The Best Peptidomimetic Strategies to Undercover Antibacterial Peptides

International Journal of Molecular Sciences

Review The Best Peptidomimetic Strategies to Undercover Antibacterial Peptides

Joanna Izabela Lachowicz 1,*, Kacper Szczepski 2, Alessandra Scano 3, Cinzia Casu 3 , Sara Fais 3, Germano Orrù 3 , Barbara Pisano 1 , Monica Piras 1 and Mariusz Jaremko 2,*

1 Department of Medical Sciences and Public Health, University of Cagliari, Cittadella Universitaria, 09042 Monserrato, Italy; [email protected] (B.P.); [email protected] (M.P.) 2 Division of Biological and Environmental Sciences and Engineering (BESE), King Abdullah University of Science and Technology (KAUST), Thuwal 23955-6900, Saudi Arabia; [email protected] 3 Department of Surgical Science, OBL Oral Biotechnology Laboratory, University of Cagliari, 09124 Cagliari, Italy; [email protected] (A.S.); [email protected] (C.C.); [email protected] (S.F.); [email protected] (G.O.) * Correspondence: [email protected] (J.I.L.); [email protected] (M.J.)

Received: 31 August 2020; Accepted: 25 September 2020; Published: 5 October 2020

Abstract: Health-care systems that develop rapidly and efficiently may increase the lifespan of humans. Nevertheless, the older population is more fragile, and is at an increased risk of disease development. A concurrently growing number of surgeries and transplantations have caused antibiotics to be used much more frequently, and for much longer periods of time, which in turn increases microbial resistance. In 1945, Fleming warned against the abuse of antibiotics in his Nobel lecture: “The time may come when penicillin can be bought by anyone in the shops. Then there is the danger that the ignorant man may easily underdose himself and by exposing his microbes to non-lethal quantities of the drug make them resistant”. After 70 years, we are witnessing the fulfilment of Fleming’s prophecy, as more than 700,000 people die each year due to drug-resistant diseases. Naturally occurring antimicrobial peptides protect all living matter against bacteria, and now different peptidomimetic strategies to engineer innovative antibiotics are being developed to defend humans against bacterial infections.

Keywords: antimicrobial peptides; antibiotics; peptidomimetics; polymers; metal coplexes

1. Introduction Antimicrobial peptides (AMPs), also known as host defense peptides (HDPs), are produced by all living matter as a critical part of the innate immune system [1–3]. Their existence was discovered in 1939, the year gramicidin was isolated from the bacteria, Bacillus brevis; some resources, however, claim that the discovery of lysozyme in the 1920s should be treated as the first AMP instance, due to lysozyme’s non enzymatic, bactericidal second mode of action [2]. As of 2019, over 3000 AMPs have been isolated across all kingdoms [4]. Most of them have variable sequence length—from 10 up to 60 amino acid residues, mainly in L-configuration [4]. AMPs are expressed in many cell types in response to the activation of the Toll-like receptor (TLR) signaling pathway [1]. They are predominantly comprised of two types of amino acids residues—cationic residues such as Arg, Lys, and His, and hydrophobic residues (mainly aliphatic and aromatic) [1]. Both cationic and hydrophobic residues engage in the antimicrobial mechanism of action. Furthermore, AMPs can be classified into three groups, based on their structures (Scheme1): α-helical, β-sheets, and extended peptides. α-helical peptides are the largest group of AMPs with characteristic qualities such as amphipathic properties, and the ability to possess a tertiary structure with a hinge in the middle of a chain [1,4]. β-sheets peptides feature one to

Int. J. Mol. Sci. 2020, 21, 7349; doi:10.3390/ijms21197349 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 2 of 44 Int. J. Mol. Sci. 2020, 21, 7349 2 of 45

chain [1,4]. β-sheets peptides feature one to five disulfide bridges that help to stabilize their bioactive ﬁveconformation. disulﬁde bridges Peptides that rich help in toamino stabilize acids their such bioactive as proline, conformation. arginine, tryptophan Peptides rich or glycine in amino usually acids suchhave asa linear proline, structure arginine, [1, tryptophan4]. or glycine usually have a linear structure [1,4].

α β Scheme 1. 1. SchematicSchematic representation representation of of α--helicalhelical and β--sheets,sheets, common secondary structure of amphipathic peptides. Hydrophobic amino acid residues are colored red, while hydrophilic residues are colored blue.blue. HDPs have a wide range of antibacterial activities, and low susceptibility to antimicrobial HDPs have a wide range of antibacterial activities, and low susceptibility to antimicrobial resistance [5–12]. Nevertheless, the use of HDPs is limited due to their low resistance to proteases, resistance [5–12]. Nevertheless, the use of HDPs is limited due to their low resistance to proteases, and their increased cost of preparation [8,13,14]. Diﬀerent peptides [15–18], peptoids [19–23], and their increased cost of preparation [8,13,14]. Different peptides [15–18], peptoids [19–23], polymethacrylate derivatives [24], polynorbornene derivatives [25], polycarbonate derivatives [26], polymethacrylate derivatives [24], polynorbornene derivatives [25], polycarbonate derivatives [26], peptide polymers [27–32], and polymers made by controlled living radical polymerization (CLRP) [33] peptide polymers [27–32], and polymers made by controlled living radical polymerization (CLRP) were developed to mimic the antibacterial properties of HDPs, and to ameliorate their shortcomings [34]. [33] were developed to mimic the antibacterial properties of HDPs, and to ameliorate their The widely developed peptide-drug research has led to the introduction of around 60 FDA shortcomings [34]. approved peptide therapeutics in the market [35]. Over 140 are in clinical trials, and over 500 are in the The widely developed peptide-drug research has led to the introduction of around 60 FDA preclinical stages. Although numerous biomimetic and de novo designed AMPs are at diﬀerent stages approved peptide therapeutics in the market [35]. Over 140 are in clinical trials, and over 500 are in of clinical trials [36,37], only eight AMPs received FDA approval, i.e., daptomycin (approved in 2003) the preclinical stages. Although numerous biomimetic and de novo designed AMPs are at different and oritavancin (approved in 2014) [37]. stages of clinical trials [36,37], only eight AMPs received FDA approval, i.e., daptomycin (approved In this review paper, we describe innovative peptidomimetics strategies and we present in 2003) and oritavancin (approved in 2014) [37]. successful examples of new antimicrobial peptide-mimicking antibiotics. The aim of this review is In this review paper, we describe innovative peptidomimetics strategies and we present to gather the latest advances in the research of new antibiotics for those who are engineering future successful examples of new antimicrobial peptide-mimicking antibiotics. The aim of this review is to antibacterial strategies. gather the latest advances in the research of new antibiotics for those who are engineering future 2.antibacterial Samsons Hair strategies. of AMPs

2. SamsonsThere are Hair three of AMPs main factors—charge, hydrophobicity and amphipathicity, which determine the activity of AMPs, and they are all related to their intrinsic properties. There are three main factors—charge, hydrophobicity and amphipathicity, which determine the The positive charge of AMPs (between 2+ and 9+) is one of the most essential factors responsible for activity of AMPs, and they are all related to their intrinsic properties. their antibacterial activity [38]. Positively charged AMPs outcompete the binding of native Mg(II) and The positive charge of AMPs (between 2+ and 9+) is one of the most essential factors responsible Ca(II) ions to lipopolysaccharides (LPS), and destabilize the outer membrane of Gram-negative for their antibacterial activity [38]. Positively charged AMPs outcompete the binding of native Mg(II) bacteria [39]. Consequently, the destabilized regions enable the peptide to pass into the cell. and Ca(II) ions to lipopolysaccharides (LPS), and destabilize the outer membrane of Gram-negative Once through, AMPs bind to the cytoplasmic membrane and cause depolarization and pore formation, bacteria [39]. Consequently, the destabilized regions enable the peptide to pass into the cell. Once resulting in cell death [40]. In the case of Gram-positive bacteria, AMPs must overcome the outer through, AMPs bind to the cytoplasmic membrane and cause depolarization and pore formation, wall with two main obstacles, peptidoglycan and teichoic acids, before an AMP interacts with the resulting in cell death [40]. In the case of Gram-positive bacteria, AMPs must overcome the outer wall cytoplasmic membrane. AMPs overcome peptidoglycan because it is able to penetrate through the with two main obstacles, peptidoglycan and teichoic acids, before an AMP interacts with the relatively porous peptidoglycan. The porous nature of peptidoglycan makes it easy for small molecules cytoplasmic membrane. AMPs overcome peptidoglycan because it is able to penetrate through the (< 50 kDa) to penetrate through it, and furthermore, it does not possess a negative charge, which could relatively porous peptidoglycan. The porous nature of peptidoglycan makes it easy for small

Int. J. Mol. Sci. 2020, 21, 7349 3 of 45 prevent the penetration of positively charged AMPs through it [40–42]. To overcome the anionic teichoic acids, they can either act as a ladder that will help positively charged AMPs to travel to the cytoplasmic membrane, or the can act as “cages” that entrap AMPs inside the bacteria, thus reducing their local concentration on the membrane The behaviors of teichoic acids vary based on the type of AMP and the type of bacteria [42]. After crossing the outer wall, AMPs can disrupt the integrity of the cytoplasmic membrane, leading to membrane disruption and dislocation of peripheral membrane proteins [42]. The hydrophobicity of AMPs is another important factor. It is assumed that hydrophobic residues help to insert peptides into the bacterial membrane and further impair the membrane [4,42]. It was also revealed that hydrophobic residues account for 40–60% of all amino acids in AMPs [38]. Moreover, the hydrophobicity is strongly correlated with antimicrobial activity, where increasing the hydrophobicity to a certain level improves antimicrobial activity [1,38]. The relationship between hydrophobicity and antimicrobial activity is also proven to be the case for peptidomimetics such as peptoids, where amino acid side chains are linked to the peptide backbone through the amide nitrogen rather than to the α-carbons [43]. However, a higher hydrophobicity also increases hemolytic activities, resulting in unwanted toxicity to eukaryotic cells [1,38]. Studies with model peptides have shown that a continuous region of 4-6 hydrophobic residues is suﬃcient to sustain the antimicrobial activity of peptides, while simultaneously reducing the hemolytic activity [44]. The amphipathicity of AMPs, created by the segregation of hydrophobic and polar residues on the opposite sites of the backbone, is another factor related to their activity [1]. Amphipathicity is considered the strongest indicator of AMP activity, and is described by the hydrophobic moment, deﬁned as the vector sum of the hydrophobicity of each amino acid located in the helix [1,45]. The mechanism by, which amphipathicity works is the result of previous factors. The positively charged regions of AMPs enable binding to the anionic phospholipid head groups of the bacterial membrane. Meanwhile, the hydrophobic regions of peptides invade the lipid bilayer and interact with the hydrophobic acyl chains of phospholipids, which results in membrane penetration [38]. Several studies have shown the importance of amphipathicity in the process of AMPs binding to membranes, and the negative impact on activity against Gram-positive bacteria and fungi, when amphipathic character of the peptide was eliminated [13,46–48].

3. AMPs: Achilles’ Heel Despite the recent advancements in AMP research, there are still major challenges to overcome. One of the main obstacles in AMP applications is the proteolytic instability of peptide drugs [49,50]. Peptides are targeted by numerous proteolytic enzymes located in bodily fluids and tissues of the host. Moreover, AMPs can be recognized as an antigen and targeted by host immune system [51,52]. One of the countermeasures for their potential immunogenicity could be glycosylating AMPs with a glycosyl profile similar to that of the host. For instance, covering AMPs with polysialic acid (derived from Neisseria meningitidis or E. coli that shares structural mimicry with host cell lectins), or its analogues, can lessen the immunological response [52]. Nevertheless, some bacteria have already developed resistance to AMPs. For example, LL-37, a human antimicrobial peptide, can be digested by proteases synthesized by P. aeruginosa, Enterococcus faecalis, Proteus mirabilis, S. aureus, and S. Pyogenes [50,53]. Synthesizing proteases to target specific antimicrobial peptides is one of the many ways bacteria participate in this evolutionary “arms race”. Other methods used by bacteria to cope with AMPs, including the use of efflux pumps, lowering the binding affinity, or reducing the anionic charge of lipopolysaccharides on their surface, are exhaustively discussed in [50,54]. The high toxicity of AMPs in eukaryotic cells is another one of their shortcomings. This high toxicity can lead to hemolysis, nephrotoxicity, and neurotoxicity [1,55–57]. More studies need to focus on the pharmacokinetics and pharmacodynamics of AMPs in order to determine a proper drug dosage that will maintain a balance between positive and negative outcomes [2]. Int. J. Mol. Sci. 2020, 21, 7349 4 of 45

The bioavailability of AMPs is rather low. Peptides are hardly absorbed by the intestinal mucosa, and their pharmacological distribution requires additional financial input. In recent years, much progress has been made in this field—e.g., nano-carriers not only increase the bioavailability, but also lower cytotoxicity and reduce degradation of a compound, leading to increased efficiency [37]. Despite different efforts, the entire process of AMPs design and discovery (costs of synthesis and screening) is relatively high and does not guarantee the expected outcome [58]. The high production cost of AMPs (as for 2006, producing 1 g of peptide using solid-phase peptide synthesis (SPPS) costs USD 100–600) limits the development and testing of new AMPs, even if recent improvements in SPPS lowered the costs of synthesis [8]. AMPs, as other peptide drugs, can induce immune responses and cause allergies [59]. Immunogenicity depends on the host immune system, but is also correlated with the peptide’s dose, duration and frequency of treatment, route of administration, and the patient’s pathologies [60–63]. Immunogenic response can affect the peptide-drug efficiency and even lead to allergy and/or hypersensitivity [63]. Bering that in mind, it is important to study the immunogenic and allergic activity of AMPs. In vitro tests together with mathematical calculations of allergens similarities, with the use of specific databases of allergenic compounds [64], i.e., http://www.allergome.org, can give nowadays reliable results [65].

4. Bacterial Membranes vs. Human Cell Membranes AMPs’ selectivity depends on the intrinsic differences in the cell wall of prokaryotes and eukaryotes [66]. Since most bacterial plasma membranes are rich in anionic phospholipids (such as phosphatidyl-glycerol (PG), cardiolipin (CL), and phosphatidylserine (PS)) and since the outer monolayers of eukaryotic membranes are composed of zwitterionic (overall neutral) lipids, the prokaryotic membranes are more electronegative than eukaryotic membranes [66]. AMPs’ selectivity towards either zwitterionic or negatively charged lipids is crucial in determining their bioactivity, toxicity, and potential use as drugs [67]. Furthermore, in some cases, the high electronegative nature of a membrane helps AMPs to adopt active conformations and interact with the membrane. The cell-walls of Gram-positive and Gram-negative bacteria vary significantly. In Gram-positive bacteria, the cell wall is comprised of multiple layers of peptidoglycan composed of a poly-[N-acetylglucosamine-N-acetylmuramic acid] backbone, with a thickness of 30–100 nm [68]. The peptidoglycan wall determines the shape of bacteria, prevents cell lysis (due to high internal osmotic pressure), and defends the cell from environmental hazards such as antibiotics. The cell wall is interspersed by two types of anionic polymers—teichoic acids, which are linked to peptidoglycan, and lipoteichoic acids that are anchored to the cell membrane. Anionic polymers play an important role in ion homeostasis, regulate cell morphology, division and autolytic activity, and protect bacteria against the host’s defense mechanisms and antibiotics [69]. When compared to Gram-negative bacteria, Gram-positive bacteria have a much higher content of negatively charged lipids, predominantly phosphatidylglycerol (PG) and cardiolipin (CL) [66]. The envelope of Gram-negative bacteria can be divided into three layers: the outer membrane (OM), the peptidoglycan cell-wall, and the cytoplasmic or inner membrane (IM). The outer membrane is composed mainly of glycolipids, and principally, of lipopolysaccharides (LPS). LPS are composed of three domains: lipid A, the core oligosaccharide, and the O-antigen [70]. LPS create a permeability barrier and protect the bacterial cell against various environmental factors such as antibiotics and bile salts. However, the LPS are primary bacterial components encountered in the host immune system and play an important role in bacterial pathogenicity (the host organism can treat them as a pathogen-associated molecular pattern; PAMP). The inhibition of LPS biosynthesis could also trigger stress response pathways and affect the assembly and function of membrane proteins [71]. In addition to LPS, the outer membrane contains lipoproteins and β-barrel proteins that can act as gated channels for vitamin transport. In Gram-negative bacteria, the peptidoglycan cell-wall plays the same role as in Gram-positive bacteria, but it is thinner—ranging from 2.5 to 7 nm (1–3 layers of peptidoglycan) [72]. Int. J. Mol. Sci. 2020, 21, 7349 5 of 45

The inner membrane of bacteria serves as a platform for all membrane-associated functions of eukaryotic organelles, which include energy production, lipid biosynthesis, protein secretion, and the transport of molecules [73]. Despite all the differences in the cell wall structure, there are three models, which describe how AMPs destabilize the bacterial membrane (Table1). In the barrel-stave model, a variable number of peptides are inserted perpendicularly into the bilayer, forming a barrel-like ring with a central lumen acting as a pore [74]. In the carpet model, peptides are positioned parallel to the bilayer, and form carpet-like structure on the membrane surface. After reaching a concentration threshold of adsorbed AMP, accumulated AMPs change membrane fluidity and reduce the barrier properties of the membrane, producing a “detergent-like” effect (formation of micelles), and resulting in membrane disruption [74]. In the last model, called toroidal-pore model, the peptides are inserted perpendicularly into the bilayer and induce a bend in the membrane. This results in a formation of a pore that constitutes partially peptides and partially phospholipid head groups. The difference between toroidal pores and barrel-stave pores is that peptides are always intercalated with the lipids head groups when forming a pore.

Table 1. Summary of the three most widely accepted mechanisms of antimicrobial peptides (AMPs).

Killing Mechanisms Bacterial Membrane Type of Interaction References AMPs are perpendicularly inserted into the membrane, Barrel-stave model [75,76] promoting peptide–peptide lateral interactions. Carpet model Binding of AMPs to the outer surface (phospholipids) of cell membrane. [77,78] Attached AMPs start aggregation and force the lipid monolayer to bend Toroidal model [79,80] incessantly through the pores

Although, AMPs’ main antimicrobial activity is membrane disruption, several studies have shown that antimicrobial peptides exhibit other mechanisms of action. AMPs can inhibit the synthesis of cell-walls, nucleic acids and proteins, and they can inhibit cell division. Moreover, AMPs are able to target organelles of eukaryotic pathogens [81]. Many of these aspects are broadly described in the literature [82]. For instance, recent results showed that IARR-Anal10, the analog of mBjAMP1, exhibits no eﬀect on the permeability or integrity of the bacterial outer membrane, and acts intracellularly rather than at the cell membrane. Moreover, IARR-Anal10 can bind bacterial DNA [82].

5. Antibacterial Peptides in the Management of Oral Infections with Biofilm Production The oral cavity is unique in the level of microbial diversity it holds, and it harbors about 1000 microbial species. Most of them have physiological functions in oral tissue, and the majority of oral microbes are considered commensals [83]. Others are associated with oral diseases and can be divided into two, ideal groups: “always” pathogenic and “potentially” pathogenic. A pathogen is a microorganism capable of causing disease in healthy individuals. It can enter the host organism, establish a niche within which to replicate, and disseminate to reach a new host. It is now possible to identify different genes associated with pathogenicity factors inside the pathogen genome. Compounds that harm the host are naturally expressed, and they are not associated with stress conditions in the oral cavity. An opportunistic pathogen does not cause disease in healthy individuals, but it can cause pathological states in “at-risk” patients, for instance, during a dysbiosis status in the oral tissues. In this context, an opportunistic bacterium could be useful or even essential during human life, but it can also become a pathogen under different chemical and biological stimuli released by the host’s tissues [84–86]. Periodontitis and periimplantitis are two of the most pervasive pathologies in dental medicine. According to recent publications and WHO reports, millions of peoples in the industrialized countries are toothless, and over hundred-and-twenty million are missing at least one tooth. Moreover, from 70 Int. J. Mol. Sci. 2020, 21, 7349 6 of 45 to 90% of people over 60 years old have dentures. Periodontitis is an infectious disease caused by microbes, the majority of which is represented by Gram-negative anaerobic rods [8], capable of harming periodontal tissues by direct products (e.g., proteases), and mark an unusual inflammatory response, which leads to the destruction of the underlying tissue and tooth loss in adults [9]. In the same way, peri-implantitis (PI) is a specific infective-inflammatory site-specific disease that occurs in dental osseointegrated implants. The onset of this infection is represented by the adherence and proliferation of the periodontal anaerobic bacteria on implant surfaces and it is strongly linked to an inflammatory process that occurs and may be limited to the soft-tissues, or is associated with the progressive loss of osseointegration, mucositis, and peri-implantitis, respectively. Peri-implantitis shows a remarkable impact on health status and related costs in industrialized countries. Recent reports indicate that the percentages of clinically evident PI range from 30 to 60%, and different efforts are undertaken to treat this infectious disease [10]. Nowadays, periodontal therapy follows two main procedures—the with or without surgery approach. The first one is used to improve access to the root surface; nevertheless, is not effective if bacteria invade periodontal tissues [11,12]. In other cases, adjunctive systemic antimicrobial therapy remains the treatment of choice [13]. However, factors such as the mode of antimicrobial action, the susceptibility of periodontal pathogens, the dosage, correct management and use in the treatment of periodontal disease, as well as the mechanism of bacterial resistance to each antimicrobial, are still discussed among dentists and microbiologists [14]. Antimicrobial resistance is a natural phenomenon of bacterial survival, but at the same time, it represents a worldwide public health risk, particularly in patients who have chronic periodontitis or periimplantitis, and who are frequently infected with multidrug-resistant strains. For instance, Prevotella intermedia isolated from oral cavity has shown resistance to clindamycin, amoxicillin, doxycycline, or metronidazole [15]. In a clinical investigation performed by Ram et al., about 70% of peri-implantitis patients exhibited strains that are drug-resistant in vitro to one or more of the tested antibiotics [16]. In this context, new approaches are required to combat the spread of drug-resistant bacteria in the oral cavity, especially given that oral microbes are also associated to degenerative systemic diseases in other tissues [17]. Oral cavity microorganisms are present either as planktonic cells in saliva, or as sessile and incorporated into a complex biological matrix called a biofilm. In the mouth, the biofilms represent the conditions of severe oral pathologies such as dental caries, periodontal diseases, and periimplantitis. The resident bacteria cells in the biofilm are encased in an exopolymeric complex matrix with amyloid-like properties. Biofilm is comprised of exopolysaccharides, lipids, proteins, and cDNA. This extracellular matrix provides mechanical rigidity and protects bacteria from the external environment, often acting as waterproof barrier (Scheme2). The resident bacteria strictly regulate the biofilm functions via a coordinated gene expression network, including growth direction, metabolic pathways, and pathogenic compounds for the host. In this way, biofilm represent an exceptional survival strategy for pathogens, and together with corresponding changes in gene expression, can protect the bacteria from disinfectant agents or antibiotics, and from the immunity system. On the other hand, biofilm sessile conditions allow bacteria to adhere to the oral tissues e.g., teeth surface, in opposing salivary flux. Int. J. Mol. Sci. 2020, 21, 7349 7 of 45 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 7 of 44

Scheme 2. Schematic structure of an oral bio bioﬁlmfilm complex. Early Early colonizer bacteria bacteria attach attach to the oral tissuestissues covered with host cell externalexternal proteins.proteins. Early Early colonizer bacteria “ “communicate”communicate” with late colonizer microorganisms microorganisms (i.e., (i.e. periodontal, periodontal pathogens) pathogens) by using by di ﬀ usingerent acceptor–receptor different acceptor interactions–receptor interacamongtions the quorum among the sensing quorum (QS) sensing pathway. (QS) pathway.

In the the oral oral cavity, cavity, we can can observe observe diverse diverse biofilms biofilms located located in in different different tissues/organs: tissues/organs: the the tongue, tongue, a dental dental apparatus, apparatus, or inor prostheses in prostheses i.e., implants i.e., implants and orthodontic and orthodontic appliances. appliances. These bacterial These complexes bacterial complexespossess great poss phenotypicess great phenotypic adaptability, adaptability, genetic resistance, genetic andresistance, as already and discussed,as already arediscussed, resistant are to resistantantimicrobial to antimicrobial treatment. It treatment. is often the It extracellularis often the extracellular biofilm matrix biofilm that physically matrix that restricts physically thedi restrictsffusion theof antimicrobial diffusion of agents, antimicrobial even if does agents, not seem even to if be does a predominant not seem mechanismto be a predominant of antimicrobial mechani resistance.sm of antimicrobialThe oral infectiveresistance. diseases are host–pathogen interactions with complicated biochemical networks, andThe in the oral last infective decade diseasesthey have are been host uncovered–pathogen with interactions metagenomic with complicated and proteomic biochemical analyses. networks,Surprisingly, and the in resident the last oral decade bacteria they can assist have the been pathogens, uncovered or theywith interact metagenomic by turning and opportunistic proteomic analysbacteriumes. Surprisingl into the pathogeny, the resident phenotype oral bacteria by a wide can assist variety the of pathogens, mechanisms, or they such interact as evasion by turning of the opportunisticimmune response, bacterium cell–cell into signaling, the pathogen metabolic phenotype interactions, by a etc. wide [87 variety]. of mechanisms, such as evasionInside of the biofilms, immune bacterial response, cells cell use–cell a signaling, communication metabolic pathway interactions, called quorumetc. [87]. sensing (QS) to coordinateInside the biofilms, population bacterial density cells and use growth a communication direction. In pathwayoral bacteria, called QS quorum also modulates sensing virulence (QS) to coordinateand pathogenic the population functions [ 88density], and studiesand growth on QS direction. signaling In molecules oral bacteria, to produce QS also a “quorum-quenching”modulates virulence andeffect pathogenic that could lead functions to bacterial [88], communication and studies on interference QS signa [ling89], and molecules new modes to produce of antibacterial a “quorum action.- quenching”Recent effect studies that have could better lead defined to bacterial the functionscommunication of antibiofilm interference peptides [89], and [26,27 new] (Scheme modes3 of). antibacterialClinical studies action. examined the expression of AMPs in oral tissues and in crevicular fluid or saliva and correlatedRecent them studies with have the clinical better defined index of the periodontal functions diseases of antibiofilm and related peptides microbiological [26,27] (Scheme analysis 3). Clinicalpathogens. studies Among examined different the expression AMPs, Cathelicidin, of AMPs inα oral- and tissuesβ-defensins and in crevicular 1–3 were stronglyfluid or saliva linked and to correlatedperiodontal them status with [90 ,the91]. clinical index of periodontal diseases and related microbiological analysis pathogens. Among different AMPs, Cathelicidin, α- and β-defensins 1–3 were strongly linked to periodontal status [90,91].

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Scheme 3. Schematic representation of two possible mode of AMPs action as antimicrobial agents: (AScheme) electrostatic 3. Schematic interference; representation (B) bacteria of two cell membranepossible mode or external of AMPs protein action alteration. as antimicrobial agents: (A) electrostatic interference; (B) bacteria cell membrane or external protein alteration. In the oral cavity, human AMPs are small cationic peptides, synthesized in the oral epithelium and salivaryIn the oral glands, cavity, and human serve AMPs as defensive are small tools. cationic After peptides, a look through synthesized the antimicrobial in the oral epithelium peptides databasesand salivary (APD) glands, [92], and over serve 40 di ffaserent defensive salivary tools. AMPs After were a look found. through These the peptides antimicrobial are, on average,peptides 43databases amino acids (APD) long, [92], with over a 40 medium different net salivary charge AMPs of 4.83. were Oral fou AMPsnd. These include peptides peptides are, ofon diaveragefferent, biological43 amino roles:acids Defensinslong, with [ 93a ],medium Histatins, net Cathelicidins, charge of 4.83. Adrenomedullin, Oral AMPs include Statherin, peptides C-C Chemokine,of different Azurocidin,biological roles: and Defensins Neuropeptides [93] , Histatins, [94,95]. Cathelicidins, Adrenomedullin, Statherin, C-C Chemokine, Azurocidin,Oral peptides and Neuropeptides have evolved [94 together,95]. with the oral microbiota and have acquired particular properties.Oral peptides Some AMPs have families evolved can togetherboth kill periodontal with the oral bacteria microbiota and neutralize and have the acquired lipopolysaccharide particular (LPS)properties. of Gram-negative Some AMPs bacteria. families Cationic can AMPs, both as kill adrenomedullin, periodontal α b-defensinsacteria and (HNP), neutralizeβ-defensins, the cathelicidin,lipopolysaccharide ll-37, histatins(LPS) of Gram 1 and-negative 3, statherin, bacteria. C–C Cationic motif AMPs, chemokine as adrenomedullin, 28 (CCL28), Azurocidin, α-defensins are(HNP), protected β-defensins, against cathelicidin, bacterial proteases ll-37, histatins (i.e., 1P. and gingivalis 3, statherin,gingipains) C–C motif by chemokine salivary proteins 28 (CCL28), [79]. OtherAzurocidin, AMPs are are involved protected in the against bacterial bacterial agglutination, proteases small (i.e. salivary, P. gingivalis mucin-7 gingipains) (MUC7) or by can salivary inhibit bacterialproteins growth[79]. Other by acting AMPs as are divalent involved cation in the scavengers bacterial agglutination, (ion chelating agents)small salivary [96]. In mucin the periodontal-7 (MUC7) aetiologicor can inhibit process, bacterial the AMPs growth mechanism by acting ofas inhibitiondivalent cation of bacterial scavengers proteases, (ion chelati MPs hBD-2ng agents) andCCL20, [96]. In orthe the periodontal peroxidase aetiologic activity is particularlyprocess, the interesting. AMPs mechanism In this process, of inhibition different of active bacterial compounds proteases, against MPs A.hBD actinomycetemcomitans-2 and CCL20, or the, P. gingivalisperoxidaseand activity oral streptococci is particularly[97,98 ] interesting. are produced. In this process, different activeDysregulation compounds of against AMPs A. in the actinomycet oral tissuesemcomitans is related, P. to gingivalis the pathogenesis and oral of streptococci periodontal [97,98] diseases. are Thisproduced. aspect is observed in the patients with systemic diseases (rheumatoid arthritis, diabetes mellitus), obesity,Dysregulation and tobacco of smokers, AMPs in all the of whichoral tissues are at is high related risk to of the periodontal pathogenesis pathologies. of periodontal It is suggested diseases. thatThis AMPs aspect may is act observed as a putative in the factor patients in these with mentioned systemic conditions diseases (rheumatoid and periodontal arthritis, diseases. diabetes mellitus),Even ifobesity, numerous andin tobacco vitro experiments smokers, all have of which shown are AMPs at high activities risk of versus periodontal oral pathogens pathologies. bacteria, It is itsuggested is not clear that if the AMPs AMPs may exert act direct as a antibacterialputative factor activity in these The concentrationmentioned conditions of AMPs inand the periodontal crevicular fluiddiseases. is lower than the MIC value, and some authors suggest that the primary role of oral AMPs is not linkedEven to if antibacterial numerous in activity, vitro experiments but inversely have may shown serve to AMPs maintain activities oral microbiota versus oral homeostasis. pathogens Futurebacteria, solutions it is not couldclear if be the antimicrobial AMPs exert peptidesdirect antibacterial cocktails that activity combat The a concentration subset of oral of pathogens AMPs in the by modulatingcrevicular fluid commensals is lower/ pathogensthan the MIC and value, by avoiding and some oral authors dysbiosis suggest [99–106 that]. the primary role of oral AMPs is not linked to antibacterial activity, but inversely may serve to maintain oral microbiota homeostasis. Future solutions could be antimicrobial peptides cocktails that combat a subset of oral pathogens by modulating commensals/pathogens and by avoiding oral dysbiosis [99–106].

6. Mimicking Amino Acid Sequence

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6. Mimicking Amino Acid Sequence The inspiration for the AMP-mimicking peptides comes mostly from nature. Temporin-SHf (FFFLSRIF;The Figure inspiration 1) is for a short the AMP-mimicking (8 amino acids peptides long) unstructured comes mostly [107] from nature.and highly Temporin-SHf hydrophobic peptide(FFFLSRIF; found Figure in the1 ) frog’s is a short skin, (8 amino with moderate acids long) antimicrobial unstructured [ activity107] and (MIC highlyE.coli hydrophobic(μM) = 25–30; E.coli µ MICS.aureuspeptide(μM found) = 12.5 in) [107] the frog’s. The skin,tuning with of its moderate antimicrobial antimicrobial properties activity has (MICled to the( identificationM) = 25–30; of MICS.aureus(µM) = 12.5) [107]. The tuning of its antimicrobial properties has led to the identiﬁcation an analogue peptide, TetraF2W-RR (WWWLRRIW; Figure 1), with enhanced antimicrobial of an analogue peptide, TetraF2W-RR (WWWLRRIW; Figure1), with enhanced antimicrobial (MICE.coli(μM) = 25; MICS.aureus(μM) = 1.6–3.1) and antibiofilm activity against methicillin-resistant (MICE.coli(µM) = 25; MICS.aureus(µM) = 1.6–3.1) and antibioﬁlm activity against methicillin-resistant StaphylococcusStaphylococcus aureus aureus (MRSA)(MRSA) in in free andand immobilized immobilized forms. forms. Substituting Substituting phenylalanine phenylalanine residues withresidues with tryptophan,tryptophan, enhanced enhanced the antimicrobialthe antimicrobial activity, activity, particularly particularly against MRSA against (MIC( MRSAµM) = (MIC1.6–3.1)(μM [108) =]. 1.6– 3.1) Horine[108]. Horine (WWWLRRRW) (WWWLRRRW) and Verine-L and (RRRWWWWL) Verine-L (RRRWWWWL) are the ultimate are uprade the ultimate of Temporine uprade of Tempo(Figurerine 1(Figure), and have 1), and also have enhanced also enhanced the antimicrobial the antimicrobial activity against activityS. aureusagainst(MIC S. aureusHorine = (MIC4 µM,Horine = 4 μM,MIC MICVerine-LVerine-=L =4 4µ μM)M) and andE. E. coli coli(MIC (MICHorineHorine= 32 = 32µM, μM, MIC MICVerine-LVerine=-L4 =µ 4M) μM) [109 [109]]. .

Figure 1. Cont.

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 9 of 44

The inspiration for the AMP-mimicking peptides comes mostly from nature. Temporin-SHf (FFFLSRIF; Figure 1) is a short (8 amino acids long) unstructured [107] and highly hydrophobic peptide found in the frog’s skin, with moderate antimicrobial activity (MICE.coli(μM) = 25–30; MICS.aureus(μM) = 12.5) [107]. The tuning of its antimicrobial properties has led to the identification of an analogue peptide, TetraF2W-RR (WWWLRRIW; Figure 1), with enhanced antimicrobial (MICE.coli(μM) = 25; MICS.aureus(μM) = 1.6–3.1) and antibiofilm activity against methicillin-resistant Staphylococcus aureus (MRSA) in free and immobilized forms. Substituting phenylalanine residues with tryptophan, enhanced the antimicrobial activity, particularly against MRSA (MIC(μM) = 1.6– 3.1) [108]. Horine (WWWLRRRW) and Verine-L (RRRWWWWL) are the ultimate uprade of Temporine (Figure 1), and have also enhanced the antimicrobial activity against S. aureus (MICHorine = 4 μM, MICVerine-L = 4 μM) and E. coli (MICHorine = 32 μM, MICVerine-L = 4 μM) [109].

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Figure 1. Primary structure of Temporin_SHf and its artiﬁcial analogues: TetraF2W-RR, Horine and

Verine-L. Amino acid side chains are colored in red (hydrophobic), blue (hydrophilic) and green (positively charged).

Frogs, such as Rana temporaria and Litoria aurea, secrete numerous closely related antimicrobial peptides as an effective chemical dermal defense [110]. Short peptides, isolated from frogs of the Rana genus (Anura), have intriguing antimicrobial activity against pathogenic microorganisms, and even drug-resistant strains. Each peptide has a heptapeptide loop, and reduction of the C-terminal disulfide bridge maintains the loop-like conformation and has no significant effect on hemolytic and antimicrobial activity [111]. In contrast, the substitution of the C-terminal cysteines and disulfide bridge by metathesized vinylic allylglycine residues resulted in increased antimicrobial potency [112]. Brevinin-1BYa (FLPILASLAAKFGPKLFCLVTKKC) was first isolated from skin secretions of the foothill yellow-legged frog Rana boylii, and showed broad-spectrum activity, and was particularly effective against opportunistic yeast pathogens [113]. Brevinin-1BYa has no secondary structure in water, while assuming a flexible helix–hinge–helix motif with C-terminal intramolecular disulfide bridge in membrane-mimicking micelles (Figure2A–D) [ 113]. Indeed, in the I-Tasser calculations [114,115] (Figure2A,B) the unfolded and partially folded structure can be obtained. Notably, residues 1, 4, 5, 7, 8, 10, 11 are potential chlorophylla binding sides, while residues 2, 3, 6, and 7 are nucleic acids binding sides. Nevertheless, the therapeutic potential of brevinin-1BYa is limited by its high hemolytic activity against human erythrocytes (LD50 = 10 µM) [116]. [C18S,C24S]brevinin-1BYa (Figure2E,F) involves substitution of the conserved cysteine residues by serine residues, which results in an acyclic analogue eightfold reduction in hemolytic activity with retained high potency against Gram-positive bacteria, including strains of S. aureus MRSA (MIC = 5 µM). However, activities against Gram-negative bacteria and yeast species were reduced [117]. The non-linear relationship between the hydrophobicity of α-helical AMPs and their hemolytic activity is well documented [118]. It is possible that the [C18S,C24S] analogue fails to maintain a non-bonded loop structure akin to that of the native peptide with reduced cysteines as the Ser24 residue is not sufficiently hydrophobic to associate with the largely hydrophobic region of the helix [116]. I-Tasser calculation show that [C18S,C24S] analogue forms a helical structure; nevertheless, the C-end remains unfolded (Figure2E,F), and the overall 3D structure of the peptide differs from that of brevinin-1BYa. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 11 of 44 Int. J. Mol. Sci. 2020, 21, 7349 11 of 45

FLPILASLAAKFGPKLFCLVTKKC A B C

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FLPILASLAAKFGPKLFSLVTKKS E

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Figure 2. 2. (A(A,B,B)) I-Tasser I-Tasser simulation simulation of brevinin-1BYa of brevinin-1BYa ternary ternary structure. structure. (C,D )( PDBC,D) (PDB: PDB 6G4U) (PDB: ternary 6G4U) ternarystructure structure of brevinin-1BYa. of brevinin (E-1BYa.,F) I-Tasser (E,F) simulation I-Tasser simulation of [C18S,C24S]brevinin-1BYa of [C18S,C24S]brevinin ternary-1BYa structure. ternary Aminostructure. acids Amino are colored acids in are red colored (hydrophobic), in red blue (hydrophobic), (hydrophilic), blue green (hydrophilic), (positively charged) green and(positively yellow (cysteine).charged) and yellow (cysteine).

Snake venom t toxinsoxins have valuable potential in the the design design and and synthesis synthesis of of novel novel antimicrobials. antimicrobials. Lys49 phospholipase A2s A2s (PLA2s) (PLA2s) are are multifunctional multifunctional snake snake toxins toxins able able to toinduce induce a huge a huge variety variety of therapeuticof therapeutic effects effects and and consequently consequently serve serve as as templates templates for for new new drug drug leads. Almeida Almeida et et al. al. synthesized five five oligopeptides mimicking regions regions of the antibacterial Lys49 PLA2 toxin (CoaTx-II(CoaTx-II isolated from Crotalus oreganus oreganus abyssus abyssus snakesnake venom). venom). The The 13 13 amino amino acid acid peptide pCpC-CoaTxII,-CoaTxII, corresponding to residues 115115-129-129 of CoaTx-IICoaTx-II (KKYRIYPKFLCKK),(KKYRIYPKFLCKK), was able to reproduce the promising bactericidal effect effect of the toxin againstagainst multi-resistantmulti-resistant clinical isolates of Gram-negativeGram-negative P. Aeruginosa µ bacteria (MIC P. Aeruginosa(μM( M)) = =5.95).5.95). Even Even though, though, the the molecular molecular dynamics dynamics (MD) (MD) simulations simulations showed

Int. J. Mol. Sci. 2020, 21, 7349 12 of 45 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 12 of 44 that pC-CoaTxII is unstructured [119], the I-Tasser calculation (Figure3) showed the peptide’s that pC-CoaTxII is unstructured [119], the I-Tasser calculation (Figure 3) showed the peptide’s susceptibility to form a helical shape. A possible antimicrobial mechanism of pC-CoaTxII is through susceptibility to form a helical shape. A possible antimicrobial mechanism of pC-CoaTxII is through strong interaction with anionic lipid membranes such as those in bacteria. In silico studies suggested strong interaction with anionic lipid membranes such as those in bacteria. In silico studies suggested formation of a water channel across the membrane upon peptide insertion, eventually leading to formation of a water channel across the membrane upon peptide insertion, eventually leading to bacterial cell disruption and death [119]. bacterial cell disruption and death [119].

Figure 3. Four models of the antibacterial synthetic peptide pC-CoaTxII structure calculated with FigureI-Tasser. 3. Four Amino models acids areof the colored antibacterial in red (hydrophobic), synthetic peptide green (positivelypC-CoaTxII charged) structure and calculated yellow (cysteine). with I- Tasser. Amino acids are colored in red (hydrophobic), green (positively charged) and yellow (cysteine).Tachyplesin-1 (TP1) is a 17 amino acid AMP (KWCFRVCYRGICYRRCR) extracted from the hemocytes of the horseshoe crab Tachypleus tridentatus [120]. It forms a β-hairpin structure (Figure4), bothTachyplesin in aqueous- solution1 (TP1) and is a in 17 lipid-mimicking amino acid AMP environments (KWCFRVCYRGICYRRCR) [121,122], and has two extracted disulfide from bridges the hemocytes(Cys3-Cys16, of the Cys7-Cys12) horseshoe thatcrab playTachypleus an important tridentat roleus [120] in broad. It forms spectrum a β-hairpin antimicrobial structure activity (Figure [123 4), ]. bothThe reductionin aqueous in solution antimicrobial and inactivity, lipid-mimicking when Cys residues environments are removed [121,122] from, and the peptide’shas two disul sequence,fide bridgesis presumably (Cys3-Cys16, linked Cys7 to the-Cys12) subsequent that play loss an of importantβ-sheet stacking role in [ broad124–126 spectrum]. TP1 exhibits antimicrobial potent activityactivity [123] against. The Gram-positive reduction in (MIC antimicrobialB. pseudomallei activity,(µM) = when62) and Cys Gram-negative residues are (MICremovedE.coli ( fromµM) = the22) peptide’sbacteria, sequence, as well as is fungi.presumably It did linked not induce to the subsequent resistance in loss short-term of β-sheet studies stacking [127 [124],– but126 caused]. TP1 exhibitsdecreased potent susceptibility activity underagainst long-term Gram-positive continuous (MIC selectionB. pseudomallei conditions(μM) = 62) [124 and]. However, Gram-negative TP1 is E.coli HEPG2 (MIChighly toxic(μM) toward= 22) bacteria, mammalian as well cells as fungi. (IC50 It did( notµM) induce= 110), resistance making itin unsuitable short-term for studies therapeutic [127], butdevelopment. caused decreased Edwards susceptibility et al. [7] prepared under long a systematic-term continuous study of aselection clear structure conditionsfunction [124]. relationship However, − TP1for each is highly amino toxic acid toward in the sequence, mammalian while cells modulating (IC50HEPG2(μM charge) = 110), and hydrophobicity making it unsuitable by residue for therapeuticmodification development. and truncation Edwards of the et al. peptide. [7] prepared They a were systematic able to study assess of a the clear eff structure−functionects of amino acid relationshipreplacements for on each antimicrobial amino acid activity, in the cytotoxicity,sequence, while and hemolyticmodulating activity. charge Moreover, and hydrophobicity they evaluated by residuethe membrane modification binding and truncation affinity and of the stability peptide. of They the most were interestingable to assess peptides. the effects Three of amino modified acid replacementspeptides: (TP1[F4A]), on antimicrobial (TP1[I11A]), activity, and (TP1[C3A,C16A]) cytotoxicity, maintained and hemolytic the β -hairpin activity. secondary Moreover, structure they evaluatedmotif (Figure the 4 membrane), and possessed binding substantially affinity and improved stability of therapeutic the most indexes interesting (26-to peptides. 64-fold) Three over modifiedthe progenitor peptides: peptide, (TP1[F4A]), exhibiting (TP1[I11A]), MICE.coli( µg and/mL) (TP1[C3A,C16A])= 0.125–4; MIC K.pneumoniae maintained(µg /mL) the β=-hairpin0.25–16; secondaryMICA.baumanii structure(µg/mL) motif= 0.25–0.5; (Figure 4), MIC andP.aeruginosa possessed(µg /substantiallymL) = 0.25–2; improved MICB.subtilis therapeutic(µg/mL) indexes= 0.125–0.25; (26- toMIC 64-S.aureusfold) over(µg/ mL)the progenitor= 2–8; MIC peptide,C.albicans(µ exhibitg/mL)ing= 2–8; MIC MICE.coli(C.neofomansμg/mL) =(µ 0.125g/mL)–4;= MIC0.25–4K.pneumoniae values(μg/mL and were) = 0.25considerably–16; MICA.baumanii less hemolyticly(μg/mL) = 0.25 toxic.–0.5; (TP1[F4A])MICP.aeruginosa and(μg/mL (TP1[I11A]),) = 0.25–2; notMIC onlyB.subtilis conserve(μg/mL) the= 0.125β-hairpin–0.25; MICsecondaryS.aureus(μg/mL structure) = 2 motif,–8; MIC butC.albicans also(μg/mL are highly) = 2 stable–8; MIC inC.neofomans mouse(μg/mL and human) = 0.25 plasma.–4 values Of noteworthy and were considerably less hemolyticly toxic. (TP1[F4A]) and (TP1[I11A]), not only conserve the β-hairpin secondary structure motif, but also are highly stable in mouse and human plasma. Of noteworthy

Int. J. Mol. Sci. 2020, 21, 7349 13 of 45 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 13 of 44 mention,mention, I-Tasser I-Tasser calculation calculation showed showed the the presence presence of of potential potential magnesium magnesium and and zinc zinc binding binding sides sides in in TP1TP1 analogues. analogues.

A KWCFRVCYRGICYRRCR

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TP1 [I11A] TP1 [F4A] TP1 [C3A; C16A]

KWCFRVCYRGACYRRCR KWCARVCYRGICYRRCR KWAFRVCYRGICYRRAR

FigureFigure 4. 4.( A(A,B,B)) Tachyplesin-1 Tachyplesin-1 (TP1) (TP1) structure structure (PDB:1WO0). (PDB:1WO0). ( C(C)) I-Tasser I-Tasser calculations calculations of of TP1 TP1 analogues analogues structure.structure. Amino Amino acids acids are are colored colored in in red red (hydrophobic), (hydrophobic), green green (positively(positively charged) charged) and and yellow yellow (cysteine).(cysteine). The design of a library of peptide antibiotics is usually done using a parent peptide (naturally The design of a library of peptide antibiotics is usually done using a parent peptide (naturally occurring or designed in the laboratory), followed by structure activity relationship (SAR) studies and occurring or designed in the laboratory), followed by structure−activity− relationship (SAR) studies ﬁne-tuning the activity of the peptide [11]. A synthetic antimicrobial peptide library based on the human and fine-tuning the activity of the peptide [11]. A synthetic antimicrobial peptide library based on the autophagy 16 polypeptide has been developed by Varnava et al. [128]. The fatty acids conjugation human autophagy 16 polypeptide has been developed by Varnava et al. [128]. The fatty acids (inspired by lipopeptide antibiotics e.g., polymyxin [129]) and N-Acetylation [130–133] strategies were conjugation (inspired by lipopeptide antibiotics e.g., polymyxin [129]) and N-Acetylation [130–133] used to enhance the activity and serum stability of AMPs. The ﬁne-tuning of structure and length of strategies were used to enhance the activity and serum stability of AMPs. The fine-tuning of structure the fatty acid component of the antimicrobial lipopeptide battacin [134] resulted in peptides library and length of the fatty acid component of the antimicrobial lipopeptide battacin [134] resulted in with enhanced potency with respect to the parent Atg1, a human autophagy polypeptide. A 21-residue peptides library with enhanced potency with respect to the parent Atg1, a human autophagy polypeptide. A 21-residue fragment of Atg16 conjugated to 4-methylhexanoic acid (K30; Figure 5) emerged as the most potent antibacterial agent (MICC.albicans(μM) = 30–60; MICP.aeruginosa(μM) = 60–120;

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Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 14 of 44 fragment of Atg16 conjugated to 4-methylhexanoic acid (K30; Figure5) emerged as the most potent antibacterial agent (MICC.albicans(µM) = 30–60; MICP.aeruginosa(µM) = 60–120; MICE.coli(µM) = 6.4–12.8; MICE.coli(μM) = 6.4–12.8; MICS.aureus(μM) = 0.9–1.8). Moreover, the relationship between the K30 S.aureus µ structureMIC (Figure( M) =5) 0.9–1.8).and function Moreover, in the bacterial the relationship membrane between was determined. the K30 structure The negatively (Figure charged5) and bacterialfunction membrane in the bacterial anchor membrane the K30 was peptide, determined. and subsequent The negatively interactions charged with bacterial the hydrophobic membrane residuesanchor the of K30the peptide, peptide, causing and subsequent the K30 peptide interactions to adopt with a the helix−loop−helix hydrophobic residues structure. of theThe peptide, folded causing the K30 peptide to adopt a helix loop helix structure. The folded structure is able to penetrate structure is able to penetrate the nonpolar− acyl− chain of the lipid molecules, where it subsequently causesthe nonpolar membrane acyl chaindisintegration of the lipid leading molecules, to cell where lysis [128] it subsequently. causes membrane disintegration leading to cell lysis [128].

RWKRHISEQLRRRDRLQRQA

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Figure 5. K30 structure calculated with I-Tasser. Amino acids are colored in red (hydrophobic), Figureblue (hydrophilic), 5. K30 structure and calculated green (positively with I-Tasser. charged). Amino acids are colored in red (hydrophobic), blue (hydrophilic), and green (positively charged). Interference with bacterial virulence is a promising alternative approach or a complementary adjunctInterference to traditional with antimicrobialbacterial virulence therapy is a [ 135promising]. Antivirulence alternative strategies approach focus or a oncomplementary pathogenicity adjunctfactors andto traditional bypass the an pressuretimicrobial on the therapy bacterium [135] to. Antivirulence develop resistance. strategies The MgtCfocus membraneon pathogenicity protein factorshas been and proposed bypass the as an pressure attractive on antimicrobial the bacterium target, to develop while itresistance. is involved The in theMgtC ability membrane of several protein major hasbacterial been pathogensproposed as (e.g., an Pseudomonasattractive antimicrobial aeruginosa) to target, survive while inside it is macrophages. involved in Moussounithe ability of et several al. [135] majordeveloped bacterial an antivirulencepathogens (e.g. strategy, Pseudomonas targeting aeruginosa MgtC, by) to taking survive advantage inside macrophages. of a natural antagonisticMoussouni etpeptide, al. [135] MgtR developed (ILFVADSLQMIPLCLRIWVALKINILFSVL). an antivirulence strategy targeting MgtC, Heterologous by taking advantage expression of of a MgtRnatural in antagonisticP. aeruginosa peptide,PAO1 was MgtR shown (ILFVADSLQMI to reduce its abilityPLCLRIWVALKINILFSVL). to survive within macrophages, Heterologous while expression exogenously of MgtRadded in synthetic P. aeruginosa MgtR PAO1 peptide was lowered shown the to intramacrophagereduce its ability survivalto survive of wild-typewithin macrophages,P. aeruginosa whilePAO1, exogenouslythus mimicking added the phenotypesynthetic MgtR of an MgtCpeptide mutant lowered as well the as intramacrophage the eﬀect of endogenously survival of produced wild-type MgtR P. aeruginosapeptide [135 PAO1,]. Similar thus to mimicking AMP-mimicking the phenotype peptides, ofMgtR an has MgtC a high mutant susceptibility as well as to form the effect a helical of endogenouslystructure. The produced crystal structure MgtR peptide not known [135]. at Similar the present to AMP time,-mimicking nevertheless, peptides, the I-TasserMgtR has structure a high susceptibilityprediction shows to form the possibility a helical to structure. form a central The crystal helix or structure a helix–strand–helix not known structure at the present (Figure time,6). nevertheless, the I-Tasser structure prediction shows the possibility to form a central helix or a helix– strand–helix structure (Figure 6).

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Figure 6. I-Tasser prediction of MgtR secondary structure. (A) one alpha-helix motif in the middle of Figure 6. I-Tasser prediction of MgtR secondary structure. Amino acids are colored in red MgtR secondary structure. (B) two short alpha-helix motifs in the middle of MgtR secondary structure. (hydrophobic), blue (hydrophilic), green (positively charged) and yellow (cysteine). Amino acids are colored in red (hydrophobic), blue (hydrophilic), green (positively charged) and yellow (cysteine). 7. Amino Acid Conjugated Polymers 7. Amino Acid Conjugated Polymers Synthetic homo- and co-polymers have low toxicity to host cells and at the same time simulate theSynthetic functions homo- of AMPs and [25,136] co-polymers. In contrast have low to AMPs, toxicity syntheti to hostc cellsantibacterial and at the polymers same time have simulate a versatile thechemical functions structure, of AMPs scalability, [25,136]. and In contrast lower synthesis to AMPs, costs synthetic [137,138] antibacterial. The polymers polymers are bilateral have— a on versatileone side chemical cationic structure, residues scalability, act with the and negatively lower synthesis charged costs bacterial [137, 138 cell].-wall, The on polymers the other are side bilateral—onhydrophobic one sidedomains cationic interact residues with act the with lipophilic the negatively layers charged of the bacterial cell-wall, cell-wall on leading the other to the sidedisruption hydrophobic of thedomains cell membrane interact with [28,139 the lipophilic–141]. Many layers peptid of thee-mimetic bacterial antibacterial cell-wall leading polymers to the are disruptionalready of reported the cell membranein the literature [28,139 in– 141recent]. Many decades, peptide-mimetic and are mainly antibacterial derivatives polymers of polymethacrylates are already reported[142,143] in the, literature polyacrylates in recent [144,145] decades,, and polynorbornenes are mainly derivatives [146,147] of polymethacrylates, poly(β-lactam)s [142 ,[28,139]143], , polyacrylatespolymaleimides [144,145 [148,149]], polynorbornenes, and polycarbonates [146,147], [150,151] poly(β-lactam)s. Extensive [28, 139studies], polymaleimides were dedicated [148 to, probing149], andthe polycarbonates correlation between [150,151 ]. antimicrobial Extensive studies activity were and dedicated polymer to probing amphiphilicity the correlation [143,152] between, structure antimicrobial(random or activity block) and[136,153] polymer, type amphiphilicity of cationic charge [143 ,152[154]],, structure molecular (random weight or[145] block), and [ 136spaced,153 ],arm type(distance of cationic from charge polymer [154], backbone molecular to weight pendant [145 cationic], and spaced center arm) [1]. (distance from polymer backbone to pendantSystematic cationic center) optimization [1]. of the (co)polymer composition, chain length, hydrophobicity, and cationicSystematic charge optimization has generated of the selected (co)polymer examples composition, that are chain also length,highly hydrophobicity,biocompatible (non and cationic-hemolytic chargeand has non generated-cytotoxic in selected vitro). examples The extensive that revieware also on highly biomimetic biocompatible antimicrobial (non-hemolytic polymers together and non-cytotoxicwith polymerin vitro chemistry). The extensivewas published review by on Ergene biomimetic et al. in antimicrobial 2018 [138], polymersand here, togetherwe discuss with some polymerrecent chemistry advances wasin effective published antimicrobial by Ergene polymer et al. in discove 2018 [138ry.], and here, we discuss some recent advancesTwo in e ffkeyective parameters antimicrobial are known polymer to discovery.affect the efficacy of AMP mimics: molecular weight and amphipathicTwo key parameters balance. Polymers are known of tolower affect molecular the efficacy weight of AMPhave mimics:greater antimicrobial molecular weight activity and [155], amphipathicwhile correct balance. amphipathic Polymers balance of lower is molecularnecessary to weight impart have both greater antibacterial antimicrobial activity activity and selectivity [155], whileversus correct bacterial amphipathic membranes balance is[25 necessary]. If the to hydrophobicity impart both antibacterial is too high, activity selectivity and selectivity is reduced, versus and bacterialeukaryotic membranes cell death [25]. occurs.If the hydrophobicity Researchers increased is too high, hydrophobicity selectivity is reduced, through and copolymerization eukaryotic cell of deathcationic occurs. monomers Researchers with increased hydrophobic hydrophobicity monomers throughwith alkyl copolymerization tails of different oflengths, cationic which monomers generally withresulted hydrophobic in increased monomers bacterial with alkyl cell tails death of di atff erent the expense lengths, which of selectivity generally [156] resulted. Multiple in increased polymer bacterialbackbone cell death structures, at the expense including of selectivity methacrylates [156]. Multiple[157], β polymer-lactams backbone[158], norbornenes structures, including [159], and methacrylatesmethacrylamides [157], β[160]-lactams, with [ 158varying], norbornenes solubility [and159 ],inherent and methacrylamides polarity have been [160 ],evaluated with varying as AMP solubilitymimics. and inherent polarity have been evaluated as AMP mimics. PositivelyPositively charged charged peptidespeptides- lysine lysine andand arginine arginine are are present present in the in theprimary primary sequence sequence of different of differentAMPs AMPs constituting constituting 6–8% 6–8%incidence incidence of each of amino each amino acid [138] acid.[ Synthetic138]. Synthetic polymers polymers containing containing moieties moietiesmimicking mimicking lysine lysineand arginine and arginine components components found in found AMPs in have AMPs been have reported been reportedto show effectiveness to show effectivenessagainst specific against bacteria. specific Exley bacteria. at al. [161] Exley created at al. [ 161a series] created of copolymers a series of containing copolymers lysine containing-mimicking aminopropyl methacrylamide (APMA) and arginine-mimicking guanadinopropyl methacrylamide

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Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 16 of 44 lysine-mimicking aminopropyl methacrylamide (APMA) and arginine-mimicking guanadinopropyl (GPMA)methacrylamide (Figure (GPMA)7). Copolymers (Figure7 were). Copolymers prepared werewith preparedvarying ratios with varying of the co ratios-monomers of the co-monomers (APMA and (APMA and GPMA), with a degree of polymerization of 35 40 and narrow molecular weight GPMA), with a degree of polymerization of 35−40 and narrow− molecular weight distribution to simulatedistribution naturally to simulate occurring naturally occurring AMPs. The AMPs. APMA The APMA homopolymer homopolymer demonstrated demonstrated the the greatest greatest antimicrobial activity against E. coli ,, S. aureus and P. aeruginosaaeruginosa (MIC((MIC(µμg/mLg/mL)) == 500) and the lowest toxicity to mammalian cells. At At the the same same time, time, the the antimicrobial antimicrobial activity of the APMA homopolymer was least aaffectedﬀected by changeschanges in salt concentrationconcentration of both type of the polymers tested [[161]161].. On the contrary, additionaddition of GPMA units in the polymer, lower the antimicrobial activity. Considering MIC 100 µg/mL as high antimicrobial activity, and MIC ranging from 500 to 1000 µg/mL as moderate ∼100 μg/mL as high antimicrobial activity, and MIC ranging from 500 to 1000 μg/mL as moderate activity, thethe APMAhomoAPMAhomo polymerpolymer showedshowed promisingpromising results,results, butbut needsneeds somesome futurefuture improvements.improvements.

FiguFigurere 7. Schematic representation of aminopropylaminopropyl methacrylamidemethacrylamide (APMA)-stat-guanadinopropyl(APMA)-stat-guanadinopropyl methacrylamide (GPMA)(GPMA) polymer. Red: hydrophobic chain, green: positively charged residues.

Brittin et et al. al. [162 [162]] optimized optimized the cationic the cationic charged charged placement, placement, amphiphilic amphiphilic balance and balance PEGylation and PEGylationcontent in synthesis content ofin acrylate-basedsynthesis of acrylate random-based ternary random copolymers ternary comprised copolymers of samecomprised center cationic,of same centeethylr and cationic, poly(oligoethyleneglycol) ethyl and poly(oligoethyleneglycol) side chains [162 side]. chains The resultant [162]. The molecules resultant (Figure molecules8) showed (Figure 8eﬀ) showedective antimicrobial effective antimicrobial activity with activity low MIC with ( lowµg/mL) MIC against (μg/mLE.) against coli and E.B. coli subtilis and ,B. ranging subtilis, between ranging between22Int. and J. Mol. 44 22 Sci. (µ andg 20/mL)20 44, 21 and(, μg/mLx FOR between PEER) and REVIEW 2.5between and 10 2.5 (µ andg/mL), 10 respectively.(μg/mL), respectively. 17 of 44 Antimicrobial polycarbonates containing primary amino groups can effectively kill bacteria as shown by Nimmagadda et al. [150]. Their single, di-block and random copolymers containing hydrophobic (0–10) and hydrophilic (10–20) units (Figure 9) were revealed to be more effective against different multidrug resistant Gram-positive bacteria strains (MICS.aureus(μM) = 5–25; MICS. epidermidis(μM) = 5–25; MICE. faecalis(μM) = 5–25) respect Maganin II (MICS.aureus(μM) = 16; MICS. epidermidis(μM)> 50; MICE. faecalis(μM)> 50). The amphiphilic units are essential for bacterial killing by bacterial membrane disruption, and random block polymers are more potent than di-block polymers, probably due to stable nanostructures of di-block polymers, which prevent them from interacting with bacterial membranes more effectively.

FigureFigure 8. 8. SchematicSchematic representation representation of of P1–P4 P1–P4 polymers. polymers. In In red: red: hydrophobichydrophobic residues, residues, in in green: green: positivelypositively charged charged chains. chains.

Antimicrobial polycarbonates containing primary amino groups can eﬀectively kill bacteria as shown by Nimmagadda et al. [150]. Their single, di-block and random copolymers containing

Figure 9. Schematic representation of the polycarbonate polymer with primary amino groups. Green: positively charged monomer; red: hydrophobic monomer.

A new frontier in host defense peptides mimicking is β-peptide polymers. Their potent antimicrobial activity and excellent biocompatibility, together with high stability upon protease associated biodegradation are well ascertained [163,164]. Recently, Chen et al. presented the new potent antimicrobial peptide polymer named 80:20 DM:Bu (Figure 10). It is composed of two subunits, one hydrophilic/cationic subunit (named DM) and one hydrophobic subunit (named Bu), to have both cationic charges and amphiphilicity as synthetic mimics of HDPs [27,28,32]. The polymer displayed fast, potent, and broad-spectrum activities upon multiple multi-drug resistant (MDR) strains of bacteria (MICS. aureus (μg/mL) = 12.5; MICs.haemolyticus (μg/mL) = 3.13; MIC P.aeruginosa (μg/mL) = 12.5; MICE.coli (μg/mL) = 50), resistant to ampicillin (MICS. aureus (μg/mL) > 200; MICs.haemolyticus (μg/mL) > 200; MIC P.aeruginosa (μg/mL) > 200; MICE.coli (μg/mL) > 200) and streptomycin (MICS. aureus (μg/mL) = 25; MICs.haemolyticus (μg/mL) > 200; MIC P.aeruginosa (μg/mL) > 200; MICE.coli (μg/mL) = 100). Moreover, peptide polymer 80:20 DM:Bu exerted satisfactory activity against K. pneumoniae (MIC (μg/mL) = 50–100) and A. baumannii (MIC (μg/mL) = 12.5–50).

Figure 10. Schematic representation of 80:20 DM:Bu polymer. Green: positively charged monomer; red: hydrophobic monomer.

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Int. J. Mol. Sci. 2020, 21, 7349 17 of 45

hydrophobic (0–10) and hydrophilic (10–20) units (Figure9) were revealed to be more e ffective against different multidrug resistant Gram-positive bacteria strains (MICS.aureus(µM) = 5–25; MICS. epidermidis(µM) = 5–25; MICE. faecalis(µM) = 5–25) respect Maganin II (MICS.aureus(µM) = 16; MICS. epidermidis(µM) > 50; MICE. faecalis(µM) > 50). The amphiphilic units are essential for bacterial killing by bacterial membrane disruption, and random block polymers are more potent than di-block polymers,Figure probably 8. Schematic due torepresentation stable nanostructures of P1–P4 polymers. of di-block In red: polymers, hydrophobic which residues, prevent in them green: from Figure 8. Schematic representation of P1–P4 polymers. In red: hydrophobic residues, in green: positively charged chains. interactingpositively with charged bacterial chains. membranes more effectively.

Figure 9. Schematic representation of the polycarbonate polymer with primary amino groups. Green: FigureFigure 9. 9. SchematicSchematic representation representation of the of polycarbonate the polycarbonate polymer polymer with primary with primary amino groups. amino groups.Green: positively charged monomer; red: hydrophobic monomer. positivelyGreen: positively charged charged monomer; monomer; red: hydrophobic red: hydrophobic monomer. monomer.

β AAA new new new frontier frontier in in host host defense defen defenses e peptides peptides peptides mimicking mimicking mimicking is is is β -peptide- βpeptide-peptide polymers. polymers. polymers. Their Their Their potent potent potent antimicrobialantimicrobialantimicrobial activity activity activity and and and excellent excellent excellent biocompatibility, biocompatibility, biocompatibility, together together together with with with high high high stability stability stability upon upon upon protease protease protease associatedassociatedassociated biodegradation biodegradationbiodegradation are areare well wellwell ascertained ascertained ascertained [163,164] [ 163[163,164],164].. Recently,. Recently,Recently, ChenChen Chen et et et al. al. al. presented presentedpresented the thethe new newnew potpotentpotentent antimicrobial antimicrobial antimicrobial peptide peptide peptide polymer polymer polymer named named named 80:20 80:20 80:20 DM:Bu DM:Bu DM:Bu (Figure (Figure (Figure 10). It 10). is 10). composed It It is is composed composed of two subunits, of of two two subunits,onesubunits, hydrophilic one one hydrophilic/cationic hydrophilic/cationic/cationic subunit (named subunit subunit DM) (named (named and one DM) DM) hydrophobic and and one one hydrophobic hydrophobic subunit (named subunit subunit Bu), (named to(named have Bu), both Bu), tocationicto have have both chargesboth cationic cationic and charges amphiphilicity charges and and amphiphilicity amphiphilicity as synthetic as mimicsas synthetic synthetic ofHDPs mimics mimics [27 of ,of28 HDP HDP,32].s [s The27 [27,28, polymer28,32,32]. ]The. The displayed polymer polymer displayedfast,displayed potent, fast, fast, and potent, potent, broad-spectrum and and broad broad-spectrum activities-spectrum upon activities activities multiple upon upon multi-drug multiple multiple multi multi resistant-drug-drug (MDR) resistant resistant strains (MDR) (MDR) of S. aureus µ S. aureus s.haemolyticuss.haemolyticusµ P.aeruginosaP.aeruginosaµ strainsbacteriastrains of of (MICbacteria bacteria (MIC (MIC( S.g aureus/mL) (μg/mL (=μg/mL12.5;) =) MIC =12.5; 12.5; MIC MICs.haemolyticus( g (/mL)μg/mL (μg/mL= )3.13; =) =3.13; 3.13; MIC MIC MIC P.aeruginosa (μg/mL (gμg/mL/mL)) =) =12.5;12.5; 12.5; E.coliE.coli µ S. aureusS. aureusµ s.haemolyticuss.haemolyticus µ MICMICMICE.coli (μg/mL ((μg/mLg/mL)) =)= =50),50), 50), resistant resistant resistant to to ampicillinampicillin ampicillin (MIC (MIC (MICS. aureus (μg/mL (μg/mLg/mL)) >>) 200;>200; 200; MICMIC MICs.haemolyticus (μg/mL ((μg/mLg/mL)) >)> >200; 200;200; P.aeruginosaP.aeruginosa µ E.coliE.coli µ S.S. aureus aureus µ MICMICMIC P.aeruginosa ( μg/mL(μg/mLg/mL)) )> >>200; 200;200; MIC MIC MICE.coli ( μg/mL(μg/mLg/mL)) )> > >200) 200)200) and and and streptomycin streptomycin streptomycin (MIC (MIC (MICS. aureus ( μg/mL((μg/mLg/mL)) )= = =25; 25;25; s.haemolyticuss.haemolyticus µ P.aeruginosaP.aeruginosa µ E.coli E.coli µ MICMICMICs.haemolyticus (μg/mL (μg/mL( g/mL)) >) >200; >200;200; MIC MIC MIC P.aeruginosa (μg/mL (μg/mL( )g >)/ mL) >200; 200; >MIC MIC200;E.coli MIC (μg/mL (μg/mL)( =) g =100)./ mL)100). Moreover,= Moreover,100). Moreover, peptide peptide µ polymerpeptidepolymer polymer80:20 80:20 DM:Bu DM:Bu 80:20 exerted DM:Bu exerted exertedsatisfactory satisfactory satisfactory activity activity activity against against against K. K. pneumoniae pneumoniaeK. pneumoniae (MIC (MIC (MIC(μg/mL (μg/mL ( g) /=)mL) =50 50–=100)–100)50–100) and and µ A.andA. baumannii baumanniiA. baumannii (MIC (MIC(MIC (μg/mL (μg/mL ( g)/ mL)=) =12.5 12.5=–12.5–50).50).–50).

Figure 10. Schematic representation of 80:20 DM:Bu polymer. Green: positively charged monomer; FigureFigure 10. 10. SchematicSchematic representation representation of of 80:20 80:20 DM:Bu DM:Bu polymer. polymer. Green: Green: positively positively charged charged monomer; monomer; red: hydrophobic monomer. rred:ed: hydrophobic hydrophobic monomer. monomer.

In 2019, Zhou et al. [165] presented a new concept of designing novel biocompatible antibacterial

copolymers and expanded the categories of next-generation antibacterial agents without inducing drug resistance. The series of di-block copolymers were inspired by Poly(ε-caprolactone) (PCL) natural AMPs. Poly(ε-caprolactone) (PCL) is a biodegradable copolymer with controlled degradability, biocompatibility, and miscibility with other polymers, properties [166–168] excellent for biomedicine applications [166]. PCL (hydrophobic component) was coupled with polylysine (Kn) to form Int.Int. J.J. Mol.Mol. Sci.Sci. 20202020,, 2211,, xx FORFOR PEERPEER REVIEWREVIEW 1818 ofof 4444

InIn 2019,2019, ZhouZhou etet al.al. [165][165] presentedpresented aa newnew conceptconcept ofof designingdesigning novelnovel biocompatiblebiocompatible antibacterialantibacterial copolymerscopolymers andand expandedexpanded thethe categoriescategories ofof nextnext--ggenerationeneration antibacterialantibacterial agentsagents withoutwithout inducinginducing drugdrug resistance. resistance. The The series series of of di di--blockblock copolymers copolymers were were inspired inspired by by Poly(ε Poly(ε--caprolactone)caprolactone) (PCL) (PCL) naturalInt.natural J. Mol. Sci.AMPs. AMPs.2020, 21 Poly(ε Poly(ε, 7349 --caprolactone)caprolactone) (PCL) (PCL) is is a a biodegradable biodegradable copolymer copolymer with with controlled controlled18 of 45 degradability,degradability, biocompatibibiocompatibility,lity, andand miscibilitymiscibility withwith otherother polymers,polymers, propertiesproperties [166[166––168168]] excellentexcellent forfor biomedicinebiomedicine applicationsapplications [166][166].. PCLPCL (hydrophobic(hydrophobic component)component) waswas coupledcoupled withwith polylysinepolylysine (K(Knn)) diblock copolymers named PCL -b-K (Figure 11). Three polymers PCL -b-K , PCL -b-K , toto formform diblockdiblock copolymerscopolymers namednamed16 PCLPCLn1616--bb--KKnn (Figure(Figure 11).11). ThreeThree polymerspolymers PCLPCL16 1616--bb--11KK1111,, PCLPCL161616--bb--KK2020,, PCL -b-K showed excellent antimicrobial activity against E. coli (MIC (µg/mL) = 8–32) and S. aureus PCLPCL1616--bb--KK2727 showedshowed excellentexcellent antimicrobialantimicrobial activityactivity againstagainst E.E. colicoli (MIC(MIC ((μg/mLμg/mL)) == 88––32)32) andand S.S. aureusaureus (MIC(MIC ((µμg/mLμg/mLg/mL))) === 88–16).8––16).16). The The membrane membrane disruption disruption mechanism mechanism and and cytoplasm cytoplasm leakage leakage were were observed observed for both E. coli and S. aureus treated with PCL -b-K copolymer. The cytotoxicity tests conducted forfor bothboth E.E. colicoli andand S.S. aureusaureus treatedtreated withwith PCLPCL161616--bb--KK2020 20 copolymer.copolymer. TheThe cytotoxicitycytotoxicity teststests conductedconducted onon on human dermal ﬁbroblasts for 72 h showed that even at 500 µg/mL PCL -b-K concentration humanhuman dermaldermal fibroblastsfibroblasts forfor 7272 hh showedshowed thatthat eveneven atat 500500 μg/mLμg/mL PCLPCL1616--bb--K16K2020 concentrationconcentration20 (fifty(fifty (ﬁftytimestimes times higherhigher higher thanthan thanMICMIC MICactivity),activity), activity), moremore more thanthan than 70%70% 70% ofof thethe of normal thenormal normal humanhuman human cellscells cells werewere were stillstill still alivealive alive [165][165] [165.. ItIt]. It was also shown that PCL -b-K copolymer vesicles exhibit the value of a potential application as waswas alsoalso shownshown thatthat PCLPCL161616--bb--KKnn copolymerncopolymer vesiclesvesicles exhibitexhibit thethe valuevalue ofof aa potentialpotential applicationapplication asas multifunctionalmultifunctional drug-carrierdrugdrug--carriercarrier systemssystems withwith antibacterialantibacterial capabilitycapability inin cancercancer therapytherapytherapy [[169][169]169]...

ε FigureFigure 11.11. SchematicSchematic representationrepresentation ofofof Poly( Poly(εPoly(ε-caprolactone)--caprolactone)caprolactone) (PCL) ((PCLPCL)16)1616-b-K--bb--KKnnn polymer.polymer. Green: Green: positively positively chargedcharged monomer;monomer; red:red: hydrophobic hydrophobic monomers. monomers.

HydrophobicityHydrophobicity modulation modulation modulation through through through incorporation incorporation incorporation of of of amino amino amino acids acids acids in in cationic cationicin cationic polymers polymers polymers can can providecanprovide provide aa significantsignificant a significant opportunityopportunity opportunity toto designdesign to newnew design aminoamino new acidacid amino conjugatedconjugated acid conjugated polymerspolymers (ACPs)(ACPs) polymers withwith (ACPs) potentpotent withantibacterialantibacterial potent activityactivity antibacterial andand minimumminimum activity andtoxicitytoxicity minimum towardtoward toxicitymammalianmammalian toward cells.cells. mammalian InIn 2019,2019, BarmanBarman cells. etet Inal.al. [170] 2019,[170] presentedBarmanpresented et al. a a class class[170] of presentedof ACPs ACPs with with a class tunable tunable of ACPs antibacterial antibacterial with tunable activity activity antibacterial through through activity a a simple simple through post post--polymerpolymer a simple-- post-polymer-functionalizationfunctionalizationfunctionalization strategy.strategy. PermanentPermanent strategy. cationiccationic Permanent chargecharge cationicwaswas prpresentesent charge inin everyevery was present repeatingrepeating in uniteveryunit (Figure(Figure repeating 12),12), wherebyunitwhereby (Figure thethe 12 hydrophobicityhydrophobicity), whereby the hydrophobicity ofof thethe entireentire moleculemolecule of the entire waswas moleculetunedtuned throughthrough was tuned incorporationincorporation through incorporation ofof differentdifferent aminoofamino different acids.acids. amino TheThe aminoamino acids. acidacid The alteration aminoalteration acid hadhad alteration aa strongstrong had influenceinfluence a strong onon influence antibacterialantibacterial on antibacterial efficacy,efficacy, andand e ffi asascacy, thethe aminoandamino as theacidacid amino sideside--chch acidainain side-chain hydrophobicityhydrophobicity hydrophobicity waswas increased,increased, was increased, bothboth thethe both antibacterialantibacterial the antibacterial activityactivity activity (against(against (against broadbroad spectrumbroadspectrum spectrum ofof bacteria)bacteria) of bacteria) andand toxicitytoxicity and toxicity increased.increased. increased. ACPACP includingincluding ACP including aa glycineglycine a glycine residueresidue residue (ACP(ACP--11 (ACP-1 (Gly),(Gly), FigureFigure (Gly), A.baumannii Figure 12) showed very good activityA.baumannii (MIC (µg/mL) = 8 16) against both drug-sensitive and 12)12) showedshowed veryvery goodgood activityactivity (MIC(MICA.baumannii ((μg/mLμg/mL)) == 8−16)8−16) againstagainst− bothboth ddrugrug--sensitivesensitive andand drugdrug-- resistantdrug-resistantresistant strains, strains, strains, including including including clinical clinical clinical isolates. isolates. isolates. Moreover, Moreover, Moreover, after after after 14 14 14 continuous continuous continuous passages passages passages there there there was was no no propensitypropensity forfor bacterialbacterial resistanceresistance developmentdevelopment againstagainstagainst thisthisthis polymer.polymer.polymer.

FigureFigureFigure 12. 12.12.Schematic SchematicSchematic representation representationrepresentation of ofof 1-10 11--1010 aminoamino acidacid conjugconjug conjugatedatedated polymerspolymers polymers ((ACPsACPs (ACPs).)).. InIn green:green: In green: permanent cationic charge. permanentpermanent cationiccationic charge.charge.

Fixing AMPs on surfaces is a new strategy to enhance their stability and increase local concentration and biological availability. Nevertheless, immobilized AMPs have lower ability to interact with multiple targets [171]. A possible solution is attachment of the peptides to the substrate Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 19 of 44 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 19 of 44 Fixing AMPs on surfaces is a new strategy to enhance their stability and increase local concentrationInt. J. Mol.Fixing Sci. 2020 AMPs and, 21, 7349biological on surfaces availability. is a new Nevertheless, strategy to enhance immobilized their AMPs stability have and lower increase ability19 localof to 45 interactconcentration with multiple and biological targets [171] availability.. A possible Nevertheless, solution is attachment immobilized of the AMPs peptides have to lower the substrate ability to viainteract a spacer, with whichmultiple increases targets [171] the flexibility. A possible and solution enhances is attachment the functional of the conformationpeptides to the of substrate AMPs, therebyviavia a a spacer, spacer, ameliorating which which increasesthe increases overall the the antimicrobial flexibility flexibility and and effect enhances enhances [172]. Such the the functional functionala strategy conformationis conformation a new horizon of of AMPs, AMPs,in the fabricationtherebythereby amelioratingameliorating of biocoatings. thethe overall overallRecently, antimicrobialantimicrobial Acosta et eal.effectff ect[172] [[172]172 proposed].. SuchSuch a aan strategystrategy effective isis aa antibiofilm newnew horizonhorizon coatings inin thethe basedfabricationfabrication on protein of of biocoatings. biocoatings.-engineered Recently, polymers Acosta Acosta and antimicrobial et et al.al. [[172]172] proposedpeptidesproposed for anan preventing e effectiveffective antibiofilm antibiofilmimplant-associated coatings coatings infectionsbasedbased ononprotein-engineered protein[172]. -engineered The unstructuredpolymers polymers GL13K andand antimicrobialantimicrobial (GKIIKLKASLKLL peptidespeptides- forNHfor preventingpreventing2) peptide implant-associated implant (Figure- associated 13) was hybridizedinfectionsinfections [ 172 with[172]]. Theelastin. The unstructured -likeunstructured recombinamers GL13K GL13K (GKIIKLKASLKLL-NH (ELRs) (GKIIKLKASLKLL and subsequently2) peptide-NH tethered2) (Figure peptide to commercially13 (Figure) was hybridized 13) pure was titaniumwithhybridized elastin-like discs, with and recombinamers elastin their-like biological recombinamers (ELRs) response and subsequently (ELRs)was successfully and tetheredsubsequently tested to commercially against tethered the to pure biofilmcommercially titanium activi discs,ty pure of oralandtitanium their microorganisms biologicaldiscs, and response their (S. gordonii biological was successfully and response P. gingivalis tested was ).successfully against This innovative the biofilm tested proteinactivity against- ofengineered the oral biofilm microorganisms molecularactivity of platform(S.oral gordonii microorganisms couldand beP. gingivalisused ( S.for gordonii).immobilization This innovative and P. gingivalisof protein-engineered different). ThisAMPs innovative on molecularan ECM protein-mimicking platform-engineered could polymer be molecular used for the for developmentimmobilizationplatform could of ofbeantibiofilm di usedfferent for AMPsimmobilizationand cytocompatible on an ECM-mimicking of different coatings AMPs polymeron titanium. on an for ECM the development-mimicking polymer of antibiofilm for the anddevelopment cytocompatible of antibiofilm coatings and on titanium. cytocompatible coatings on titanium. GKIIKLKASLKLL GKIIKLKASLKLL

Figure 13. SchematicSchematic representation representation of of the GL13K peptide hybridized with cyclooctyne-modiﬁedcyclooctyne-modified elastinelastin-likeFigure-like 13. recombinamer Schematic representation ( (ELR)ELR) (spacer). (spacer). of the GL13K GL13K GL13K secondary secondary peptide structure structurehybridized was was with calculated calculated cyclooctyne with with -Imodified I-Tasser.-Tasser. Inelastin blue: blue:- hydrophiliclike hydrophilic recombinamer amino amino acids; (ELR acids; )in (spacer). red: in red: hydropho GL13K hydrophobicbic secondary amino amino acids; structure acids; in green: inwas green: positively calculated positively charged with chargedI- aminoTasser. acids.aminoIn blue: acids. hydrophilic amino acids; in red: hydrophobic amino acids; in green: positively charged amino acids. 8. Mimicking Mimicking the the Structure Structure of AMPs 8. Mimicking the Structure of AMPs Peptoids are non-natural,non-natural, sequence sequence-speciﬁc-specific peptidomimetic oligomers based on a protein-likeprotein-like backbone,Peptoids but withare non a side -natural, chain sequence appendage-specific at the amidepeptidomimetic nitrogen (Scheme(Sche oligomersme4 4)[) 23[based23].]. on a protein-like backbone, but with a side chain appendage at the amide nitrogen (Scheme 4) [23].

Scheme 4.4. Chemical structures of peptides ((left)left) and peptoids (right). (right). Scheme 4. Chemical structures of peptides (left) and peptoids (right). Synthetic polymers mimicking antimicrobial peptides have have huge therapeutics potential; nevertheless,Synthetic designing designing polymers an an ideal mimicking ideal peptoid peptoid antimicrobial drug drug is complicated is complicated peptides because have because there huge there are therapeutics many are many variables variables potential; that influencethatnevertheless, influence the activity thedesigning activity and an and function ideal function peptoid of the of the drugoligomer. oligomer. is complicated The The most most importantbecause important there factors factors are manyto to consider consider variables are are thethat the influence the activity and function of the oligomer. The most important factors to consider are the cationic charge, conformational constraint via macrocyclization, and hydrophobicity, which are directly correlated with membrane interactions and biological activities in vitro. Peptoid optimization strategies Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 20 of 44 cationic charge, conformational constraint via macrocyclization, and hydrophobicity, which are directlyInt. J. Mol. correlated Sci. 2020, 21, 7349 with membrane interactions and biological activities in vitro. Peptoid20 of 45 optimization strategies in use are in vitro trial-and-error approaches [173], high-throughput ligand screening on large peptoid libraries [174], and computational approaches with prediction of peptoid efficacyin use arein in vitro vitro [175trial-and-error–177]. The approaches synchrotron [ 173 liquid], high-throughput surface X-ray ligand scattering screening studies on oflarge molecule peptoid structurelibraries– [activity174], and relationships computational have approaches a new impact with on prediction studies of ofthe peptoid mechanism efficacy of actionin vitro by[ 175peptoid–177 ]. antimicrobials,The synchrotron and liquid suggest surface optimization X-ray scattering strategies studies for future of molecule therapeutics structure–activity based on peptoids relationships [178]. Forhave instance, a new impactsubstitution on studies of lysine of theto mechanismguanidine groups, of action increases by peptoid the cationic antimicrobials, charge and guides suggest peptideoptimization–peptoid strategies chimeras for toward future phosphatidylglycerol therapeutics based on in peptoids bacterial [178membranes]. For instance, [179]. Macrocyclic substitution peptoidsof lysine reveal to guanidine superior groups, in vitro increasesefficacy over the linear cationic peptoids, charge andwith guidesidentical peptide–peptoid monomer composition, chimeras againsttoward Gram phosphatidylglycerol-positive and Gram in bacterial-negative membranes bacteria [180] [179. The]. Macrocyclic optimal hydrophobic peptoids reveal parameters, superior balancingin vitro e ffimembranecacy over specificity linear peptoids, and at with the identicalsame time monomer low cytotoxicity, composition, have against been proposed Gram-positive to form and helicalGram-negative peptoids [ bacteria20]. [180]. The optimal hydrophobic parameters, balancing membrane specificity andOf at thenoteworthy same time mention, low cytotoxicity, peptoids adopt have been a helical proposed structure to form [181] helical, and are peptoids resistant [20 to]. proteolytic degradationOf noteworthy [182]. Helical mention, peptidomimetic peptoids adopt oligomers a helical (foldamers) structure [181 with], and a structure are resistant similar to proteolyticto that of linear,degradation cationic, [182 facially]. Helical amphipathic peptidomimetic helical antibacterial oligomers (foldamers)peptides such with as magainins a structure (class similar of AMPs to that foundof linear, in the cationic, African facially clawed amphipathic frog Xenopus helical laevis [183] antibacterial) have garnered peptides particular such as magaininsinterest [181] (class. For of example,AMPs found certain in the amphipathic African clawed β-peptide frog Xenopus helices laevis are [ comparable183]) have garnered to magainins particular (Figure interest 14) [ 181 in ]. antibacterialFor example, activity certain and amphipathic selectivityβ [163]-peptide. Parameters helices are that comparable may have to an magainins influence (Figure on activities 14) in (oligomerantibacterial length, activity charge, and hydrophobicity, selectivity [163 chirality,]. Parameters secondary that structure may have and an side influence chain nature), on activities the mode(oligomer of action length, of these charge, peptide hydrophobicity,-mimetics and chirality, their selectivity secondary for structurebacteria versus and side mammalian chain nature), cells havethe modebeen scrutinized of action of theseover the peptide-mimetics last ten years [20,173,184 and their selectivity–186]. for bacteria versus mammalian cells have been scrutinized over the last ten years [20,173,184–186]. A GIGKFLHSAKKFGKAFVGEIMNS

Turn 180◦

Figure 14. Structure of Magainin-2 (PDB: 2LSA) as a representative of the magainin protein family. Figure(A) Ribbon 14. Structure model (ofB) Magainin Surface model:-2 (PDB: left 2LSA) and rightas a representative side. In blue: of hydrophilic the magainin amino protein acids, family. in green: In blue:positively hydrophilic charged amino amino acids, acids; in ingreen red:: hydrophobicpositively charged amino amino acids. acids; in red: hydrophobic amino acids. The structure–activity relationship among 22 cationic amphipathic peptoids was studied by Mojsoska et al. [187]. All studied peptoids had an overall net charge of +4 and were 8 to 9 residues The structure–activity relationship among 22 cationic amphipathic peptoids was studied by long; however, the hydrophobicity and charge distribution along the abiotic backbone varied. Mojsoska et al. [187]. All studied peptoids had an overall net charge of +4 and were 8 to 9 residues Changes, such as replacing Ntrp with monomers with specific aromatic side chains, rearranging the long; however, the hydrophobicity and charge distribution along the abiotic backbone varied. charge distribution along the peptoid backbone, and shortening the length, significantly impacted the Changes, such as replacing Ntrp with monomers with specific aromatic side chains, rearranging the overall hydrophobicity profiles of the peptoids. Peptoids with high hydrophobicity do not always charge distribution along the peptoid backbone, and shortening the length, significantly impacted appear as the most potent against E. coli and P. aeruginosa, while against S. aureus, there is a linear the overall hydrophobicity profiles of the peptoids. Peptoids with high hydrophobicity do not always relationship between their hydrophobicity and potency. In addition, increased hydrophobicity caused

Int.Int. J.J. Mol.Mol. Sci.Sci. 20202020,, 2211,, xx FORFOR PEERPEER REVIEWREVIEW 2121 ofof 4444 appearappear asas thethe mostmost potentpotent againstagainst E.E. colicoli andand P.P. aeruginosaaeruginosa,, whilewhile againstagainst S.S. aureusaureus,, therethere isis aa linearlinear Int. J. Mol. Sci. 2020, 21, 7349 21 of 45 relationshiprelationship between between their their hydrophobicity hydrophobicity and and potency. potency. In In addition, addition, increased increased hydrophobicity hydrophobicity causedcaused byby introducingintroducing highlyhighly aromaticaromatic residues,residues, suchsuch asas thethe NN--(2,2(2,2--diphenylethyl)glycinediphenylethyl)glycine (Ndpe)(Ndpe) monomer,bymonomer, introducing isis stronglystrongly highly correlated aromaticcorrelated residues, withwith aa lossloss such ofof as aantibacterialntibacterial the N-(2,2-diphenylethyl)glycine specificity,specificity, resultingresulting inin (Ndpe) highhigh toxicitytoxicity monomer, inin mammalianismammalian strongly correlated cells.cells. with a loss of antibacterial specificity, resulting in high toxicity in mammalian cells. BarronBarron andand and colleaguescolleagues colleagues showedshowed showed thatthat that thethe the threefoldthreefold threefold periodicityperiodicity periodicity ofof thethe of polypoly the- poly-proline-prolineproline typetype I typeI--likelike I-like(PPI(PPI-- like)(PPI-like)like) helix helix helix of of α α of--peptoidspeptoidsα-peptoids [188][188] [188 enabledenabled] enabled mimicry mimicry mimicry of of of the the the magainin magainin magainin helical helical helical structure structure structure [[ [232323]].].. The The The new new amphipathicamphipathic helixhelix waswas built built by by periodic periodic incorporation incorporation ofof aa cationic cationic sideside chain chain (NLys)(NLys) every every three three residues,residues, thethe the remainingremaining remaining positionspositions positions beingbeing being occupiedoccupied occupied byby thethe by aromatic thearomatic aromatic aa--chiralchiral a-chiral (S)(S)--phenylethylphenylethyl (S)-phenylethyl sideside chainchain side (Nspe),chain(Nspe), (Nspe), which which which helps helps helpsto to format format to format the the helix helix the helixstructu structu structurerere and and provides provides and provides hydrophobic hydrophobic hydrophobic helical helical helical faces. faces. faces. The The dodecamerThedodecamer dodecamer (NLys(NLys (NLys-Nspe-Nspe)--NspeNspe--Nspe)Nspe)44 (Figure(Figure4 (Figure 15)15) showedshowed 15) showed excellentexcellent excellent broadbroad--spectrumspectrum broad-spectrum antibacterialantibacterial antibacterial activitiesactivities E.coli E.coli (MICactivities(MICE.coli ((μM (MICμM)) == 3.53.5––(14),µ14),M) butbut= 3.5–14), alsoalso displayeddisplayed but also potentpotent displayed cytotoxicitycytotoxicity potent cytotoxicitytowardtoward carcinomacarcinoma toward cellscells carcinoma (LC(LC5050(breast(breast cells cancer)(LCcancer)50(breast((μMμM)) = cancer)(= 5;5; LC LC50µ50(prostateM)(prostate= 5; cancer)LC cancer)50(prostate((μMμM)) == cancer)(5;5; LC LC5050(ovarianµ(ovarianM) = 5; cancer) cancer) LC50(ovarian((μMμM)) == 6;6; cancer)( LC LC5050(fetal(fetalµM) lung lung= 6; fibroblast)LCfibroblast)50(fetal(( lungμMμM)) fibroblast)(== 8;8; LCLC5050(primary(primaryµM) = 8;dermaldermal LC50(primary fibroblast)fibroblast) dermal((μMμM)) == fibroblast)( 8)8) [189][189].. µM) = 8) [189].

Figure 15. Chemical structure of (NLys-Nspe-Nspe)4 peptoid. In green: positively charged side chain; Figure 15. Chemical structure of (NLys-Nspe-Nspe)(NLys-Nspe-Nspe)44 peptoid. In In green: green: positively positively charged charged side side chain; chain; inin red: red: hydrophobichydrophobic side side chain. chain.

FollowingFollowing this this mimicking mimicking trend, trend, Shyam Shyam et et al. al. preparedprepared aa seriesseries ofof 1,2,31,2,3 1,2,3-triazolium-based--triazoliumtriazolium--basedbased cationiccationic amphipathicamphipathic peptoid peptoid oligomers oligomers that that mimic mimic antimicrobial antimicrobial helical helical peptides peptides [190] [[190]190]... They They explored explored the the potentialpotential of of of the the the triazolium triazolium triazolium group g grouproup as a cationic as as a a cationic cationic moiety and moiety moiety helix and and inducer helix helix to inducerdevelop inducer potent to to develop develop antimicrobial potent potent antimicrobialhelicalantimicrobial peptoids. helicalhelical Several peptoids.peptoids. triazolium-based SeveralSeveral triazoliumtriazolium oligomers,--basedbased even oligomers,oligomers, of short length, eveneven ofof selectively shortshort length,length, killed selectivelyselectively bacteria killedoverkilled mammalian bacteriabacteria overover cells. mammalianmammalian Among cells. testedcells. AmongAmong oligomers testedtested H-(Naetm-Nspe-Nspe) oligomersoligomers HH--(Naetm(Naetm4--Nspe-NHNspe2--Nspe)Nspe)(Figure44--NH NH16)22 was (Figur(Figur theee E.coli E.fecalis S.aureus 16)most16) waswas eﬀ ectivethethe mostmost (MIC effectiveeffective(µ (MICM)(MIC= E.coliE.coli6.3–50; ((μMμM MIC)) == 6.36.3––50;50; MIC(MICµM)E.fecalisE.fecalis= 11; ((μMμM MIC)) == 11;11; MICMIC(µM)S.aureusS.aureus= 10).((μMμM)) == 10).10).

FigureFigure 16.16. ChemicalChemical structurestructure ofofof H-(Naetm-Nspe-Nspe)HH--(Naetm(Naetm--NspeNspe--Nspe)Nspe)444-NH--NHNH22 peptoid.peptoid. InIn green:green: positivelypositively charged charged sideside chain; chain; in in red: red: hydrophobichydrophobic side side chain. chain. The amino acids—leucine and lysine—have strong helix-promoting abilities [191] and have TheThe aminoamino acidsacids——leucineleucine andand lysinelysine——havehave strongstrong helixhelix--promotingpromoting abilitiesabilities [191][191] andand havehave beenbeen been used for the construction of prototype α-helical antimicrobial peptides, usually referred to usedused forfor thethe constructionconstruction ofof prototypeprototype αα--helicalhelical antimicrobialantimicrobial peptides,peptides, usuallyusually referredreferred toto asas ““LKLK as “LK peptides”. Among them, 14- and 15-mer peptides with amphipathic helix, were found to peptidespeptides””.. AmongAmong them,them, 1414-- andand 1515--mermer peptidespeptides withwith amphipathicamphipathic helix,helix, wewerere foundfound toto bebe thethe mostmost be the most effective against bacteria [44,192], while shorter peptides were inactive. Attaining a effectiveeffective against against bacteria bacteria [44,192][44,192],, while while shorter shorter peptides peptides were were inactive. inactive. Attaining Attaining a a secondary secondary secondary structure and amphipathicity are two determining factors for the antimicrobial activity structurestructure andand amphipathicityamphipathicity areare twotwo determiningdetermining factorsfactors forfor thethe antimicrobialantimicrobial activityactivity ofof thethe LKLK of the LK peptides. Indeed, Monroc et. al. demonstrated that 4 10-residue-long LK peptides have − antimicrobial activity only when cyclized [193], due to structural rigidity, and increased binding Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 22 of 44 Int. J. Mol. Sci. 2020, 21, 7349 22 of 45 peptides. Indeed, Monroc et. al. demonstrated that 4−10-residue-long LK peptides have antimicrobial activity only when cyclized [193], due to structural rigidity, and increased binding to the bacteria membranes.to the bacteria Additionally membranes., the presence Additionally, of numerous the presence Trp residues of numerous stabilize Trp the residueshelical structure stabilize [194] the andhelical increase structure the affinity [194] andof the increase naturally the occurring affinity ofAMPs the peptide naturally for occurring the membrane AMPs [195] peptide. Even for single the tryptophanmembrane [substitution195]. Even singleat certain tryptophan positions substitution of inactive atfragments certain positionsof the am ofphipathic inactive helical fragments AMPs of canthe amphipathicsignificantly enhance helical AMPs their antimicrobial can significantly activity enhance [195] their. Following antimicrobial these instructions, activity [195 and]. Following keeping inthese mind instructions, the role of C and-terminal keeping amidation in mind theon the role activity of C-terminal of AMPs amidation and the prevalence on the activity of tryptophan of AMPs residuesand the prevalencein several natural of tryptophan and synthetic residues AMPs, in several Pandit naturalet al. prepared and synthetic short peptides, AMPs, Pandit cheaper et in al. synthesis,prepared shortand just peptides, as effective cheaper as long in synthesis,AMPs [196] and, with just P5 as peptide effective (MIC as longE.coli (μM AMPs) = 10; [196 MIC], withP.aeruginosa P5 E.coli P.aeruginosa K. pneumonie S.typhi (peptideμM) = 10; (MIC MICK. ( pneumonieµM) =(μM10;) MIC= 10; MICS.typhi(µ(μMM) =) =10; 7.5; MIC MICS.aureus(μM(µ)M) = =50;10; M MICICC. albicans((μMµM)) = 7.5;50; S.aureus C. albicans C.grubii MICC.grubii(μM(µM)) = =15)50; slightly MIC more effective(µM) = 50; than MIC the P4 peptide(µM) = 15)(MIC slightlyE.coli (μM more) = 50; eff ectiveMICP.aeruginosa than the (μM P4) E.coli P.aeruginosa K. pneumonie S.typhi =peptide 50; MIC (MICK. pneumonie(µ(μMM) )= =50; 50; MIC MICS.typhi(μM(µ)M) = =15;50; MIC MICS.aureus(μM) (µ=M) 50;= 50; MIC MICC. albicans(μM(µM)) == 50;15; S.aureus C. albicans C.grubii MICC.grubii(μM(µM)) = 25).= 50; Moreover, MIC the (peptidesµM) = 50; P4 MIC(LKWLKKL(µ-M)NH=2) and25). P5 Moreover, (LRWLRRL the-NH peptides2) (Figure P4 17)(LKWLKKL-NH were found to2 )be and non P5-hemolytic (LRWLRRL-NH and non-2cytotoxic) (Figure to17 human) were cell found lines. to Surprisingly, be non-hemolytic these small and peptidesnon-cytotoxic did not to adopt human any cell specific lines. secondary Surprisingly, structure these in small the free peptides or sodium did dodecyl not adopt sulphate any specific (SDS) micellarsecondary bound structure state, in theand free do or not sodium need dodecyl secondary sulphate structures (SDS) to micellar exhibit bound antimicrobial state, and activity. do not Electrostaticneed secondary interaction structures is to enough exhibit for antimicrobial P4 and P5 activity. to attach Electrostatic to the membrane, interaction and is enoughits successive for P4 deformationand P5 to attach and tolysis the [196] membrane,. and its successive deformation and lysis [196].

2 2 Figure 17. ChemicalChemical s structurestructures of P4 (LKWLKKL-NH (LKWLKKL-NH2)) and and P5 (LRWLRL-NH(LRWLRL-NH2)) peptides. In In red: red: hydrophobic side chains; in green: positively charged side chains.

Cationic antimicrobial antimicrobial poly( poly(αα-amino-amino acid)s acid)s (APAAs) (APAAs) mimic mimic structures structures and antimicrobial and antimicrobial properties propertiesof the AMPs of the were AMPs extensively were extensi describedvely described in a recent in a recent review review of Shen of Shen et al.et al. [197 [197]].. APAAsAPAAs are are easy to synthesize, and and have have prolonged antimicrobial antimicrobial activity, activity, low cytotoxicity, and enhanced stability to to protease protease degradation. degradation. A A series series of of random random co co-polypeptides-polypeptides with with various various chain chain lengths lengths (5 to(5 200 to 200 residues) residues and) and hydrophobic hydrophobic contents contents (1– (1–5050 mol%) mol%) synthesized synthesized by Wyrsta by Wyrsta et al. et [198] al. [198 mimic] mimic the cationicthe cationic and and amphiphilic amphiphilic nature nature (Figure (Figure 18) 18 of) of many many natural natural antimicrobial antimicrobial peptides. peptides. The The most reactivereactive samples samples have have high high hydrophobic hydrophobic contents contents and and intermed intermediateiate chain chain lengths, lengths, and and in in particular, particular, peptides consisting of Lys/Leu Lys/Leu with α--helixhelix conformation exhibit good selectivity and activity to the microbialmicrobial-mimicked-mimicked membrane membrane..

Int. J. Mol. Sci. 2020, 21, 7349 23 of 45 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 23 of 44 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 23 of 44

Figure 18. Schematic representation of cationic antimicrobial poly(α-amino acid)s (APAAs) mimic Figure 18. Schematic representation of cationic antimicrobial poly(α-amino acid)s (APAAs) Figure 18.1 Schematic representation+ - 2 of cationic antimicrobial poly(α-amino acid)s (APAAs) mimic structures. R (green) = -(CH1 2)4NH3 Br ; R (red) = -+CH3; -CH(CH2 3)2; -CH(CH3)CH2CH3; -CH2CH(CH3)2; mimicstructures. structures. R1(green) R =(green) -(CH2)4=NH-(CH3+Br2-); 4RNH2(red)3 Br = −-CH;R 3(red); -CH(CH= -CH3)2;3 -;CH(CH -CH(CH3)CH3)2;2CH -CH(CH3; -CH32)CHCH(CH2CH3)32;; -CH2C6H5. -CH-CH22CH(CHC6H5. 3)2; -CH2C6H5. Moreover, the surface-initiated N-Carboxyanhydride (NCA) polymerization strategies used for Moreover,Moreover, thethe surface-initiatedsurface-initiated N-CarboxyanhydrideN-Carboxyanhydride (NCA)(NCA) polymerizationpolymerization strategiesstrategies usedused forfor APAA synthesis have also been proposed to generate dense polypeptides brushes on surfaces of a APAAAPAA synthesis synthesis have have also also been been proposed proposed to generateto generate dense dense polypeptides polypeptides brushes brushes on surfaces on surfaces of a wide of a wide range of organic and inorganic substrates, including gold, carbon nanotubes, macroporous rangewide ofrange organic of organic and inorganic and inorganic substrates, substrates, including including gold, carbon gold, nanotubes, carbon nanotubes, macroporous macroporous polymeric polymeric templates, magnetite nanoparticles, and silica nanoparticles [199]. Thus, surface-grafting templates,polymeric magnetitetemplates, nanoparticles, magnetite nanoparticles, and silica nanoparticles and silica nanoparticles [199]. Thus, surface-grafting[199]. Thus, surface technologies-grafting technologies could be used to fabricate APAAs coatings on biomedical devices, and solve the critical couldtechnologies be used could to fabricate be used APAAs to fabricate coatings APAAs on biomedical coatings on devices, biomedical and devices, solve the and critical solve problem the critical of problem of device-related infections [200]. device-relatedproblem of device infections-related [200 infections]. [200]. Recently, short peptides combining α-helix and β-turn sequence-motif in a symmetric-end Recently,Recently, short short peptides peptides combining combining α α-helix-helix and and β β-turn-turn sequence-motif sequence-motif in in a a symmetric-end symmetric-end template showed enhanced cell selectivity, antibacterial activity (MICE.coliE.coli(μM) = 2–32; templatetemplate showed showed enhanced enhanced cell cell selectivity, selectivity, antibacterial antibacterial activity activity (MIC (MICE.coli(µμMM)) == 2–32;2–32; MICB.pyocyaneumB.pyocyaneum(μM) = 2–16; MICS.pullorumS.pullorum(μM) = 2–32; MICS.aureus(μMS.aureus) = 8–32; MICS.faecalis(μMS.faecalis) = 8–32) and MICMICB.pyocyaneum(μM(µM)) = =2–2–16;16; MIC MICS.pullorum(μM(µ)M) = 2=–32;2–32; MIC MICS.aureus(μM()µ =M) 8–=32;8–32; MIC MICS.faecalis(μM)( µ=M) 8–32)= 8–32) and salt-resistance. Two peptides PQ (IHKFWRCRRRFCRWFKHI-NH2) and PP (IHKFWRPGRWFKHI- andsalt-resistance. salt-resistance. Two peptides Two PQ (IHKFWRCRRRFCRWFKHI peptides PQ (IHKFWRCRRRFCRWFKHI-NH-NH2) and PP (IHKFWRPGRWFKHI2) and PP- NH2) tended to form an α-helical structure upon α interacting with a membrane-mimicking (IHKFWRPGRWFKHI-NHNH2) tended to form an2 ) α tended-helical to structure form an upon-helical interacting structure with upon a membrane interacting-mimicking with a environment (Figure 19). According to I-Tasser calculation, PP has a manganese binding site (K3, R6, membrane-mimickingenvironment (Figure 19). environment According (Figure to I-Tasser 19). Accordingcalculation, to PP I-Tasser has a manganese calculation, PPbinding has a si manganesete (K3, R6, P7 residues), while PQ has a magnesium binding site (R10, C12 and R13 residues). Moreover, they bindingP7 residues), site (K3,while R6, PQ P7 has residues), a magnesium while binding PQ has site a magnesium (R10, C12 and binding R13 residues). site (R10, Moreover, C12 and they R13 showed significant cell selectivity toward bacterial cells over eukaryotic cells. Their activities were residues).showed significant Moreover, cell they selectivity showed toward signiﬁcant bacterial cell selectivity cells over toward eukaryotic bacterial cells. cellsTheir over activities eukaryotic were mostly maintained in the presence of different conditions (salts, serum, heat, and pH), which cells.mostly Their maintained activities were in the mostly presence maintained of differe in thent presenceconditions of di (salts,ﬀerent serum, conditions heat, (salts, and serum, pH), which heat, indicated their stability in vivo [201]. andindicated pH), which their stability indicated in theirvivo stability[201]. in vivo [201].

IHKFWRPGRWFKHI IHKFWRPGRWFKHI

PPPP

IHKFWRCRRRFCRWFKHI IHKFWRCRRRFCRWFKHI

PQPQ

Figure 19. Two possible secondary structures of PP and PQ peptides calculated with I-Tasser. In blue: Figure 19. Two possible secondary structures of PP and PQ peptides calculated with I-Tasser. In blue: hydrophilicFigure 19. Two amino possible acids; secondary in green: positivelystructures chargedof PP and amino PQ peptides acids; in calculated red: hydrophobic with I-Tasser. amino In acids. blue: hydrophilic amino acids; in green: positively charged amino acids; in red: hydrophobic amino acids. hydrophilic amino acids; in green: positively charged amino acids; in red: hydrophobic amino acids. Synthetic peptides with high antibacterial activity and low toxicity can be identiﬁed with Synthetic peptides with high antibacterial activity and low toxicity can be identified with high highSynthetic accuracy peptides using cheminformatics with high antibacterial and machine activity learning, and low and toxicity without can be the identified use of an with original high accuracy using cheminformatics and machine learning, and without the use of an original template accuracy using cheminformatics and machine learning, and without the use of an original template sequence [202]. To achieve that, Fjell et al. [203] used a quantitative structure–activity relationship sequence [202]. To achieve that, Fjell et al. [203] used a quantitative structure–activity relationship

Int. J. Mol. Sci. 2020, 21, 7349 24 of 45

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 24 of 44 template sequence [202]. To achieve that, Fjell et al. [203] used a quantitative structure–activity relationship (QSAR) approach utilizing artificial neural networks (ANN), and built computational (QSAR) approach utilizing artificial neural networks (ANN), and built computational models of models of peptide activity based on data from over 1400 random sequences, biased to contain amino peptide activity based on data from over 1400 random sequences, biased to contain amino acids acids believed from substitution analyses to be important for antibacterial activity. In addition, believed from substitution analyses to be important for antibacterial activity. In addition, they posed they posed an innovative method of generating candidate peptide sequences using the heuristic an innovative method of generating candidate peptide sequences using the heuristic evolutionary evolutionary programming method of genetic algorithms (GA), which provided a large (19-fold) programming method of genetic algorithms (GA), which provided a large (19-fold) improvement in improvement in identification of novel antibacterial peptides. To demonstrate the effectiveness identification of novel antibacterial peptides. To demonstrate the effectiveness of these techniques in of these techniques in identifying drug candidates, an in silico screening of 100,000 peptides was identifying drug candidates, an in silico screening of 100,000 peptides was performed. The 10 most performed. The 10 most active peptides from this optimization were selected for in vivo study, active peptides from this optimization were selected for in vivo study, all having a positive charge of all having a positive charge of 14, a uniform chain length of 9 amino acids and at least three tryptophan 14, a uniform chain length of 9 amino acids and at least three tryptophan residues [204]. Potent broad residues [204]. Potent broad spectrum activity was observed for GN-2 (RWKRWWRWI-CONH2), spectrum activity was observed for GN-2 (RWKRWWRWI-CONH2), GN-4 (RWKKWWRWL- GN-4 (RWKKWWRWL-CONH2), and GN-6 RKRWWWWFR-CONH2 peptides (Figure 20) against CONH2), and GN-6 RKRWWWWFR-CONH2 peptides (Figure 20) against E. coli (MICGN−2(μL/mL) = E. coli (MICGN 2(µL/mL) = 6.2; MICGN 4(µL/mL) = 6.2; MICGN 6(µL/mL) = 12.5), P. aeruginosa 6.2; MICGN−4(μL/− mL) = 6.2; MICGN−6(μL/− mL) = 12.5), P. aeruginosa− (MICGN−2(μL/mL) = 3.1; (MICGN 2(µL/mL) = 3.1; MICGN 4(µL/mL) = 3.1; MICGN 6(µL/mL) = 6.2), and S. aureus MICGN−4(−μL/mL) = 3.1; MICGN−6(μL/mL− ) = 6.2), and S. aureus (MIC−GN−2(μL/mL) = 3.1; MICGN−4(μL/mL) (MICGN 2(µL/mL) = 3.1; MICGN 4(µL/mL) = 3.1; MICGN 6(µL/mL) = 3.1). = 3.1; MIC− GN−6(μL/mL) = 3.1). − −

Figure 20. ChemicalChemical structures structures of GN GN-2,-2, GN GN-4-4 and GN GN-6-6 peptides. In In green: green: positively positively charged charged amino acids; in red: hydrophobic hydrophobic amino acids.

From an an AMP AMP database database [205] [205 ]with with a total a total of of2619 2619 AMP AMP sequences, sequences, AMPs AMPs have have an average an average of +3.2 of +net3.2 positive net positive charges charges and 32.7 and amino 32.7 amino acids, acids, with withglycine glycine (G), lysine (G), lysine (K), and (K), leucine and leucine (L) being (L) beingthe most the abundantmost abundant amino amino acids, acids, and α and-helicesα-helices and β and-sheetsβ-sheets being being the most the mostcommon common secondary secondary struc structures.tures. For theFor development the development of potent of potent and biocompatible and biocompatible AMPs, AMPs, α-helicalα-helical cationic cationic AMPs AMPs (αCAMPs) (αCAMPs) have havebeen the most popular species for investigation [36]. Extensive studies have focused on developing new AMPs by either modifying natural ones or following the strategy of artificial design to produce efficient and cost-effective versions. Among other approaches, one key factor lies in altering amino

Int. J. Mol. Sci. 2020, 21, 7349 25 of 45 been the most popular species for investigation [36]. Extensive studies have focused on developing Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 25 of 44 new AMPs by either modifying natural ones or following the strategy of artificial design to produce acideffi cientsequences and cost-e to produceffective AMPs versions. with Among different other amphiphilicities approaches, one [206 key–208 factor]. Following lies in altering this trend, amino a acid sequences to produce AMPs with different amphiphilicities [206–208]. Following this trend, series of surfactant-like αCAMPs based on the general formula of G(IIKK)nI-NH2 (n = 2−4, and the a series of surfactant-like αCAMPs based on the general formula of G(IIKK)nI-NH (n = 2 4, and the AMPs are denoted as G2, G3, and G4, respectively) [209] were synthesized. Among2 them (Figure− 21), AMPs are denoted as G2, G3, and G4, respectively) [209] were synthesized. Among them (Figure 21), G3 is optimal with potent bioactivity (MICE.coli(μM) = 8; MICB. subtilis(μM) = 2) and low cytotoxicity (IC50 E.coli µ B. subtilis µ (μMG3) is = optimal15 (HeLa with cells); potent = 25 bioactivityHL60 cells) (MIC to the host( M) mammalian= 8; MIC cells. Hydrophobic( M) = 2) and modifications low cytotoxicity of µ G3(IC have50 ( M)been= 15created (HeLa by cells); replacing= 25 HL60the amino cells) acid to the residues host mammalian at the peptide cells. N Hydrophobic-terminals or modificationsC-terminals orof the G3 side have chains been created [210,211] by. replacingThe high theselectivity aminoacid and residuesassociated at thefeatures peptide are N-terminals attributed to or two C-terminals design tactics:or the sidethe use chains of Ile [210 residues,211]. The rather high than selectivity Leu and and the associated perturbation features of the are hydrophobic attributed to face two of design the helicaltactics: structure the use of with Ile residues the insertion rather of than a positively Leu and the charged perturbation Lys resid of theue. hydrophobic Moreover, studies face of for the anticancerhelical structure applications with the have insertion demonstrated of a positively that charged the stronger Lys residue. the hydrophobicity Moreover, studies—the for higher anticancer the bioactivity,applications but have also demonstratedthe higher the toxicity that the [209] stronger. the hydrophobicity—the higher the bioactivity, but also the higher the toxicity [209].

G2 G3 G4

GIIKKIIKKI-NH2 GIIKKIIKKIIKKI-NH2 GIIKKIIKKIIKKIIKKI-NH2

Figure 21. Secondary structure of G2, G3 and G4 peptides calculated with I-Tasser. In green: positively Figure 21. Secondary structure of G2, G3 and G4 peptides calculated with I-Tasser. In green: positively charged amino acids; in red: hydrophobic amino acids. charged amino acids; in red: hydrophobic amino acids. Potent in vitro activity of AMPs often does not translate into in vivo effectiveness due to the interferencePotent in of vitro the hostactivity microenvironment of AMPs often withdoes peptidenot translate stability into/availability. in vivo effectiveness Hence, mimicking due to the the interferencecomplex environment of the host microenvironment found in biofilm-associated with peptide infections stability/availability. is essential to Henc predictinge, mimicking the clinical the complexpotential environment of novel AMP-based found in antimicrobialsbiofilm-associated [212 infections]. The antibiofilm is essential activity to predicting of the semisynthetic the clinical potential of novel AMP-based antimicrobials [212]. The antibiofilm activity of the semisynthetic peptide lin-SB056-1 (KWKIRVRLSA-NH2; Figure 22) and its dendrimeric derivative (lin-SB056-1)2-K peptide lin-SB056-1 (KWKIRVRLSA-NH2; Figure 22) and its dendrimeric derivative (lin-SB056-1)2-K ([KWKIRVRLSA]2-K) against Pseudomonas aeruginosa in an in vivo-like three-dimensional lung epithelial ([KWKIRVRLSA]cell model, and an2-K)in against vitro wound Pseudomonas model (consisting aeruginosa ofin an an artificial in vivo dermis-like three and-dimensional blood components lung epithelialat physiological cell model, levels) and gave an in rewarding vitro wound results. model [212 (consisting]. When alone, of an Lin-SB056-1 artificial dermis was moderatelyand blood componentseffective (MIC at P.aeruginosa physiological(µM) = levels)38.5) in gave reducing rewardingP. aeruginosa results. biofilm[212]. When formation alone, in 3D Lin lung-SB056 epithelial-1 was P.aeruginosa moderatelycells, but its effective antibiofilm (MIC activity( wasμM) significantly= 38.5) in reducing increased P. aeruginosa in association biofilm with formation the chelating in 3D agentlung epithelialEDTA. The cells, combination but its antibiofilm of lin-SB056-1 activity was at significantly 38.5 mM with increa EDTAsed in (0.3 association to 1.25 mM),with the know chelating metal agentchelating EDTA. agent, The resultedcombination in the of reductionlin-SB056- of1 at the 38.5 initial mM bacterial with EDTA inoculum (0.3 to to1.25 the mM), limit know of detection metal chelating agent, resulted in the reduction of the initial bacterial inoculum to the limit of detection (10 (10 CFU/mL). The dimeric derivative (lin-SB056-1)2-K demonstrated an enhanced biofilm-inhibitory CFU/mL).activity (MIC TheP.aeruginosa dimeric (µ derivativeM) = 19.25) (lin as-SB056 compared-1)2-K demonstrated to both lin-SB056-1 an enhanced and the biofilm lin-SB056-1-inhibitory/EDTA P.aeruginosa activitycombination, (MIC reducing(μM the) = number 19.25) of as biofilm-associated compared to both bacteria lin-SB056 up- to1 3-Log and the units lin at-SB056 concentrations-1/EDTA combination,causing less thanreducing 20% cellthe number death [212 of ].biofilm-associated bacteria up to 3-Log units at concentrations causing less than 20% cell death [212].

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 26 of 44 Int. J. Mol. Sci. 2020, 21, 7349 26 of 45 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 26 of 44 KWKIRVRLSA KWKIRVRLSA

Figure 22. Secondary structure of lin-SB056-1 calculated with I-Tasser. In blue: hydrophilic amino Figure 22. Secondary structure of lin-SB056-1 calculated with I-Tasser. In blue: hydrophilic amino Figureacids; 22. in Secondarygreen: positively structure charged of lin amino-SB056 acids;-1 calculated in red: hydrophobicwith I-Tasser. amino In blue: acids. hydrophilic amino acids; in green: positively charged amino acids; in red: hydrophobic amino acids. acids; in green: positively charged amino acids; in red: hydrophobic amino acids. 9. Unnatural Amino Acids 9. Unnatural Amino Acids 9. UnnaturalIntroduction Amino Acids of fluorinated amino acids or other non-natural amino acids such as α- Introduction of ﬂuorinated amino acids or other non-natural amino acids such as α-aminoisobutyric aminoisobutyric acid (Figure 23) in AMPs were proved to improve their resistance to protease acid (FigureIntroduction 23) in AMPs of fluorinated were proved amino to improve acids or their other resistance non-natural to protease amino [213 acids,214]. such A possible as α- [213,214]. A possible mechanism is that the abnormal structure of AMPs can impede the access of mechanismaminoisobutyric is that acid the abnormal (Figure 23) structure in AMPs of AMPs were proved can impede to improve the access their of protease resistance to to the protease amide protease to the amide backbone. In addition, many peptidomimetics were introduced in order to backbone.[213,214]. A In possible addition, mechanism many peptidomimetics is that the abnormal were introduced structure of in AM orderPs can to avoidimpede the the undesired access of avoid the undesired degradation of the α-polypeptides in vivo. For example, Gellman and co- degradationprotease to ofthe the amideα-polypeptides backbone. Inin vivoaddition,. For example,many peptidomimetics Gellman and co-workers were introduced designed in aorder series to workers designed a series of cationic and amphiphilic β-amino acid oligomers and polyamides to ofavoid cationic the and undesired amphiphilic degradationβ-amino of acid the oligomersα-polypeptides and polyamides in vivo. For to example, mimic the Gellman natural and AMPs, co- mimic the natural AMPs, which exhibited low hemolysis, high antibacterial activity, and extreme whichworkers exhibited designed low a hemolysis, series of cationic high antibacterial and amphiphilic activity, β and-amino extreme acid oligomers stability [215 and]. polyamides to mimicstability the [215]natural. AMPs, which exhibited low hemolysis, high antibacterial activity, and extreme stability [215].

FigureFigure 23. Examples23. Examples of unnatural of unnatural amino amino acids acids used used for thefor the synthesis synthesis of AMPs. of AMPs. Figure 23. Examples of unnatural amino acids used for the synthesis of AMPs. AlkeneAlkene and maleic and maleic anhydride anhydride copolymer copolymer mimic amphiphilic mimic amphiphilic structure and structure antimicrobial and antimicrobialproperties of naturalpropertiesAlkene antimicrobial of and natural maleic antimicrobial peptides. anhydride Szkudlarek peptides. copolymer Szkudlarek et mimic al. used amphiphilic et 4-methyl-1-penteneal. used 4 structure-methyl- 1 and- (Figurepentene antimicrobial 23(Figure) as a 23) hydrophobicpropertiesas a hydrophobic of co-monomer natural co antimicrobial-monomer (structure (structure similaritypeptides. similarity withSzkudlarek leucine) with et andleucine) al. maleic used and 4 anhydride- methylmaleic -anhydride1- (leavespentene ample (leaves(Figure space ample 23) foras space furthera hydrophobic for design further ofco -design themonomer hydrophilic of the (structure hydrophilic part bysimilarity means part ofwithby chemicalmeans leucine) of modiﬁcation) chemicaland male icmodification) anhydride at constant (leaves at 1:1 constant ratio ample to 1:1 ensurespaceratio for the to further hydrophobicensure designthe hydrophobic and of the cationic hydrophilic and part cationic similar part part by to thosemeans similar in of Leu:Lysto chemical those 1:1in modification)Leu:Lys LK-peptides 1:1 LK [at216- peptidesconstant]. The C3 [216]1:1 . E.coli copolymerratioThe to ensure C3 showed copolymer the rewardinghydrophobic showed antimicrobial and cationic rewarding activity part similar antimicrobial against to Gram-negativethose in activity Leu:Lys (MIC against1:1 LK- (peptidesµGg/rammL)-negative =[216]20). E.coli S.epidermidisi S.epidermidisi andThe(MIC Gram-positive C3 (μg/mL copolymer) bacteria= 20) and showed (MIC Gram -positive rewarding(µ bacteriag/mL) antimicrobial= (MIC20). activity(μg/mL ) = against20). Gram-negative (MICLabellingE.coliLabelling(μg/mL with) =with 20) the andthe paramagnetic paramagneticGram-positive amino amino bacteria acidacid (MIC TOAC S.epidermidisi (Figure (Figure(μg/mL 24) 24 ))is = isa20). new a new strategy strategy in the in studies the studiesof Labelling conformation, of conformation, with the dynamics, paramagnetic dynamics, orientation, orientation, amino acid and andTOAC physicochemical physicochemical (Figure 24) is a propertiesnew strategy of of in AMPs AMPsthe studies [217 [217]]. . Unexpectedly,of Unexpectedly, conformation, labelling labelling dynamics, with withTOAC or TOACientation, increases increases activity and activity physicochemical against againstGram-positive Gram properties-positive bacteria, of bacteria,as it AMPs was shown as[217] it was. forUnexpectedly,shown Tritrpticin for (TRP3;Tritrpticin labelling Figure (TRP3; with 24 ), TOAC Figure an AMP increases24), against an AMP activity bacteria against against and bacteria fungi Gram [ 218and-positive– 223fungi]. TRP3 [218 bacteria,– is223 13-residues] . asTRP3 it was is 13 - longshownresidue (VRRFPWWWPFLRR) fors Tritrpticinlong (VRRFPWWWPFLRR) (TRP3; with Figure a net 24), positivewith an aAMP net charge positive against of charge (bacteria+4) at of physiological and (+4) fungiat physiological [218 pH.–223 It] exhibits. pH.TRP3 It exhibitis ion 13- s channel-likeresidueion channels long activity (VRRFPWWWPFLRR)-like activity in planar in membranes,planar membranes,with anda net permeabilizes positive and permeabilizes charge bacterial of (+4) bacterial cytoplasmicat physiological cytoplasmic membranes pH. Itmembranes exhibit [218],s leadingion[218] channel, to leading leakage-like toactivity leakage of the in cell of planar the contents cell membranes, contents [222,224 [222,224] ].and Recombinational permeabilizes. Recombinational bacterial studies studies cytoplasmic showed showed that membranesthat replacing replacing both[218]both P, leading or P residuesor residues to leakage by A,by or A,of removal theor removalcell contents of P5 of ledP5 [222,224] led to peptide to peptide. Recombinational conformational conformational studies changes, changes, showed higher higher that membrane replacing membrane permeabilization,bothpermeabilization, P or residues and by and ultimately,A, ultimatelyor removal increased, increasedof P5 led antimicrobial toantimicrobial peptide activityconformational activity [222 [222,223,225],223 ,changes,225]. Labelling. Labellinghigher TRP3membrane TRP3 with with TOACpermeabilization,TOAC (TOAC-VRRFPWWWPFLRR (TOAC-VRRFPWWWPFLRR and ultimately, increased (T1) (T1) and andantimicrobial VRRF-TOAC-WWWPFLRR VRRF-TOAC activity-WWWPFLRR [222,223,225] (T2)) (T2)) led. Labelling led to theto the increase TRP3 increase with in in TOAC (TOAC-VRRFPWWWPFLRR (T1) and VRRF-TOAC-WWWPFLRR (T2)) led to the increase in

Int.Int. J. J. Mol. Mol. Sci. Sci. 20202020, ,2211, ,x 7349 FOR PEER REVIEW 2727 of of 44 45 antibacterial activity against M. luteus (MICT1(μM) < 0.38; MICT2(μM) < 0.39) and E.coli (MICT1(μM) = antibacterial activity against M. luteus (MICT1(µM) < 0.38; MICT2(µM) < 0.39) and E.coli (MICT1(µM) = 4; 4; MICT2(μM) = 1.3), respect to TRP3 (MICM.luteus(μM) = 1.1; MICE.coli(μM) = 1.9) TOAC presented a MICT2(µM) = 1.3), respect to TRP3 (MICM.luteus(µM) = 1.1; MICE.coli(µM) = 1.9) TOAC presented a greater freedom of motion at the N-terminus rather than at the internal position. Analogously to greater freedom of motion at the N-terminus rather than at the internal position. Analogously to TRP3, TRP3, both TOAC-labelled peptides, showed prominent cation selectivity [217]. both TOAC-labelled peptides, showed prominent cation selectivity [217].

VRRFPWWWPFLRR

Figure 24. Chemical structure of TOAC (left) and secondary structure of TRP3 calculated with I-Tasser. FigureIn green: 24. positivelyChemical charged structure amino of TOAC acids; (left) in red: and hydrophobic secondary aminostructure acids. of TRP3 calculated with I- Tasser. In green: positively charged amino acids; in red: hydrophobic amino acids. Most AMPs targeting membrane bilayers are cationic antimicrobial peptides (CAMPs) [226] withMost numerous AMPs arginine targeting or membrane lysine residues bilayers (generally are cationic from antimicrobial 2 to 9), which peptides account (CAMPs)for the positive [226] withnet charge. numerous CAMPs arginine have or an lysine amphiphilic residues structure,(generally in from which 2 to cationic 9), which and account hydrophobic for the positive residues net are charge.clustered CAMPs in diﬀerent have spatial an amphiphilic regions. Nevertheless, structure, in CAMPs which are cationic highly and sensitive hydrophobic to proteases. residues In order are clusteredto overcome in different their short spatial half-life, regions. diﬀerent Nevertheless, CAMP-mimicking CAMPs are peptides highly were sensitive protected to proteases. on N- and In order C-end toby over 6-aminohexanoiccome their short acid half residues-life, different (Figure CAMP 22). All-mimicking peptides peptides display highwere activityprotected toward on N- aand broad C- endspectrum by 6-aminohexanoic of pathogens (MIC acidE.coli residues(µM) (Figure= 10, MIC 22).P.aeruginosa All peptides(µM) display= 1.25–20; high MIC activityB.spizizenii toward(µM) a= broad10–40; spectrumMICS.aureus of( µpathogensM) = 5–40) (MIC andE.coli have (μM high) = stability 10, MIC [227P.aeruginosa]. (μM) = 1.25–20; MICB.spizizenii (μM) = 10–40; MICS.aureusA clever (μM) solution= 5–40) and for have protease high resistancestability [227] is the. replacement of L-amino acids with their D counterparts.A clever solution This strategy for protease was successfully resistance is used the by replacement Qui et al. of [228 L-]amino who replaced acids with the their amino D counterparts.acid residues This or the strategy cationic was lysine successfully residue withused theby Qui corresponding et al. [228] who D-amino replaced acids the in amino protonectin acid residues(Prt; Figure or the25). cationic Protonectin lysine (ILGTILGLLKGL–NH residue with the corresponding2) was originally D-amino isolated acids from in protonectin the venom of(Prt; the Figureneotropical 25). Protonectin social wasp (ILGTILGLLKGLAgelaia pallipes [229–NH], and2) was has originally potent antifungal isolated and from antibacterial the venom activity of the neotropicalwith membrane social activewasp Agelaia action pallipes mode [[229]230,231, and]. has Qui potent et al. antifungal showed that and bothantibacterial the D-enantiomer activity with of membraneprotonectin active (D-prt) action and D-Lys-protonectin mode [230,231]. Qui (D-Lys-prt) et al. showed have strongthat both antimicrobial the D-enantiomer activity againstof protonectin bacteria (D(MIC-prt)S.aureus and( µ DM)-Lys= -4–128;protonectin MICE.coli (D(-µLysM)-prt)= 2–8; have MIC strongB.subtilis ( antimicrobialµM) = 8–128; activityMICS.epidermis against(µM) bacteria= 8–16; (MICMICK.penumoniaeS.aureus(μM)( µ=M) 4–=128;8–258) MIC andE.coli(μM fungi) = (MIC 2–8;Sakazaii MIC(B.subtilisµM) =(μM8–64).) = 8 Moreover,–128; MIC D-prtS.epidermis showed(μM) = strong8–16; MICstabilityK.penumoniae against(μM) trypsin, = 8–258) chymotrypsin and fungi (MIC andSakazaii the(μM human) = 8–64). serum, Moreover, while D-Lys-prt D-prt showed only strong showed stability strong againststability trypsin, against chymotrypsin trypsin. D-Lys-prt and the still hum keptan typical serum,α -helicalwhile D structure-Lys-prt only in the showed membrane strong mimicking stability againstenvironment, trypsin. while D- D-prtLys-prt showed still left kept hand typicalα-helical α-helical structure. structure In addition, in the all D-amino membrane acid mimicking substituted environment,analogues or partially while D D-amino-prt showed acid leftsubstituted hand α analogues-helical structure. could act In on addition, bacteria with all D mechanism-amino acid of substitutedprotonectin,Int. J. Mol. Sci. anal 20 and20ogues, disrupt21, x FOR orthe PEERpartially integrity REVIEW D-amino of membrane acid substituted while leading analogues to the cellcould death act [228on ].bacteria 28with of 44 mechanism of protonectin, and disrupt the integrity of membrane while leading to the cell death [228]. ILGTILGLLKGL

Figure 25. Secondary structure of protonectin (PDB: 6N68). In blue: hydrophilic amino acids; in green: positivelyFigure 25. charged Secondary amino structure acids; of in protonectin red: hydrophobic (PDB: amino6N68). acids.In blue: hydrophilic amino acids; in green: positively charged amino acids; in red: hydrophobic amino acids.

10. Mimicking Peptide Bonds Peptidomimetics can mimic primary, secondary, and even tertiary structures of peptides and

proteins, and therefore they have been developed for biomolecular recognition and modulation of protein interactions. In addition, peptidomimetics display advantages over conventional peptides, including resistance to enzymatic hydrolysis, improved bioavailability, and enhanced chemo- diversity [232]. The past decade showed the fast progress in developing biomimetic oligomers, including β-peptides [233] peptoids [234], α-aminoxy-peptides [235], α/β-peptides [236], azapeptides [237], oligoureas [238], aromatic oligoamides [239]. Nonetheless, the development and application of peptidomimetics is still limited due to the availability of backbones and molecular frameworks. A new class of peptidomimetics, “γ-AApeptides”, based on the chiral γ-PNA backbone was developed by Shi et al. [17]. These γ-AApeptides are oligomers of γ-substituted-N-acylated-N- aminoethyl amino acids resistant to proteolytic degradation and possess the potential to enhance chemo-diversity γ-AApeptides could mimic the primary structure of peptides, as they project the same number of side chains as peptides of the same lengths. Moreover, γ-AApeptides can fold into discrete secondary structures, such as helical and β-turn-like structures, and they can mimic host- defense peptides and display potent and broad-spectrum activity toward a panel of drug resistant bacterial pathogens. Linear γ-AApeptides link amphiphilic building blocks (containing one hydrophobic and one cationic group in each building block) together, and the sequence may adjust the conformation to adopt a globally amphipathic structure on the surface of bacterial membranes. Indeed, γ-AA16 (Figure 26) kills bacteria (MICE.coli, E.faecalis(μg/mL) = 5) through the disruption of bacterial membranes. Sulfono-γ-AApeptides form helical structures [240], that mimic cationic host- defense peptides e.g., magainin 2. Among them, γ-AA22 (MICE.coli, E.faecalis(μg/mL) = 2) and γ-AA23 (MICE.coli(μg/mL) = 4; (MICE.faecalis(μg/mL) = 2) (Figure 26) possess excellent antimicrobial activity [241]. Cyclic γ-AApeptides successfully mimic cyclic peptide antibiotics. This is particularly seen for γ- AA24 (MIC E.faecalis(μg/mL) = 5) (Figure 26), which displays broad-spectrum antimicrobial activity [18].

Int. J. Mol. Sci. 2020, 21, 7349 28 of 45

10. Mimicking Peptide Bonds Peptidomimetics can mimic primary, secondary, and even tertiary structures of peptides and proteins, and therefore they have been developed for biomolecular recognition and modulation of protein interactions. In addition, peptidomimetics display advantages over conventional peptides, including resistance to enzymatic hydrolysis, improved bioavailability, and enhanced chemo-diversity [232]. The past decade showed the fast progress in developing biomimetic oligomers, including β-peptides [233] peptoids [234], α-aminoxy-peptides [235], α/β-peptides [236], azapeptides [237], oligoureas [238], aromatic oligoamides [239]. Nonetheless, the development and application of peptidomimetics is still limited due to the availability of backbones and molecular frameworks. A new class of peptidomimetics, “γ-AApeptides”, based on the chiral γ-PNA backbone was developed by Shi et al. [17]. These γ-AApeptides are oligomers of γ-substituted-N-acylated-N-aminoethyl amino acids resistant to proteolytic degradation and possess the potential to enhance chemo-diversity γ-AApeptides could mimic the primary structure of peptides, as they project the same number of side chains as peptides of the same lengths. Moreover, γ-AApeptides can fold into discrete secondary structures, such as helical and β-turn-like structures, and they can mimic host-defense peptides and display potent and broad-spectrum activity toward a panel of drug resistant bacterial pathogens. Linear γ-AApeptides link amphiphilic building blocks (containing one hydrophobic and one cationic group in each building block) together, and the sequence may adjust the conformation to adopt a globally amphipathic structure on the surface of bacterial membranes. Indeed, γ-AA16 (Figure 26) kills bacteria (MICE.coli, E.faecalis(µg/mL) = 5) through the disruption of bacterial membranes. Sulfono-γ-AApeptides form helical structures [240], that mimic cationic host-defense peptides e.g., magainin 2. Among them, γ-AA22 (MICE.coli, E.faecalis(µg/mL) = 2) and γ-AA23 (MICE.coli(µg/mL) = 4; (MICE.faecalis(µg/mL) = 2) (Figure 26) possess excellent antimicrobial activity [241]. Cyclic γ-AApeptides successfully mimic cyclic peptide antibiotics. This is particularly seen for γ-AA24 Int.(MIC J. Mol.E.faecalis Sci. 20(µ20g/,mL) 21, x= FOR5) (Figure PEER REVIEW26), which displays broad-spectrum antimicrobial activity [18]. 29 of 44

Figure 26. Cont.

Figure 26. Chemical structures of linear γ-AA16, γ-AA22, γ-AA23 and cyclin γ-AA16 peptides. In green: positively charged amino acids; in red: hydrophobic amino acids.

11. Enhancing AMPs Activity with Metal Ions Enhancement of antibiotic activity through complexation with metal ions is well ascertained. Copper (II), zinc (II) and silver (I) metal adducts with different antibiotics were extensively studied

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 29 of 44

Int. J. Mol. Sci. 2020, 21, 7349 29 of 45

FigureFigure 26. 26. ChemicalChemical structures structures of oflinear linear γ-AA16,γ-AA16, γ-AA22,γ-AA22, γ-AA23γ-AA23 and and cyclin cyclin γ-AA16γ-AA16 peptides. peptides. In green:In green: positively positively charged charged amino amino acids; acids; in red: in red: hydrophobic hydrophobic amino amino acids. acids.

11.11. Enhancing Enhancing AMPs AMPs Activity Activity with with Metal Metal Ions Ions EnhancementEnhancement of of antibiotic antibiotic activity activity through through complexation complexation with with metal metal ions ions is is well well ascertained. ascertained. CopperCopper (II), (II), zinc zinc (II) (II) and and silver silver (I) (I) metal metal a adductsdducts with with different different antibiotics antibiotics were were extensively extensively studied studied in recent years [242–245]. Cu(II) and Ag(I) themselves have antimicrobial activity [246], but together with antibiotics e.g., ampicillin, penicillin G and Cefuroxime, they present synergistic effects against Gram-positive (MRSA) bacteria (S. aureus). Zn(II) is an essential cofactor for metallo-β-lacamases (enzyme resistant to β-lactan antibiotics), and together with ampicillin and penicillin G shows a high synergistic effect [245]. Not surprisingly, AMPs also bind metal ions (30% of proteins in the living cell coordinate at least one metal ion [247]) and their activity against bacteria can be enhanced upon metal binding. AMPs with a metal chelating ability can simply compete with bacteria for their essential metal ions Int. J. Mol. Sci. 2020, 21, 7349 30 of 45 and lead to the bacteria’s death under starvation. Psoriasin enters bacterial cells and sequesters Zn(II) ions [248], while Microplusin binds copper(II) ions, making them unavailable for the pathogen [249]. Metal binding can also have toxic effects. For instance, copper(II)/Colistin adducts cleave RNA molecules and have dangerous side effects [250]. AMPs change the structure and charge upon metal binding, leading to acquisition and/or enhancement of antibacterial activity [248]. Binding Zn(II) to Calcitermin increases the positive charge and facilitate the interactions with negatively charged bacterial membranes [251]. A similar effect can be observed in zinc(II)/Histidine rich glycoprotein (HRG) abundant in human plasma [252]. Histatins compose a wide group of short, cationic salivary peptides secreted by the parotid and sub-mandibular salivary gland. The C-terminal 16 amino acids fragment is rich in histidine residues, giving an opportunity for complex formation with copper(II) and zinc ions. Indeed, histatin 1 and 3 complexes with metal ions were already established [253], even if the exact functioning of metal adducts is still under study. Mimicking a metal binding motif is a new strategy in enhancing AMPs activity. One of the most studied strategies is the use of Amino Terminal Copper and Nickel (ATCUN) binding motif. ATCUN is a three-amino acid sequence, finishing with histidine residues (XXH), with high affinity to divalent transition metal ions. The ATCUN motif is present in human albumin, the most abundant metal and drug carrier in plasma [254], but is also found in other metal-binding proteins (e.g., histatins) [255]. Recently, Agbale et al. [256] engineered ATCUN motifs into the native sequence of two AMPs: CM15 and citropin1.1. The incorporation of metal binding motifs changed the antimicrobial activity of the peptides against a panel of carbapenem-resistant enterococci (CRE) bacteria, including carbapenem-resistant Klebsiella pneumoniae (KpC+) and Escherichia coli (KpC+). The antimicrobial activity was modulated according to the type of ATCUN variant utilized. For instance, CM15 modulated by incorporation of the GGH and VIH ATCUN motifs had 4-fold and 8-fold increased potency against carbapenem-resistant K. pneumoniae (KpC+ 1825971), respectively, with respect to the potency of the original peptide. It is noteworthy that there is no need to incorporate Cu(II) metals within the ATCUN-AMP complex drugs since the motif is able to scavenge metal ions in the plasma or target organism. This should avoid many regulatory bottlenecks associated with the use of metals in drugs [257]. Incorporation of mimosine (MIM) residues in the peptide backbone is another rewarding strategy in enhancing antimicrobial activity upon metal binding. Mimosine is a non-protein amino acid with various properties, such as antibacterial, anti-inflammatory, anti-cancer, and anti-virus. Due to its structural similarity with deferiprone (DFP), mimosine is an excellent chelator of Cu(II), Cd(II), Al(III), Fe(III), Ga(III), Gd(III), and In(III) metal ions, as well as actinides and lanthanides. Recent studies showed that in silico design of mimosine-containing peptides is an effective tool in prediction of metal/complexes [258], which increases the antimicrobial activity of free mimosine peptides. For instance, iron(III) complex with 6-amino acid peptide with three MIM residues (H-Mim-Gly-ProGly-Mim-Gly-Gly-Mim-OH) showed 15 times higher activity against S. aureus and B. cereus strains relative to the free peptide [96].

12. Bacteriocins Bacteriocins comprise a large and functionally diverse family of AMPs produced by a variety of bacteria and play a critical role in mediating microbial interactions and in maintaining microbial diversity [259]. Most Gram-positive bacteria produce bacteriocins, which inhibit the growth of alike or closely related bacterial strains with a narrow spectrum of activity. Some bacteriocins have activity against pathogenic and opportunistic bacteria (including multidrug-resistant species), not discriminating between antibiotic resistant and sensitive strains [260]. Moreover, bacteriocins shown antiviral, antiprotozoal and anticancer activity [261]. Therefore, bacteriocins are considered as alternatives to conventional antibiotics, and represent promising candidates as antibiotic synergists, or alternatives to enhancing the therapeutic eﬀects of current infection treatments and decrease the prevalence of resistant strains. Int. J. Mol. Sci. 2020, 21, 7349 31 of 45

The term bacteriocins is referred to as a ribosomally-produced peptides composed of 20-60 amino acids, mostly cationic and hydrophobic [262,263]. Initially, bacteriocins were divided into three classes differing in function, molecular weight, amino acid sequence and physicochemical properties. Class I bacteriocins consist of small (<10 kDa) and heat-stable peptides that are post-translationally modified, resulting in the non-standard amino acids, such as lanthionine and β-methyllanthionine [264]. Class II bacteriocins are small (<10 kDa), temperature- and pH-resistant peptides. Subsequently, class II bacteriocins are divided into subclasses based on structure and modifications: subclass IIa bacteriocins (known as pediocin-like bacteriocins, consist of the anti-listerial one-peptide with one or two disulfide bonds, and a conservative N-terminal motif). Subclass IIb bacteriocins are two-peptide bacteriocins, subclass IIc bacteriocins are cyclic bacteriocins. Class III bacteriocins (>30 kDa) are heat-sensitive, protein-like bacteriocins produced by both Gram-positive and Gram-negative bacteria [263,265]. A fourth class of bacteriocins, initially described by Klaenhammer 1993 [266], has been aborted and renamed as bacteriolysins, which comprise large complexes with carbohydrate and lipid moieties such as leuconocin S and lactococcin 27 [267]. Given the complexity and diversity of bacteriocin, as well as the identification of novel ones, the classification scheme is constantly evolving. Bacteriocins act in different modes, but most of them interact initially with a specific receptor on the target cell and form pores in bacterial cell-membrane, resulting in a dissipation of proton-motive force that leads to cell death [264]. Class I bacteriocins kill target microorganisms by pore formation in the cell membrane and by inhibition of cell-wall peptidoglycan biosynthesis. Class II bacteriocins, such as pediocin and lactococcin, bind to a membrane protein component of the mannose phosphotransferase system to interact with the membranes. The membrane permeabilization allows bacteriocins to enter the cytoplasm, where they destabilize DNA/RNA integrity, protein and cell wall synthesis and enzyme activity. In some cases, Gram-negative bacteria resist bacteriocins due to the outer membrane, which acts as an effective barrier. In such cases, some metal chelating agents and environmental conditions (acidic pH, high salt concentration, temperature variations) may destabilize the outer membrane and enables bacteriocins to act towards Gram-negative bacteria [268]. The use of bacteriocins in association with chemical compounds or physical treatments extends their activity spectrum on Gram-negative bacteria and counteract the emergence of resistant bacterial strains [269]. Bacteriocins are already used in our daily life. Lactic acid bacteria (LAB) produce bacteriocins applied in food preservation alone, or in combination with other preservation methods (hurdle technology), to increase the shelf-life and the safety of the foods. Nisin, produced by Lactococcus lactis, is authorized as a food additive in the EU (E234) under Annex II of Regulation (EC) 1333/2008 for use in several food categories (clotted cream, mascarpone, ripened and processed cheese and cheese products, pasteurized liquid eggs and semolina and tapioca puddings and similar products). Nisin inhibits different Gram-positive pathogens alone, among them are the particularly dangerous Clostridium botulinum, Listeria monocytogenes, Staphylococcus aureus, Bacillus and Enterococcus. When used in combination with other antimicrobials, Nisin is also active towards Gram-negative bacteria [270]. Bacteriocins are promising defenses against pathogenic bacteria, but before market introduction, there is still much to know about their biosynthesis and mode of action. Genomics, peptidomics and proteomics are used to understand the molecular mechanistic of their production, regulation, immunity, and mode of action, and help to decrease cytotoxic effects and increase the target selectivity.

13. Conclusions In front of Fleming’s fearsome prophecies about resistant bacteria, we can find consolation in Pasteur’s optimism “If it is a terrifying thought that life is at the mercy of the multiplication of these minute bodies it is a consoling hope that Science will not always remain powerless before such enemies.” The antimicrobial peptides provide new and promising therapeutic approaches, especially in chronic diseases, like oral cavity pathologies, where they can effectively interfere in the early steps of biofilm Int. J. Mol. Sci. 2020, 21, 7349 32 of 45 formation, and at the same time block the inflammatory effects of bacterial toxins. Human antimicrobial peptides are often unable to exhibit toxicity at physiological concentrations, while other organisms’ AMPs express toxicity in the human body. Clever AMPs-mimicking strategies lead to the development of innovative antimicrobial peptides that express antibacterial activity without harmful side effects. In this review, we gathered the most successful AMPs-mimicking strategies of the last decade to be used as a manual for the future synthesis of new peptides.

Author Contributions: J.I.L. coordinated project and described sections dedicated to different peptidomimicking strategies, K.S. described sections dedicated to known AMPs, A.S., C.C., S.F. and G.O. presented growing potential of AMPs in oral infections and revised entire manuscript, B.P. presented bacteriocins as a new class of AMPs, M.P. described sections dedicated to the selectivity of AMPs, M.J. coordinated project and revised entire manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Acknowledgments: K.S. and M.J. would like to thank King Abdullah University of Science and Technology (KAUST) for financial support. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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