the mechanisms of action are not completely well-defined and they are partially overlapping [51]. Moreover, all these models are based on the membrane perturbation but, then, the killing effect is not always enough to provide antimicrobial activity [52]. Besides membrane disruption, recent studies showed how peptides could act on other targets as well (Figure 1b) [53]. Some AMPs have shown their efficacy by binding some components and receptors on the extracellular side of the membrane and wall, thus destabilizing the permeability and/or activating intracellular signaling pathways that have, as a response, the inhibition or the activation of several functions. The inhibitors of the nucleic acid biosynthesis seem to have a high binding affinity for both DNA and RNA because they share with nucleic acid-binding enzymes or substrates, homologous fragments of their sequences; an interesting example is represented by DNA-binding protein histone H2A [54]. Other mechanisms use the inhibition of the enzymes involved in the DNA/RNA biosynthesis, like DNA topoisomerase I preventing DNA relaxation [55], RNA polymerase blocking the transcription [56] and gyrase impairing the supercoiling of DNA. [57] As a result, DNA/RNA degradation is induced and consequentially also cell death. There are several inhibitors of protein biosynthesis which alter the transcription and the translation but also the correct folding and the degradation of the protein. Usually, the AMPs that act on the protein biosynthesis target the ribosome subunits [58] but some others can interfere with the incorporation of histidine, uridine and thymidine [59][60], the amino acid synthesis pathways [61], the release factors on the ribosome [62], the regulation of sigma factors [63], the nucleotide and coenzyme transport [61] and the degradation of DNA-replication-associated proteins [64]. Some peptides influence protein folding, in particular, DnaK, the major Hsp70 of the chaperone pathway in Escherichia coli, which has been seen as an optimal target to prevent the refolding of misfolded proteins [65]. Another approach is linked to the inhibition of matrix metalloproteases, essential enzymes in microbial cell growth and homeostasis, i.e., serine protease, trypsin-like protease, elastase and chymotrypsins [66][67][68]. There are also inhibitors of cell division that block DNA replication or the mechanisms essential for the repair of DNA damages, then resulting in the block of the cell cycle, in the impairment of the chromosome separation, in the failure of septation, in the alteration of mitochondrial activity and in a substantial change in the cell morphology with clearly visible blebbing and elongation towards a filamentous shape [69][70]. Cell wall synthesis is another suitable target. Some AMPs act on lipid II by sequestrating it from the functional site [71][72] or by binding D-Ala-D-Ala residues of its precursor preventing the addition of N-acetylglucosamine and N-acetylmuramic acid in the structure, hence the peptidoglycan elongation [73].