**2. General Features, Mechanism of Action and Possible Clinical Application of MF-AMPs**

AMPs are effective agents for killing or blocking the growth of free microorganisms due to the self-endogenous origins and natural factors produced by the organisms [4,22]. In addition, the endogenous AMPs are not toxic at high concentrations, and some of these are reported as multifunctional agents with immunomodulatory activity without generating pathogen resistance [22]. Among these, cationic AMPs target bacterial cell membranes in a non-specific manner for pathogens. Still, they are not toxic for host cells due to the specific electrostatic interaction with microbial membrane compounds, such as negatively charged lipids or specific microbial ligands for antimicrobial peptides [23,24]. These agents belong to the host defense response of vertebrates' innate immune system [23,24]. More than 2000 AMPs are known to be derived from several organisms and catalogued in the AMPs Database [25–27]. The peptides are classified based on four different biochemical characteristics: 1. net charge (anionic, cationic and neutral); 2. length of the primary structure (more or less than 100 amino acids); 3. typology of secondary or tertiary structure (linear with β-sheet, α-helix, extended loop and very complex structure, such as cyclic peptides); and 4. hydrophobicity (amphipathic, hydrophobic and hydrophilic) [23,28,29] (Figure 1).

These molecules' general mechanism of action consists of an initial phase with membrane targeting based on electrostatic interactions with lipids and membrane ligands [23,28]. After achieving a threshold concentration, the molecular interactions between the peptides and membrane induce a conformational phase transition of these molecules. This effect is characterized by a modification from the disordered structure of peptides, typical in aqueous environments, to α-helical or β-sheet conformation upon interaction with phospholipids in the pathogen membrane [23,28]. Then, the peptides can destabilize the membrane inducing electrostatic tension. Finally, membrane disintegration is obtained by pore formation with micellization or lipid segregation (Figure 1). This phenomenon is caused by different molecular mechanisms, such as self-association and multimerization, barrel-stave mechanism, toroid pore or wormhole mechanism and carpet mechanism [23,24,28]. In addition, due to their lipophilic profile, some AMPs do not degrade the membrane but may

translocate across it. These peptides exert a cell-penetrating peptides (CPPs)-like function. They induce inhibition of intracellular functions by blocking the cytoskeleton growth, causing membrane dysfunction inside the cell or inhibiting extracellular biopolymers and DNA/RNA/protein synthesis [22,23,28] (Figure 1). In recent years, these agents have acquired an increasing interest in clinical applications. They may represent a novel and promising therapeutic alternative to conventional antibiotics for preventing and eradicating resistant pathogens. AMPs also prevent pathogens' biofilm formation due to antimicrobial activity [20,29]. In addition, AMPs are elective to destabilize existing biofilms targeting matrix proteins or signaling pathways for growth and essential metabolic processes or compounds [20,29]. In these aspects, another therapeutic utilization of AMPs is preventing infection in the surgical sites and microbial biofilms on biomaterials often associated with the onset of nosocomial infections. Different biomaterials functionalized with peptides and peptidomimetics agents are available for clinical applications. As a few examples, in addition to medically implanted devices, they can be used orally and for wound sites in the surgical area [30,31]. Other vital interests have been reported for endogenous MF-AMPs based on their properties to modulate systemic and tissue inflammation and immune cell activation [21], supporting future clinical applications as immunomodulators. On the other hand, numerously exogenous MF-AMPs are reported as anti-proliferative against several cancer cells with potential clinical application in cancer therapy [14].

**Figure 1. Antimicrobial Mechanisms of Action of Multifunctional Antimicrobial Peptides** (**MF-AMPs**). Based on their biochemical characteristics, MF-AMPs can interact with the membrane lipids, inducing the pathogens' instability and rigidity of the cell membrane. Upon reaching the minimum inhibitory concentration (MIC) values, they cause pore formation and cell lysis. Some MF-AMPs exert a cellpenetrating peptides-like function. They can penetrate the membrane, blocking pathogens' growth by internal membrane dysfunction, cytoskeleton interaction and interference of biomolecules production. Deoxyribonucleic acid (DNA); ribonucleic acid (RNA).
