The Road from Host-Defense Peptides to a New Generation of Antimicrobial Drugs

molecules

Review The Road from Host-Defense Peptides to a New Generation of Antimicrobial Drugs

Alicia Boto *, Jose Manuel Pérez de la Lastra and Concepción C. González Instituto de Productos Naturales y Agrobiología del CSIC, CSIC-Spanish Research Council, Avda. Astrofísico Fco. Sánchez, 3-38206 La Laguna, Tenerife, Spain; [email protected] (J.M.P.d.l.L.); [email protected] (C.C.G.) * Correspondence: [email protected]; Tel.: +34-922-260112

Received: 27 December 2017; Accepted: 30 January 2018; Published: 1 February 2018

Abstract: Host-defense peptides, also called antimicrobial peptides (AMPs), whose protective action has been used by animals for millions of years, fulfill many requirements of the pharmaceutical industry, such as: (1) broad spectrum of activity; (2) unlike classic antibiotics, they induce very little resistance; (3) they act synergically with conventional antibiotics; (4) they neutralize endotoxins and are active in animal models. However, it is considered that many natural peptides are not suitable for drug development due to stability and biodisponibility problems, or high production costs. This review describes the efforts to overcome these problems and develop new antimicrobial drugs from these peptides or inspired by them. The discovery process of natural AMPs is discussed, as well as the development of synthetic analogs with improved pharmacological properties. The production of these compounds at acceptable costs, using different chemical and biotechnological methods, is also commented. Once these challenges are overcome, a new generation of versatile, potent and long-lasting antimicrobial drugs is expected.

Keywords: antimicrobial; antibiotic; peptides; AMP; cathelicidins; site-selective; peptide modification; tag-and-modify; customizable units; gene mining

1. Introduction Host-defense peptides, also called antimicrobial peptides (AMPs), have been used by animals and plants for millions of years as defense against pathogens [1–6]. There are several AMP families, such as defensins, dermaseptins, cathelicidins, temporins, plant systemins, and others [2–6]. Although there is a considerable structural variety, in general these compounds are short amphiphilic peptides (generally less than 50 residues), with net cationic charge. Interestingly, some microorganisms produce related antimicrobials composed by hydrophobic and cationic aminoacids [7] (e.g., polymyxins [8], octapeptins [9], gramicidin [10]). These peptides probably allow the producing microorganism to get rid of competitors or to protect their plant or animal hosts. Thus, Paenibacillus polymyxa is a nitrogen-fixing bacteria that grows on plant roots; it produces the antibiotic polymyxins that are able to disrupt biofilms of pathogens such as Pseudomonas aeruginosa or Staphylococcus aureus [11]. AMPs present many advantages as potential antimicrobial drugs: they display a broad spectrum of activity, induce very little resistance, have a synergic effect with conventional antibiotics, and can modulate the immune system and neutralize endotoxins [12–20]. They are usually classified in three groups: α-helical, β-sheet, and extended peptides; the latter are usually enriched with certain aminoacids (e.g., proline, lysine). Many AMPs form α-helical structures with cationic and hydrophobic residues oriented on opposite faces of the helix; this facial amphiphilicity is described as “amphipathicity” [21]. Their amphiphilic structure with segregated hydrophobic and cationic regions favours their interaction with anionic bacterial cytoplasmic membranes, followed by their disruption [21–26].

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According to the “barrel-stave” and “toroidal pore” models, the AMPs form pores in the membrane, while in the “carpet” model, the peptides form micelles with the membrane components, causing membrane destruction. At high concentrations, the peptides can behave as a detergent. Other less disruptive (non-pore) models have been proposed, such as membrane thinning, depolarization or aggregation. In any case, the mechanism may change according to the peptide concentration, pH, or the temperature [21,22]. The selectivity of AMPs for bacterial membranes over eukaryotic membranes is due to their different composition. Bacterial membranes contain many anionic lipids (cardiolipin, phosphatidylglycerol), in contrast to eukaryotic membranes, which contain zwitterionic lipids (such as sphingomyelin and phosphatidylcholine), and also cholesterol [21]. The cationic peptides are attracted by electrostatic forces to the negatively-charged bacterial membrane, and after adsorption to the cell surface, the peptide hydrophobic residues insert into the membrane (permeation effect) [21–26]. Membrane disruption is not the only mechanism of action of these peptides. They can also entangle microbes [27], affect many cytoplasmic processes, such as cell wall, protein and nucleic acid synthesis [1,2,16,19], and hinder the formation of bacterial biofilms [28,29]. In addition, they can act as immunomodulating agents, controlling inflammation and other septic responses to infections [30–32]. It has been suggested that many AMPs act on the bacterial invader causing different stresses, until their combined action causes the cell death [16]. In any case, due to their promiscuous mode of action and lack of specificity, AMPs induce little resistance. This fact, together with their broad activity spectrum and synergy with other antimicrobials [12,33], has drawn the attention of the pharmaceutical industry and much work has been carried out to develop AMP drugs [12,13,34–36]. However, some drawbacks for their clinical use have limited the number of approved AMPs: poor selectivity, hemolytic activity and host toxicity, low stability to protease degradation in vivo, low hydrosolubility and other biodistribution problems, and cost of production [1,12,13]. In addition, although several AMPs have reached advanced clinical trials, most were not approved because they were not superior to the current treatments, as in the case of magainin 2 analogue pexiganan, which was developed for the prevention of diabetic foot ulcers [34–37]. Currently, there are some AMPs approved for clinical use [12,13,34–36]. Gramicidins, isolated from Bacillus brevis, are used in ophthalmic drops and for the treatment of wound infections and genital ulcers. However, their haemolytic activity prevents their use in systemic infections [10,38–40]. Polymyxin B, isolated from P. polymyxa, is prescribed to treat eye infections; it is also useful to remove endotoxins, but its nephrotoxic effects limit its applications. Polymyxin E (colistin, and prodrug colomycin) is used to treat infected wounds, but also as last-resort drug against some Pseudomonas aeruginosa infections, particularly recurrent ones that affect the lungs in cystic fibrosis [8,41]. The anti-Candida drug caspofungin (Cancidas®, Merck, USA) is a cyclic hexapeptide with several cationic chains [42]. Caspofungin is a semisynthetic compound derived from fungal echinocandins [43]. However, the natural peptides such as echinocandin B were unsuitable for clinical use, due to their hemolytic activity. To overcome this problem, semisynthetic analogs were developed, but the first drug to enter clinical trials (cilofungin) was discarded due to the toxicity of the vehicle (solvent needed for administration). Finally, caspofungin was approved by FDA for antifungal prophylaxis in stem cell transplant and febrile neutropenia, and in the treatment of refractory aspergillosis and yeast infection. Its toxicity is remarkably low, presents few interactions with other drugs, and as an additional advantage, has a long half-life. However, its oral bioavailability is poor and, therefore, it is administered by intravenous infusion [42,43]. More examples that are in advanced clinical trials (>phase III) are iseganan, derived from pig protegrin, for the treatment of oral mucositis [44,45], and omiganan (ox indolicidin) for the prevention of catheter infections and acne [46,47]. Other AMPs and mimetics are also in clinical trials, such as the defensin mimetic Brilacidin® (Innovation Pharmaceuticals, USA), in Phase II to fight acute skin and Molecules 2018, 23, 311 3 of 26 soft tissue infections such as oral mucocitis [48,49]. The human defensin Novexatin® (NovaBiotics Ltd., Aberdeen, UK) has been formulated as a brush-on treatment for fungal nail infection, and Phase II studies have shown that is safe and the infection disappears after just one month of daily application [50]. Human LL-37 (also called OP145) is under development (phase II) to treat chronic middle ear infection, and is also studied for manufacturing of biomedical devices [51,52]. The human lactoferrin fragment hLF1-11 is in phase II clinical trials for the treatment of bacterial and fungal infections, and in phase I for prophylaxis in hematopoietic stem cell transplantation [53,54]. PAC-113 (histatin 3) is a 12-amino acid peptide derived from natural histatin found in human saliva; it is used in oral mouth rinse formulation to treat candida infections (Phase II) [55–57]. One of the peptides under development (IDR1) is a small synthetic peptide derived from bovine bactenecin [58,59]. Interestingly, the innate defense regulator IDR-1 displayed no antimicrobial properties in vitro, but was highly active in vivo against methicillin-resistant Staphylococcus aureus (MRSA) in invasive peritonitis mouse models. This result suggested that IDR-1 presented immunomodulatory properties. In effect, it was observed that the peptide increased the levels of monocyte chemokines, and therefore recruited monocytes and macrophages to fight the bacteria. In addition, it regulated pro-inflammatory cytokine responses to appropriate levels. Other bactenecin derivatives such as IDR-1002 and 1018 have displayed improved properties, and a synergic effect in live infection models [60]. The IDRs highlight the efforts to develop AMPs not only for the treatment of topical infections, but also for systemic ones. Another example in Phase II development is WAP-8294A2 (Lotilibcin), derived from Lysobacter sp., for the treatment of systemic infections produced by Gram-(+) bacteria, particularly MRSA and vancomycin-resistant enterococci (VRE) [61]. Other antimicrobial peptides in preclinical phase, with promising activities in animal models, will soon enter clinical studies against a variety of fungal and bacterial infections. Some of them are natural peptides or directly derived therefrom (Buforin II, Temporin 10, BacBc) [17,34–36], while others are synthetic peptides generated by rational drug design. For instance, Novamycin® (NovaBiotics Ltd., Aberdeen, UK) is a synthetic cationic peptide active against invasive fungal infections caused by Aspergillus and Candida [62], while Novarifyn® can fight bacterial infections caused by difficult pathogens such as MRSA, Pseudomonas aeruginosa, Clostridium difficile and Acinetobacter baumannii [63]. Although at this early stage it is difficult to predict how many of these new-generation peptides will arrive to the market, their promising activity against pathogens which do not respond to current treatments, together with their improved pharmacological properties, allow us to expect that some AMPs will join the list of approved drugs in the next years [34–36].

2. Discovery of New AMPs

2.1. Biotechnological Discovery Strategies In the traditional approach to discover new bioactive products, they have been isolated from their natural sources by processing a sample, extraction of the metabolites (e.g., by partition with different solvents), and bioguided characterization of the extracts (Scheme1,[ 64]). However, this procedure has limitations. For AMPs, the isolation techniques depend on the amount of peptide available [64–68]. When the peptide is detected in microorganisms, scale-up of the culture and conventional extraction may be possible. However, for the isolation of gene-encoded AMPs, other strategies are preferred. For example, the cDNA encoding the biosynthetic precursor of Dermaseptin-PH was identified from the skin of the south-american frog Pithecopus hypochondrialis [65]. The skin secretion underwent initial rapid amplification of complementary DNA (cDNA) ends by PCR (RACE-PCR), which allowed identification of the cDNA encoding the biosynthetic precursor of Dermaseptin-PH. The predicted primary structure was confirmed by identification in the skin secretion by reverse-phase high-performance liquid chromatography (RPHPLC) and MS/MS fragmentation. Then the chemical synthesis of Dermaseptin-PH was carried out, followed by the biological assays. The peptide inhibited the growth of Gram-(+) and Gram-(−)-bacteria, and the fungus Candida albicans. Molecules 2018, 23, 311 4 of 26 Molecules 2018, 23, x FOR PEER REVIEW 4 of 24

SchemeScheme 1. 1. BioguidedBioguided search search of of AMPs. AMPs.

An important limitation in natural products search is that many interesting products are not An important limitation in natural products search is that many interesting products are not expressed expressed by the organism, and their genes remain in a ‘silent’ state until a change in the by the organism, and their genes remain in a ‘silent’ state until a change in the environment promotes their environment promotes their transcription. For instance, some actynomycetes do not produce transcription. For instance, some actynomycetes do not produce antibiotics until they are co-cultured with antibiotics until they are co-cultured with competing bacteria or until the culture medium is competing bacteria or until the culture medium is provided with activators such as goadsporin [69]. provided with activators such as goadsporin [69]. Clearly, a method to track these ‘cryptic’ or ‘silent’ natural products would be desirable [70,71]. Clearly, a method to track these ‘cryptic’ or ‘silent’ natural products would be desirable [70,71]. Bioinformatic techniques have been used to predict cryptic products codified in the known genome of Bioinformatic techniques have been used to predict cryptic products codified in the known genome different organisms, from microbes to animals. of different organisms, from microbes to animals. For instance, Brady et al. have described the discovery of new antibiotics from microorganisms For instance, Brady et al. have described the discovery of new antibiotics from microorganisms by bioinformatic screening of primary sequences of the biosynthetic gene clusters (Scheme2,[ 72]). by bioinformatic screening of primary sequences of the biosynthetic gene clusters (Scheme 2, [72]). The structures were then obtained by chemical synthesis to afford synthetic-bioinformatic natural The structures were then obtained by chemical synthesis to afford synthetic-bioinformatic natural products (syn-BNPs). This approach allowed bypass the culture steps. Brady identified cryptic peptides products (syn-BNPs). This approach allowed bypass the culture steps. Brady identified cryptic that could be produced by non-ribosomal peptide synthetases, among them a potent broad-spectrum peptides that could be produced by non-ribosomal peptide synthetases, among them a potent antibiotic, active against MRSA, and which induced negligible resistance. Another peptide with broad-spectrum antibiotic, active against MRSA, and which induced negligible resistance. Another activity against several fungal pathogens was also discovered, and proved a promising agent for the peptide with activity against several fungal pathogens was also discovered, and proved a treatment of vaginal, ear, hand and central nervous system infections. promising agent for the treatment of vaginal, ear, hand and central nervous system infections.

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Scheme 2. Identification of ‘cryptic’ or ‘silent’ products by bioinformatics techniques. Scheme 2. IdentificationIdentification of ‘cryptic’ oror ‘silent’‘silent’ products byby bioinformaticsbioinformatics techniques.techniques. It is also possible to carry out this genome mining from other natural sources. Our group has ItIt is also possible possible to to carry carry out out this this genome genome mining mining from from other other natural natural sources. sources. Our Our group group has carried out work in the discovery of AMPs (cathelicidins) from animal origin using bioinformatic hascarried carried out work out in work the discovery in the discovery of AMPs of(cathelicidins) AMPs (cathelicidins) from animal from origin animal using bioinformatic origin using techniques, and then produced them in transformed microalgae (the eukaryotic Chlamydomonas bioinformatictechniques, and techniques, then produced and then them produced in transformed them inmicroalgae transformed (the microalgae eukaryotic (theChlamydomonas eukaryotic reindhartii and the prokaryotic Synechococcus elongatus), as summarized in Scheme 3 [73]. Chlamydomonasreindhartii and the reindhartii prokaryoticand the Synechococcus prokaryotic Synechococcuselongatus), as summarized elongatus), as summarizedin Scheme 3 [73]. in Scheme 3[ 73].

Scheme 3. Identification of ‘cryptic’ or ‘silent’ products from animal sources by bioinformatics Scheme 3. Identification of ‘cryptic’ or ‘silent’ products from animal sources by bioinformatics techniques,Scheme 3. andIdentification production ofin ‘cryptic’microalgae. or ‘silent’ products from animal sources by bioinformatics techniques,techniques, andand productionproduction inin microalgae.microalgae. Indeed, among the AMPs, cathelicidins are particularly interesting, since they contain highly Indeed, among the AMPs, cathelicidins are particularly interesting, since they contain highly conservedIndeed, domains among ( theN-terminal AMPs, cathelicidinscathelin-domain), are particularly preserved interesting,across evolutionary since they distant contain species. highly conserved domains (N-terminal cathelin-domain), preserved across evolutionary distant species. Usingconserved this domain domains to (searchN-terminal into genome cathelin-domain), databases, novel preserved cathelicidins across evolutionarywere found, from distant reptiles species. to Using this domain to search into genome databases, novel cathelicidins were found, from reptiles to batsUsing and this birds domain [74–76]. to search Predicted into genomecathelicidins databases, were identified novel cathelicidins by screening were genome found, fromdatabases reptiles [77] to bats and birds [74–76]. Predicted cathelicidins were identified by screening genome databases [77] usingbats and the birdsBLAST [74 tool–76 for]. Predicted DNA sequences cathelicidins [78]. were identified by screening genome databases [77] using the BLAST tool for DNA sequences [78]. usingThe the complete BLAST tool sequence for DNA of sequencescathelicidins [78 ].was assembled by joining the exons identified in key The complete sequence of cathelicidins was assembled by joining the exons identified in key contigsThe using complete GeneScan sequence [79] and of cathelicidins GeneMarK was[80] assembledprograms. byEach joining DNA thesequence exons identifiedthus assembled in key contigs using GeneScan [79] and GeneMarK [80] programs. Each DNA sequence thus assembled wascontigs translated using GeneScan into amino [79 acids] and GeneMarKand the resultin [80] programs.g protein was Each subjected DNA sequence to PSI-BLAST thus assembled to confirm was was translated into amino acids and the resulting protein was subjected to PSI-BLAST to confirm thattranslated it was intoindeed amino a cathelicidin. acids and the The resulting initial protein waswas subjectedprocessed to to PSI-BLAST release the to active confirm peptide, that it that it was indeed a cathelicidin. The initial protein was processed to release the active peptide, separatingwas indeed ita from cathelicidin. inactive Thefragments. initial protein was processed to release the active peptide, separating it separating it from inactive fragments. fromThe inactive AMPs fragments. were subjected to in silico analysis of their antimicrobial activity using several The AMPs were subjected to in silico analysis of their antimicrobial activity using several bioinformaticThe AMPs tools were [21,81–87]. subjected For toexample, in silico the analysis online tool of their APD3 antimicrobial calculates the activity different using parameters several bioinformatic tools [21,81–87]. For example, the online tool APD3 calculates the different parameters relatedbioinformatic to the possible tools [21 ,antimicrobial81–87]. For example, activity theof th onlinee peptides tool APD3 (e.g., calculatesnet charge, the length, different percentage parameters of related to the possible antimicrobial activity of the peptides (e.g., net charge, length, percentage of hydrophobicrelated to the residues, possible helicity) antimicrobial [81,82]. activity On the ofother the hand, peptides the (e.g.,bioinf netormatics charge, tool length, AMPA percentage can predict of hydrophobic residues, helicity) [81,82]. On the other hand, the bioinformatics tool AMPA can predict antimicrobialhydrophobic regions residues, on helicity) any protei [81,n82 by]. Onassigning the other an hand,antimicrobial the bioinformatics index to each tool residue AMPA [83,84]. can predict antimicrobial regions on any protein by assigning an antimicrobial index to each residue [83,84]. antimicrobialIn addition, regions the antimicrobial on any protein activity by assigning of the anpeptides antimicrobial was tested index ag toainst each Gram-positive residue [83,84]. and In addition, the antimicrobial activity of the peptides was tested against Gram-positive and Gram-negativeIn addition, microorganisms, the antimicrobial using activity the ofminima the peptidesl inhibitory was tested concentration against Gram-positive assay [88]. The and Gram-negative microorganisms, using the minimal inhibitory concentration assay [88]. The citotoxicityGram-negative (hemolytic microorganisms, activity) of using the thepeptides minimal was inhibitory assesed concentrationas well. The assaymost [promising88]. The cytotoxicity peptides citotoxicity (hemolytic activity) of the peptides was assesed as well. The most promising peptides were(hemolytic selected activity) for their of the recombinant peptides was production assesed as well.in microorganisms, The most promising in orde peptidesr to obtain were selected adequate for were selected for their recombinant production in microorganisms, in order to obtain adequate amountstheir recombinant for other productionassays. in microorganisms, in order to obtain adequate amounts for other assays. amounts for other assays. TheThe bacteria bacteria E. coli andand yeastyeast werewereconsidered considered first, first, since since their their physiology physiology is well-known,is well-known, there there are The bacteria E. coli and yeast were considered first, since their physiology is well-known, there arenumerous numerous protocols protocols for their for transformation,their transformation, and vectors and vectors that contain that acontain great diversitya great ofdiversity promoters of are numerous protocols for their transformation, and vectors that contain a great diversity of promotersand selection and markers selection are markers readily available are readily [89]. availabl Specifice and[89]. well-characterized Specific and well-characterized strains can achieve strains high promoters and selection markers are readily available [89]. Specific and well-characterized strains canlevels achieve of expression. high levels However, of expression. these microorganisms However, these also microorganisms present disadvantages, also present such asdisadvantages, their potential can achieve high levels of expression. However, these microorganisms also present disadvantages, suchsusceptibility as their topotential the harmful susceptibility antimicrobial to peptides.the harmful Another antimicrobial drawback wouldpeptides. be theAnother need to drawback purify the such as their potential susceptibility to the harmful antimicrobial peptides. Another drawback wouldpeptides be from the theneed extract, to purify in case the that peptides the peptide from is the trapped extract, as insolublein case that form the in inclusionpeptide is bodies, trapped and as in would be the need to purify the peptides from the extract, in case that the peptide is trapped as insoluble form in inclusion bodies, and in some cases, the removal of endotoxins. Synthetic biology insoluble form in inclusion bodies, and in some cases, the removal of endotoxins. Synthetic biology techniques facilitate the availability of the genetic material that codes for antimicrobial peptides with techniques facilitate the availability of the genetic material that codes for antimicrobial peptides with

Molecules 2018, 23, 311 6 of 26 Molecules 2018, 23, x FOR PEER REVIEW 6 of 24 somethe codon cases, optimization the removal of of endotoxins. the host and Synthetic it also biology allows techniquesthe incorporation facilitate of the signal availability peptides, of the epitopes genetic materialfor the detection that codes of for the antimicrobial final product, peptides or the inclus with theion codon of appropriate optimization targets of the for host proteolytic and it also enzymes, allows thetags incorporation or tails to facilitate of signal purification peptides, epitopes[90]. for the detection of the final product, or the inclusion of appropriateAn alternative targets for for proteolytic an expression enzymes, system tags or of tails antimicrobial to facilitate purification peptides [without90]. contaminating endotoxinsAn alternative could be forthe transformation an expression of system eukaryotic of antimicrobial cells from animal peptides or human without origin. contaminating In this case, endotoxinsit may not couldbe necessary be the transformationto purify or decontamin of eukaryoticate the cells extracts. from animalHowever, or humanthe cost origin. of producing In this case,recombinant it may not proteins be necessary would tobe purify considerably or decontaminate higher than the using extracts. conventional However, microorganisms the cost of producing [91]. recombinantSeveral proteinsgroups wouldhave shown be considerably that a production higher than system using conventional based on microalga microorganisms could [91be]. an alternativeSeveral to groups the conventional have shown thatfermentation a production syst systemems that based use onbacteria, microalga yeast, could or mammalian be an alternative cells to[73,92–95]. the conventional First, the fermentation antimicrobial systems peptide that is less use like bacteria,ly to be yeast, toxic or to mammalian this production cells [system.73,92–95 Besides,]. First, themicroalgae antimicrobial allow peptidegreater isscaling-up less likely volumes, to be toxic spre toad this over production large areas, system. making Besides, these microalgaeproduction allowsystems greater very scaling-upprofitable. In volumes, spite of spreadsmaller over producti largeon areas, yields, making microalgae these offer production a higher systems benefit/cost very profitable.ratio compared In spite to of conventional smaller production systems. yields, Another microalgae asset offeris that a higher some benefit/costmicroalgae ratioare considered compared toGRAS conventional (Generally systems. Regarded Another as Safe), asset and isin thatthese some cases microalgae and for certain are consideredcommercial GRAS uses, purification (Generally Regardedprocesses asmay Safe), not and be in necessary. these cases andFor forexample, certain commercialapplications uses, for purificationcosmetics, topical processes medicines, may not bepesticides necessary. for Foragriculture, example, food applications conservation, for cosmetics, etc. would topical not require medicines, peptide pesticides purification. for agriculture, food conservation,There are already etc. wouldnumerous not requiretools for peptide the efficien purification.t transformation microalgae. For example, in the caseThere of are the already microalga numerous Chlamydomonas tools for the reindhartii efficient, transformationit is possible to microalgae. transform Forthe example,three genomes: in the casenuclear, of the chloroplastic microalga Chlamydomonas and mitochondrial. reindhartii ,Once it is possible transformed, to transform microalgae the three can genomes: be grown nuclear, in chloroplasticphotobioreactors, and mitochondrial.in controlled conditions Once transformed, and free microalgaeof pathogens. can On be grownthe other in photobioreactors,hand, controlled inconditions controlled would conditions avoid and release free of of the pathogens. transforme Ond the microorganism other hand, controlled into the environment, conditions would and would avoid releaseimprove of the biosecurity transformed of microorganism the final product into [92]. the environment, and would improve the biosecurity of the finalPerez product de Lastra [92]. et al. have used the eukaryotic microalgae Chlamydomonas reindhartii and the prokaryoticPerez de Synechococcus Lastra et al. elongatus have used as theexpression eukaryotic systems. microalgae Both theChlamydomonas prokaryotic and reindhartii the eukaryoticand the prokaryoticmicroalga haveSynechococcus strong, endogenous elongatus as promotors expression and systems. can introduce Both the 6xHi prokaryotics Tags for and the thepurification eukaryotic of microalgathe recombinant have strong, proteins endogenous (Figure 1). promotorsUsing the comm and canercially introduce available 6xHis kits Tags for formicroalga the purification expression, of thethe recombinanttwo microalgae proteins were (Figuretransformed1). Using for the the recomb commerciallyinant expression available kitsof several for microalga cathelicidins. expression, Some theof the two isolated microalgae cathelicidins were transformed were particularly for the recombinant effective against expression both of Gram-(+) several cathelicidins. and Gram-( Some−) bacteria, of the isolatedand besides, cathelicidins displayed were low particularly haemolytic effective activity against in human both Gram-(+) erythrocytes and Gram-( [73]. −These) bacteria, results and highlight besides, displayedtheir potential low haemolytic for use activity in different in human formulatio erythrocytesns [73and/or]. These pharmaceutical, results highlight theircosmetic potential and/or for usephytosanitary in different formulationscompositions. and/or pharmaceutical, cosmetic and/or phytosanitary compositions.

Figure 1. (Left) Transformed S. elongatus growing in BG-11 agar plates containing spectinomycin (5 Figure 1. (Left) Transformed S. elongatus growing in BG-11 agar plates containing spectinomycin µg/mL). Negative control with no insert (Left) and containing the bird cathelicidin peptide (5 µg/mL). Negative control with no insert (Left) and containing the bird cathelicidin peptide Mm_KR-26; (Right) Comparison of the microalga vectors pChlamy_4 and pSyn_6 (ThermoFisher) Mm_KR-26; (Right) Comparison of the microalga vectors pChlamy_4 and pSyn_6 (ThermoFisher) used used for the recombinant expression of cathelicidin antimicrobial peptides into the microalga C. for the recombinant expression of cathelicidin antimicrobial peptides into the microalga C. reindhartii andreindhartiiS. elontgatus and S., respectively.elontgatus, respectively.

2.2. Chemical Synthesis in Discovery Strategies 2.2. Chemical Synthesis in Discovery Strategies Antimicrobial peptides are usually isolated in very small amounts from the natural sources. Antimicrobial peptides are usually isolated in very small amounts from the natural sources. Therefore, a chemical synthesis is often implemented to obtain adequate amounts for proper peptide Therefore, a chemical synthesis is often implemented to obtain adequate amounts for proper peptide characterization and for the biological assays [96–99]. Moreover, the chemical synthesis can provide analogues of natural peptides with improved properties, such as superior potency, selectivity, stability or biodistribution, and is a key technology in drug discovery and development programs

Molecules 2018, 23, 311 7 of 26 characterization and for the biological assays [96–99]. Moreover, the chemical synthesis can provide analoguesMolecules of2018 natural, 23, x FOR peptides PEER REVIEW with improved properties, such as superior potency, selectivity,7 stability of 24 or biodistribution, and is a key technology in drug discovery and development programs [1,16,100]. The[1,16,100]. following The sections following dealwith sections the advancesdeal within the synthetic advances methodologies in synthetic methodologies to provide suitable to provide amounts of AMPs,suitable and amounts also toof diversifyAMPs, and peptide also to diversify libraries. peptide Then, libraries. the use ofThen, libraries the use for of thelibraries determination for the of structure-activitydetermination of structure-activity relationships (SAR) relationships will be discussed,(SAR) will andbe discussed, how this and knowledge how this hasknowledge led to the developmenthas led to ofthe semi-synthetic development of or semi-synthetic synthetic analogues or synthetic with betteranalogues pharmacological with better pharmacological profiles. profiles. 2.2.1. Advances in Synthetic Methodologies: Providing Scarce AMPs and Selected Analogues 2.2.1. Advances in Synthetic Methodologies: Providing Scarce AMPs and Selected Analogues When an antimicrobial peptide is scarce in nature, chemical synthesis can provide in a relatively short timeWhen suitable an antimicrobial amounts forpeptide its complete is scarce in characterization nature, chemical synthesis and for biologicalcan provide screening in a relatively [98, 99]. short time suitable amounts for its complete characterization and for biological screening [98,99]. In In addition, the synthesis of selected analogues is also possible. addition, the synthesis of selected analogues is also possible. The standard solution and solid phase synthesis of small and medium-size peptides is The standard solution and solid phase synthesis of small and medium-size peptides is well-known [96–100], but the preparation of difficult sequences can pose many problems, and has been well-known [96–100], but the preparation of difficult sequences can pose many problems, and has reviewedbeen reviewed by Albericio by Albericio et al. [101 et]. Inal. the[101]. case In of the cyclic case peptides, of cyclic thepeptides, cyclization the cyclization may be troublesome, may be andtroublesome, different approaches and different have beenapproaches developed, have frombeen dilutiondeveloped, conditions from dilution to the developmentconditions to ofthe new couplingdevelopment reagents, of usenew of coupling enzymes, reagents, use of nativeuse of peptide enzymes, ligation use methodologies,of native peptide etc. ligation [102–105 ]. Schememethodologies,4 shows the etc. solid-phase [102–105]. Sc synthesisheme 4 shows and cyclizationthe solid-phase of ansynthesis antimicrobial and cyclization BPC precursor of an (conversionantimicrobial1→4 BPC[106 precursor]). (conversion 1→4 [106]). OtherOther synthetic synthetic challenge challenge is the is productionthe production of disulfide of disulfide bonds, bonds, which which is usually is usually carried carried out atout the at end of thethe synthesis, end of the as synthesis, will be shown as will later be forshown the synthesis later for ofthe snakins synthesis [107 of] andsnakinsθ-defensins [107] and [108 θ-defensins]. As a result, peptide[108]. analogues As a result, with peptide disulfide analogues bond surrogateswith disulfide (such bond as C-Csurrogates or C-S (such bonds) as haveC-C or been C-S developed bonds) have [109 ]. beenFor thedeveloped generation [109]. of large peptides, several small or medium-size fragments are synthesized and then attached.For the Usual generation ligation of large methods peptides, are solution several orsmall solid-phase or medium-size fragment fragments condensation are synthesized (Scheme 5), and then attached. Usual ligation methods are solution or solid-phase fragment condensation and native chemical ligation (Scheme6). In Scheme5, the pentapeptide 6 is produced by solid-phase (Scheme 5), and native chemical ligation (Scheme 6). In Scheme 5, the pentapeptide 6 is produced by synthesis, and then coupled to fragment 7 to give a precursor 8 of a polymyxin analogue [110]. solid-phase synthesis, and then coupled to fragment 7 to give a precursor 8 of a polymyxin analogue In the final analogue, the Dab-3 residue was replaced by a Dap-3 unit, and a synthetic, more polar [110]. In the final analogue, the Dab-3 residue was replaced by a Dap-3 unit, and a synthetic, more − side-chainpolar side-chain was introduced. was introduced. The analogue The analogue was was active activein in vitro vitroagainst against Gram-(Gram-(−) )bacteria, bacteria, such such as as AcinetobacterAcinetobacter baumanii baumaniiand andPseudomonas Pseudomonas aeruginosa aeruginosa[ [8,41,110].8,41,110].

Scheme 4. SPPS in the preparation of peptides with non-proteinogenic units, and cyclization of a Scheme 4. SPPS in the preparation of peptides with non-proteinogenic units, and cyclization of a BPC precursor. BPC precursor.

Molecules 2018, 23, 311 8 of 26 Molecules 2018, 23, x FOR PEER REVIEW 8 of 24 Molecules 2018, 23, x FOR PEER REVIEW 8 of 24

Scheme 5. Ligation of peptides in the synthesis of a precursor of polymixin E. Reaction conditions: (a) Scheme 5. LigationLigation of of peptides peptides in in the the synthe synthesissis of of a aprecursor precursor of ofpolymixin polymyxin E. Reaction E. Reaction conditions conditions: (a): Fmoc-SPPS; (b) HATU, DIEA, DMF, 45 min. (Fmoc-SPPS;a) Fmoc-SPPS; (b) (HATU,b) HATU, DIEA, DIEA, DMF, DMF, 45 45min. min.

i-vi O i-vi Gly1-Cys29 OS R O S H2N R Gly1-Cys29 S R peptide OS S peptide H2N 10 + R+ peptide H3N peptide NH S O 2 10 HS + +H N NH 123 O 2 HS 30 63 9 Cys -Pro CO2H 12 H2N 30 63 Cys -Pro CO2H 9 vii-viii H2N 11 Thioester vii-viii 11 exchangeThioester exchange O NH2 NH O S 2 20 10 1 O R D20 A L G A K S C R L 10K C K S D C F N S G1 NH2 S C 13 O R D A L G A K S C R L K C K S D C F N S G NH2 LC 13 30 40 LK S N acyl transfer Y C G I C 30C E E C KTC V P S G 40 G K Y S N acyl transfer G I C C C P S G N Y C E E C KTV Y G HS ligation site NK H OHS ligation site K HOOC P C K S K G K S N K K D R Y C P C EH N O O HOOC P C K 60S K G K S N K K D R 50Y C P C E HN 50 H O 60 15 14 15 Disulfide formation/folding 14 Disulfide formation/folding Snakin-1 Snakin-1 Scheme 6. Synthesis of a precursor of snaking-1. Reaction conditions: (i) Boc-Ala-Pam, DIC, CH2Cl2, 1 Scheme 6. Synthesis of a precursor of snaking-1. Reaction conditions: (i) Boc-Ala-Pam, DIC, CH2Cl2, 1 Schemeh; (ii) TFA; 6. (iii)Synthesis TrtSCH of2CH a2 precursorCO2H, HBTU, of snaking-1. DIPEA, ReactionDMF, 16 conditions min; (iv): (i)TFA, Boc-Ala-Pam, TIPS, H2O; DIC,(v) h; (ii) TFA; (iii) TrtSCH2CH2CO2H, HBTU, DIPEA, DMF, 16 min; (iv) TFA, TIPS, H2O; (v) CHBoc-Cys-(4-MeBn)-OH,2Cl2, 1 h; (ii) TFA; (iii)HBTU, TrtSCH DIPEA,2CH 2DMF,CO2H, 1 h; HBTU, (vi) Boc DIPEA, SSP; (vii) DMF, Boc-Pro-Pam, 16 min; (iv) DIC, TFA, CH TIPS,2Cl2, H 12 O;h; Boc-Cys-(4-MeBn)-OH, HBTU, DIPEA, DMF, 1 h; (vi) Boc SSP; (vii) Boc-Pro-Pam, DIC, CH2Cl2, 1 h; (v)(viii) Boc-Cys-(4-MeBn)-OH, Boc SSPS. TIPS HBTU,= triisopropylsilane; DIPEA, DMF, 1 h; (vi)DIC Boc = SSP;diisopropylcarbodiimide; (vii) Boc-Pro-Pam, DIC, CHDIPEA2Cl2, 1 h;= (viii) Boc SSPS. TIPS = triisopropylsilane; DIC = diisopropylcarbodiimide; DIPEA = (viii)diisopropylethylamine; Boc SSPS. TIPS = triisopropylsilane; DMF = N,N DIC-dimethylformamide; = diisopropylcarbodiimide; HBTU DIPEA= O-benzotriazole- = diisopropylethylamine;N,N,N′,N′- diisopropylethylamine; DMF = N,N-dimethylformamide; HBTU = O-benzotriazole-0 0 N,N,N′,N′- DMFtetramethyluronium = N,N-dimethylformamide; hexafluorophosphate; HBTU Pam = = phenylacetamidomethyl;O-benzotriazole-N,N,N Trt,N =-tetramethyluronium triphenylmethyl. hexafluorophosphate;tetramethyluronium hexafluorophosphate; Pam = phenylacetamidomethyl; Pam = phenylacetamidomethyl; Trt = triphenylmethyl. Trt = triphenylmethyl. For the synthesis of snakin-1, an antimicrobial peptide produced by several plants, such as For the synthesis of snakin-1, an antimicrobial peptide produced by several plants, such as potato,For rice, the maize, synthesis soybean, of snakin-1, and strawberry, an antimicrobial a native chemical peptide producedligation method by several was used. plants, As such shown as potato, rice, maize, soybean, and strawberry, a native chemical ligation method was used. As shown potato,in Scheme rice, 6, maize, two fragments soybean, and10 and strawberry, 11 were a produced native chemical by solid-phase ligation method synthesis, was and used. then As ligated shown in Scheme 6, two fragments 10 and 11 were produced by solid-phase synthesis, and then ligated in(conversion Scheme6, 12 two→14 fragments) to give the10 andsnakin11 precursorwere produced 15. Oxidation by solid-phase of the cysteine synthesis, units and generated then ligated six (conversion 12→14) to give the snakin precursor 15. Oxidation of the cysteine units generated six (conversioninternal disulfide12→14 bonds,) to give providing the snakin the folded precursor snakin-115. Oxidation in about 50% of the yield cysteine [107]. The units native generated chemical six internal disulfide bonds, providing the folded snakin-1 in about 50% yield [107]. The native chemical internalligation has disulfide also been bonds, used providing to cyclize the peptides, folded snakin-1 as in the in case about of θ 50%-defensins yield [107 [108].]. The native chemical ligation has also been used to cyclize peptides, as in the case of θ-defensins [108]. ligationThe has advances also been in the used chemical to cyclize synthesis peptides, have as inalso the affected case of theθ-defensins generation [108 of]. peptide libraries for The advances in the chemical synthesis have also affected the generation of peptide libraries for SAR Thestudies. advances Thus, in in the the chemical traditional synthesis way haveto prepare also affected peptide the libraries, generation each of peptidelibrary librariesmember foris SAR studies. Thus, in the traditional way to prepare peptide libraries, each library member is SARprepared studies. de Thus,novo. in theThe traditionalintroduction way toof preparea new peptideamino libraries,acid involves each library two membersteps, namely is prepared the prepared de novo. The introduction of a new amino acid involves two steps, namely the dedeprotection novo. The of introduction the terminal of unit a new and amino the couplin acid involvesg of the two new steps, residue. namely When the deprotectionlarge peptides of theare deprotection of the terminal unit and the coupling of the new residue. When large peptides are terminalsynthesized, unit or and when the couplingdifficult ofsequences the new or residue. macrocycles When largeneed peptidesto be prepared, are synthesized, the conventional or when synthesized, or when difficult sequences or macrocycles need to be prepared, the conventional difficultprocess can sequences pose problems or macrocycles and be needcostly to in be time prepared, and materials. the conventional In addition, process since can for posemany problems activity process can pose problems and be costly in time and materials. In addition, since for many activity andstudies be costlyonly ina small time andpart materials. of the peptide In addition, needs since modification, for many activityan alternative studies onlystrategy a small would part be of studies only a small part of the peptide needs modification, an alternative strategy would be thepreferrable, peptide needswhere modification,a few starting anpeptides alternative undergo strategy a site-selective would be preferrable,modification where of certain a few positions, starting preferrable, where a few starting peptides undergo a site-selective modification of certain positions, peptideswhich does undergo not affect a site-selective to the other modificationpositions [111]. of certain positions, which does not affect to the other which does not affect to the other positions [111]. positionsAlthough [111]. this strategy represents a synthetic challenge, due to the similar reactivity of the Although this strategy represents a synthetic challenge, due to the similar reactivity of the amino acid units, in the last years a variety of methods have allowed the selective modification of amino acid units, in the last years a variety of methods have allowed the selective modification of peptides and proteins. In the tag-and-modify approach, a functional group in a residue (tag) is peptides and proteins. In the tag-and-modify approach, a functional group in a residue (tag) is

Molecules 2018, 23, 311 9 of 26

Although this strategy represents a synthetic challenge, due to the similar reactivity of the aminoMolecules acid2018, units,23, x FOR in PEER the REVIEW last years a variety of methods have allowed the selective modification9 of 24 ofMolecules peptides 2018, and23, x FOR proteins. PEER REVIEW In the tag-and-modify approach, a functional group in a residue (tag)9 of 24 is replaced or transformedtransformed intointo otherother ones.ones. For in instance,stance, amino acids with haloaryl units undergoundergo sp2-sp2replaced couplings couplingsor transformed [ 112[112].]. Click intoClick chemistryother chemistry ones. provides For provides instance, a practical a practicalamino method acids method for with the for introductionhaloaryl the introduction units of differentundergo of groupsdifferentsp2-sp2 and couplingsgroups even and the[112]. even ligation theClick ligation of chemistry peptide of peptide fragments,provides fragments, a as practical shown as shown bymethod the by synthesisthe for synthesis the byintroduction Planasby Planas et al.ofet ofal.different cyclicof cyclic peptidotriazolesgroups peptidotriazoles and even derivedthe derived ligation from from of thepeptide the antimicrobial an timicrobialfragments, peptide peptideas shown BPC194 BPC194 by the (Scheme (Schemesynthesis7 ,7, conversionby conversion Planas et 4al.→ of16 ,,[cyclic [113]).113]). peptidotriazoles Some Some of of them them presented presented derived from potent potent the activity activity antimicrobial against against plantpeptide plant pathogens, pathogens, BPC194 (Schemefrom from bacteria 7, conversion to fungi, fungi, → while4 16 , producing [113]). Some lowlow of hemolysis, hemolysis,them presented andand werewere potent quitequite activity stablestable against towardstowards plant proteaseprotease pathogens, cleavagecleavage from [[106,113,114].106 bacteria,113,114 to]. fungi, while producing low hemolysis, and were quite stable towards protease cleavage [106,113,114].

Scheme 7. Peptide ligation using click’ chemistry. Scheme 7. Peptide ligation using click’ chemistry. In a second approach for the generation of peptide libraries by site-selective modification, the In a second approach for the generation of peptide libraries by site-selective modification, the core core Inof athe second starting approach amino foracid the (customizable generation of unit) pept undergoeside libraries a scissionby site-selective process modification,and is converted the of the starting amino acid (customizable unit) undergoes a scission process and is converted into intocore aof verythe startingdifferent amino one (e.g.,acid (customizableby cleavage of unit) the undergoeslateral chain a scission and attachment process and of ais newconverted one). a very different one (e.g., by cleavage of the lateral chain and attachment of a new one). Recently, Recently,into a very our different group onehas (e.g., introduced by cleavage new ofcustomizable the lateral chain units and that attachment allow the of site-selectivea new one). our group has introduced new customizable units that allow the site-selective modification of peptides modificationRecently, our of grouppeptides has and introduced the creation new of bothcustomizable cationic or unitshydrophobi that callow residues the [111,115–119].site-selective and the creation of both cationic or hydrophobic residues [111,115–119]. Peptide ligation is also Peptidemodification ligation of peptidesis also possible, and the as creation shown inof theboth Scheme cationic 8. orThis hydrophobi strategy hasc residues allowed [111,115–119]. the synthesis possible, as shown in the Scheme8. This strategy has allowed the synthesis of peptide libraries with ofPeptide peptide ligation libraries is also with possible, arylglycine, as shown 4-aminohomoalanines, in the Scheme 8. This dehydroamino strategy has allowed acid units, the synthesis β- and arylglycine, 4-aminohomoalanines, dehydroamino acid units, β- and γ-amino acid residues, and many γof-amino peptide acid libraries residues, with and arylglycine, many other 4-aminohomoalanines, residues. Some of thes dehydroaminoe compounds acid have units, displayed β- and a other residues. Some of these compounds have displayed a promising activity against plant and promisingγ-amino acid activity residues, against and plant many and otheranimal residues. pathogens. Some of these compounds have displayed a animalpromising pathogens. activity against plant and animal pathogens.

Scheme 8. Site-selective modification of peptides in the discovery of new AMPs. Scheme 8. Site-selective modification modification of peptidespeptides in the discovery of new AMPs. 2.2.2. AMP Libraries for SAR Studies 2.2.2. AMP Libraries for SAR Studies Once a promising antimicrobial peptide has been identified, SAR studies are carried out to determineOnce athe promising structural antimicrobial features which peptide are keyhas beenfor the identified, activity. SARSince studies a polymyxin are carried derivative, out to colistin,determine is theone structuralof the AMPs features in clinical which use,are differentkey for theanalogues activity. have Since been a polymyxin produced derivative,(Figure 2, [8,41,110]).colistin, is Inone the of Pfizer the AMPs 5x, MicuRx in clinical and CB-182,804 use, different analogs, analogues the fatty have acid been group produced in the lateral (Figure chain 2, is[8,41,110]). replaced Inby the other Pfizer acyl 5x, or MicuRx carbamate and CB-182,804protecting analogs,groups. Inthe the fatty Monash acid group FADDI-, in the Queensland-, lateral chain is replaced by other acyl or carbamate protecting groups. In the Monash FADDI-, Queensland-,

Molecules 2018, 23, 311 10 of 26

2.2.2. AMP Libraries for SAR Studies Once a promising antimicrobial peptide has been identified, SAR studies are carried out to determine the structural features which are key for the activity. Since a polymyxin derivative, colistin, is one of the AMPs in clinical use, different analogues have been produced (Figure2, [ 8,41,110]). In the Pfizer 5x, Molecules 2018, 23, x FOR PEER REVIEW 10 of 24 MicuRx and CB-182,804 analogs, the fatty acid group in the lateral chain is replaced by other acyl or carbamateCantab-, and protecting Northern groups. Antibiotic In the analogs, Monash the FADDI-, terminal Queensland-, acyl group Cantab-,and the amino and Northern acids in Antibioticthe cyclic analogs,core are thechanged. terminal In addition, acyl group in and the theBarcelona amino acidsanalogs, in the a disulfide cyclic core group are changed. is introduced In addition, in the incyclic the Barcelonacore. Some analogs, of these a analogs disulfide displayed group is introduceda potent anti inbacterial the cyclic activity. core. Some A similar of these study analogs was displayedcarried out a potentfor the antibacterial structurally activity. related A octapeptins similar study [9]. was In carried related out studies for the for structurally other family related ofoctapeptins antimicrobial [9]. Inpeptides, related studiesInoue has for othercarried family out ofthe antimicrobial synthesis of peptides, lysocin InoueE, preparing has carried enantiomeric, out the synthesis epimeric, of lysocin and E,N-demethylated preparing enantiomeric, analogues, epimeric, and also and Ndifferent-demethylated amino analogues, acid residues and also [120]. different In aminothis way, acid residuesstructure-activity [120]. In this(SAR) way, relationships structure-activity have (SAR)been determined, relationships such have beenas the determined, influence of such cationic, as the influencehydrophobic of cationic, and aromatic hydrophobic units on and the aromatic antibacterial units on activity the antibacterial [121]. activity [121].

Figure 2.2. Structures of colistin A, its analog CB-182,804,CB-182,804, the Cantab analogs CA-824 and CA-1049, and the Barcelona disulfidedisulfide analogs.analogs. The positions were changeschanges werewere mademade areare displayeddisplayed inin colors.colors.

These studies have shown that the ratio between cationic and hydrophobic units is essential for These studies have shown that the ratio between cationic and hydrophobic units is essential for the antimicrobial activity but also for the selectivity. Thus, Fattorusso has reported a new analogue the antimicrobial activity but also for the selectivity. Thus, Fattorusso has reported a new analogue of temporins [122], the defense peptides isolated from frogs (Rana temporaria). Temporins are small of temporins [122], the defense peptides isolated from frogs (Rana temporaria). Temporins are small peptides (8–14 residues) with amides at the C-terminal position, and a small positive charge at peptides (8–14 residues) with amides at the C-terminal position, and a small positive charge at neutral neutral pH. The analogue presented one point mutation (G6A) in the temporine sequence and two pH. The analogue presented one point mutation (G6A) in the temporine sequence and two extra extra lysines at the N-terminal position. This peptide was active against Gram-positive and lysines at the N-terminal position. This peptide was active against Gram-positive and Gram-negative Gram-negative bacteria, and displayed no hemolytic activity. On interaction with bacterial bacteria, and displayed no hemolytic activity. On interaction with bacterial membranes, the unnatural membranes, the unnatural peptide used one Lys residue to anchor the peptide to the bacterial peptide used one Lys residue to anchor the peptide to the bacterial membrane, while the other Lys membrane, while the other Lys unit allowed penetration of the micellar structure. unit allowed penetration of the micellar structure. This understanding has prompted the development of peptide analogues and peptidomimetics This understanding has prompted the development of peptide analogues and peptidomimetics where where different ratios of hydrophobic and cationic residues were tested. With the right balance, different ratios of hydrophobic and cationic residues were tested. With the right balance, truncated sections truncated sections of AMPs and even very small peptides can display antimicrobial activity of AMPs and even very small peptides can display antimicrobial activity [28,29,123–130]. [28,29,123–130]. Thus, structural simplification by truncation has been used with human cathelicidin LL-37 [123], Thus, structural simplification by truncation has been used with human cathelicidin LL-37 generating several fragments which outperformed the antibiofilm effect of LL-37 against MRSA [123], generating several fragments which outperformed the antibiofilm effect of LL-37 against strains. Besides, truncation of LL-37 decreases its cytotoxic effects while the antimicrobial activity is MRSA strains. Besides, truncation of LL-37 decreases its cytotoxic effects while the antimicrobial activity is maintained. Montesinos and Bardají have reviewed the shortening of linear AMPs [124], including magainin (pexiganan, MSI-99, ESFs), protegrins (iseganan), cecropins (D4E1) and tachyplesins (TPY). A similar effect was observed when linear analogues of the cyclic pepide battacin were produced. Conjugation of a linear analog with a smaller fatty acid, 4-methylhexanoic acid, gave a lipopeptide with superior antibacterial activity and selectivity. In addition, antibiofilm activity against S. aureus, P. aeruginosa and P. syringae was observed [28].

Molecules 2018, 23, 311 11 of 26 maintained. Montesinos and Bardají have reviewed the shortening of linear AMPs [124], including magainin (pexiganan, MSI-99, ESFs), protegrins (iseganan), cecropins (D4E1) and tachyplesins (TPY). A similar effect was observed when linear analogues of the cyclic pepide battacin were produced. Conjugation of a linear analog with a smaller fatty acid, 4-methylhexanoic acid, gave a lipopeptide with superior antibacterial activity and selectivity. In addition, antibiofilm activity against S. aureus, Molecules 2018, 23, x FOR PEER REVIEW 11 of 24 P. aeruginosa and P. syringae was observed [28]. Plant systemins served as inspirationinspiration for synthetic small-size peptides such as FRLKFHFFRLKFHF that have beenbeen developed developed as as pesticides pesticides in agriculturein agriculture [124 ].[124]. In another In another example, example, natural natural sapecin Bsapecin inspired B ainspired short synthetic a short peptide synthetic BF2 (RWRLLLLKKH)peptide BF2 (RWRLLLLKKH) with a potent activity with againsta potent vancomycin-resistant activity against enterococcivancomycin-resistant at low micromolar enterococci range, at low also micromolar displaying activityrange, inalso vivo displaying. The peptide activity was in non-toxic vivo. The to mammalianpeptide was cellsnon-toxic [125]. to mammalian cells [125]. An even shorter peptide NapFFKK 28 (Figure 33,, onon thethe left),left), withwith aa terminalterminal naphthalenenaphthalene ring,ring, displayed a potent antimicrobial activity [[130].130]. TheThe peptide self-assembled and its hydrogels greatly reduced thethe formation formation of ofS. epidermidisS. epidermidisbiofilms. biofilms. Interestingly, Interestingly, it was it observed was observed that reducing that reducing the α-chain the lenghtα-chain at lenght the cationic at the cationic residues, residues, the biofilm the biofilm activity activity was reduced. was reduced. The authors The authors suggested suggested that with that shorterwith shorter chains, chains, the amine the groupamine did group not interactdid not wellinte withract well the negatively with the chargednegatively bacterial charged membranes. bacterial Amembranes. good activity/citotoxicity A good activity/citotoxicity ratio was observed. ratio was observed.

Figure 3. StructuresStructures of of AMP AMP analogues: analogues: (Left (Left) the) theNApFFKK NApFFKK peptide; peptide; (Center (Center) Synthetic) Synthetic AMPs AMPs with withferrocene ferrocene or rutacene or rutacene groups; groups; (Right) (RightUnnatural) Unnatural tryptophan tryptophan units in unitsa ferritin-inspired in a ferritin-inspired peptide, peptide,LTX-109. LTX-109.

Some synthetic antimicrobial peptides alternate residues with aromatic and with cationic Some synthetic antimicrobial peptides alternate residues with aromatic and with cationic chains. chains. In a recent work, the structure-activity relationships of the synthetic AMPs 29 (Figure 3, In a recent work, the structure-activity relationships of the synthetic AMPs 29 (Figure3, center) was center) was explored using a ferrocene scan [131]. explored using a ferrocene scan [131]. The introduction of unnatural hydrophobic and cationic residues into the peptides can improve The introduction of unnatural hydrophobic and cationic residues into the peptides can improve the antimicrobial activity and the in vivo stability [132]. After systemic truncation and optimization the antimicrobial activity and the in vivo stability [132]. After systemic truncation and optimization of peptides derived from lactoferricin, novel synthetic antimicrobial peptidomimetics (SAMPs) of peptides derived from lactoferricin, novel synthetic antimicrobial peptidomimetics (SAMPs) were were identified as drug candidates, and one of them (LTX-109 (30), Figure 3, right), is in clinical identified as drug candidates, and one of them (LTX-109 (30), Figure3, right), is in clinical trials against trials against bacterial topical infections (Lytix Biopharma AS) [133]. In addition, LTX-109 displayed bacterial topical infections (Lytix Biopharma AS) [133]. In addition, LTX-109 displayed high antifungal high antifungal activity against candidiasis and onychomycosis, superior to the current reference activity against candidiasis and onychomycosis, superior to the current reference drugs, and high drugs, and high selectivity [134]. selectivity [134]. The stereochemistry of the residues can also influence the antimicrobial activity. The The stereochemistry of the residues can also influence the antimicrobial activity. The introduction introduction of residues with D-amino acids can increase the proteolytic stability. The replacement of of residues with D-amino acids can increase the proteolytic stability. The replacement of L- by D-amino L- by D-amino acids in human cathelicidin LL-37 decreased degradation by proteases and increased acids in human cathelicidin LL-37 decreased degradation by proteases and increased the antimicrobial the antimicrobial activity [135]. Besides, this replacement can increase drug selectivity; thus, activity [135]. Besides, this replacement can increase drug selectivity; thus, peptides composed solely peptides composed solely of L-units have usually high helicity and propensity to form pores both in of L-units have usually high helicity and propensity to form pores both in bacterial and eukaryotic bacterial and eukaryotic membranes. When some of the L-units are replaced by D-residues, the membranes. When some of the L-units are replaced by D-residues, the helicity decreases and the helicity decreases and the peptides selectively interact with the bacterial membranes, causing less peptides selectively interact with the bacterial membranes, causing less hemolysis while maintaining hemolysis while maintaining the antibacterial activity [136]. the antibacterial activity [136]. The introduction of D-amino acids can also favor the formation of secondary structures such as β-turns. The development of β-hairpin epitome mimetics has been reviewed by Robinson [137]; in Figure 4, a L-Pro-D-Pro unit is vital for the formation of a β-hairpin mimetic scaffold incorporating cationic and hydrophobic units. The L-Pro-D-Pro unit can be found in murepavadin 31 (Figure, left), which has recently completed phase-II clinical trials for the treatment of life-threatening lung infections caused by Pseudomonas sp., and is starting Phase III studies. Interestingly, murepavadin was developed by optimization of a hit, compound L27-11 (32), which displayed a potent and selective anti-pseudomonas activity [138]. The mechanism of action of L27-11 was new, since the peptide selectively interacted with the β-barrel protein LptD in Pseudomonas spp., which is involved in lipopolysaccaride (LPS) transport to the membrane [139]. However, L27-11 was not

Molecules 2018, 23, 311 12 of 26

The introduction of D-amino acids can also favor the formation of secondary structures such as β-turns. The development of β-hairpin epitome mimetics has been reviewed by Robinson [137]; in Figure4, a L-Pro-D-Pro unit is vital for the formation of a β-hairpin mimetic scaffold incorporating cationic and hydrophobic units. The L-Pro-D-Pro unit can be found in murepavadin 31 (Figure4, left), which has recently completed phase-II clinical trials for the treatment of life-threatening lung infections caused by Pseudomonas sp., and is starting Phase III studies. Interestingly, murepavadin was developed by optimization of a hit, compound L27-11 (32), which displayed a potent and selective anti-pseudomonas activity [138]. The mechanism of action of L27-11 was new, since the peptide β Moleculesselectively 2018, interacted 23, x FOR PEER with REVIEW the -barrel protein LptD in Pseudomonas spp., which is involved12 of in24 lipopolysaccaride (LPS) transport to the membrane [139]. However, L27-11 was not suitable as a drug, suitablesince it was as rapidlya drug, degraded since it bywas proteases rapidly in degraded human serum. by proteases Structural in optimization human serum. led to compoundStructural optimization33 (POL7001), led where to compound many of 33 the (POL7001), arginine (R) where or lysine many (K) of the residues arginine were (R) replaced or lysine by (K) Dab residues units were(diaminobutyric replaced by acid), Dab resultingunits (diaminobutyric in greater stability acid), against resulting proteases. in greater Further stability optimization against proteases. provided Furthermurepavadin optimization31. The provided research onmurepavadin other analogues 31. The provided research a different on otherβ-hairpin analogues peptidomimetic provided a differentJB-95 (34 )β which-hairpin was peptidomimetic active against EscherichiaJB-95 (34) which coli [140 was]. active against Escherichia coli [140].

H2N NH HN H 2 H2N N

O O H HN O N O NH H2N O CH D NH 3 R R Dab Dab R W HN O K W K W I K NH2 H H CH3 N HN O O NH K K Dab Dab R R H N 2 O L NH L K I Dab I OH HN O CH3 W A W A R R H CH3 HN O R O NH TK T Dab W H O O L D OH LP DP LP DP P P N N H

31 Murepavadin 32 L27-11 33 POL7001 34 JB95

Figure 4. ((LeftLeft)) Murepavadin Murepavadin ( (31), an anti-pseudomonas peptidepeptide in Phase II trials, with a LL--Pro-Pro-D--ProPro unit thatthat inducesinduces a aβ -turnβ-turn in in the theβ-hairpin β-hairpin structure; structure; (Center (Center) The) 12-residueThe 12-residueβ-hairpin β-hairpin mimetic mimetic L27-11 L27-11(32) with (32 a) potentwith a andpotent specific and antipseudomonasspecific antipseudomonas activity; activity; optimization optimization of this hit of generated this hit generated POL7001 POL7001(33) (Dab =(33 L-2,4-diaminobutyric) (Dab = L-2,4-diaminobutyric acid); (Right) Anotheracid); ( syntheticRight) Another peptidomimetic synthetic antibiotic peptidomimetic JB-95 (34), antibioticactive against JB-95E. (34 coli),. active against E. coli.

Alternating D- and L-units in 6–8-membered cyclic peptides 35 (Figure 5) induce the formation Alternating D- and L-units in 6–8-membered cyclic peptides 35 (Figure5) induce the formation of tubular structures which disrupt the bacterial membrane [141,142]. The combination of L-and of tubular structures which disrupt the bacterial membrane [141,142]. The combination of L- and D-residues generate flat rings with the lateral chains oriented on the outside, which can form D-residues generate flat rings with the lateral chains oriented on the outside, which can form intermolecular hydrogen bonds very efficiently. The presence of two or three basic chains gave intermolecular hydrogen bonds very efficiently. The presence of two or three basic chains gave derivatives with micromolar activity against MRSA, while acid groups reduced the activity due to derivatives with micromolar activity against MRSA, while acid groups reduced the activity due to electrostatic repulsion of the negatively charged bacterial membrane. electrostatic repulsion of the negatively charged bacterial membrane.

Figure 5. Cyclic peptides with alternating D, L-units self-assemble and form pores in the bacterial membrane.

Cyclic peptides, as those in the previous example, are more resistant than linear peptides to proteolytic cleavage. Thus, the cyclic peptide BPC194 [cyclo(KKLKKFKKLQ)] hardly shows degradation by proteinase K after 45 min, while the linear analog BP100 (KKLFKKILKYL-NH2)

Molecules 2018, 23, x FOR PEER REVIEW 12 of 24 suitable as a drug, since it was rapidly degraded by proteases in human serum. Structural optimization led to compound 33 (POL7001), where many of the arginine (R) or lysine (K) residues were replaced by Dab units (diaminobutyric acid), resulting in greater stability against proteases. Further optimization provided murepavadin 31. The research on other analogues provided a different β-hairpin peptidomimetic JB-95 (34) which was active against Escherichia coli [140].

H2N NH HN H 2 H2N N

O O H HN O N O NH H2N O CH D NH 3 R R Dab Dab R W HN O K W K W I K NH2 H H CH3 N HN O O NH K K Dab Dab R R H N 2 O L NH L K I Dab I OH HN O CH3 W A W A R R H CH3 HN O R O NH TK T Dab W H O O L D OH LP DP LP DP P P N N H

31 Murepavadin 32 L27-11 33 POL7001 34 JB95

Figure 4. (Left) Murepavadin (31), an anti-pseudomonas peptide in Phase II trials, with a L-Pro-D-Pro unit that induces a β-turn in the β-hairpin structure; (Center) The 12-residue β-hairpin mimetic L27-11 (32) with a potent and specific antipseudomonas activity; optimization of this hit generated POL7001 (33) (Dab = L-2,4-diaminobutyric acid); (Right) Another synthetic peptidomimetic antibiotic JB-95 (34), active against E. coli.

Alternating D- and L-units in 6–8-membered cyclic peptides 35 (Figure 5) induce the formation of tubular structures which disrupt the bacterial membrane [141,142]. The combination of L-and D-residues generate flat rings with the lateral chains oriented on the outside, which can form intermolecular hydrogen bonds very efficiently. The presence of two or three basic chains gave

Moleculesderivatives2018 ,with23, 311 micromolar activity against MRSA, while acid groups reduced the activity due13 of to 26 electrostatic repulsion of the negatively charged bacterial membrane.

D, L Figure 5. 5. CyclicCyclic peptides peptides with with alternating alternating -unitsD, L -unitsself-assemble self-assemble and form and pores form in poresthe bacterial in the bacterialmembrane. membrane.

Cyclic peptides, as those in the previous example, are more resistant than linear peptides to Cyclic peptides, as those in the previous example, are more resistant than linear peptides proteolytic cleavage. Thus, the cyclic peptide BPC194 [cyclo(KKLKKFKKLQ)] hardly shows toMolecules proteolytic 2018, 23, cleavage.x FOR PEER REVIEW Thus, the cyclic peptide BPC194 [cyclo(KKLKKFKKLQ)] hardly shows13 of 24 degradation by proteinase K after 45 min, while the linear analog BP100 (KKLFKKILKYL-NH2) degradation by proteinase K after 45 min, while the linear analog BP100 (KKLFKKILKYL-NH2) underwent 44% degradation in that time [124]. [124]. The ring size [143] [143] and the flexibility flexibility [144] [144] of the ring backbone also affect the bioactivity. In summary,summary, thethe generation generation of of peptide peptide libraries libraries and and the the subsequent subsequent SAR SAR studies studies have have shed shed light onlight the on structural the structural features features required required for antimicrobial for antimicrobial activity. activity. As a result, As a result, completely completely synthetic synthetic AMPs (SAMPs)AMPs (SAMPs) and then and peptidomimetics then peptidomimetics have been have developed been developed in an effort in an to effort improve to improve selectivity, selectivity, stability, bioavailabilitystability, bioavailability and alsoto and reduce also costs.to reduce The generationcosts. The ofgeneration SAMPs has of beenSAMPs commented has been before. commented In the nextbefore. section, In the the next development section, the ofdevelopment peptidomimetics of peptidomimetics will be discussed. will be discussed.

2.2.3. SAR Studies: Natural AMPs as Inspiration for Peptidomimetics In a furtherfurther stage,stage, chemicalchemical synthesis has allowed the preparationpreparation of peptidepeptide analoguesanalogues where at least part part of of the the αα-amino-amino acid acid units units have have been been replaced replaced by byother other structural structural elements. elements. However, However, the theprinciple principle that that hydrophobic hydrophobic and and cationic cationic structures structures must must be balanced be balanced is still is still applied. applied. For instance, Mor et al. have designed acyllysine oligomers (OAKs) with alternating acyl chain (A) and cationic lysine (K) units (such as compounds 36 and 37, Figure6 6))[ [145].145]. The design intended to avoid the formation of stable secondarysecondary structures, while allowing the generation of the active structures on interaction with cell membranes [[114646].]. The The studies studies on on the variation of the A chain lenght, and the number of A and K units, finallyfinally afforded peptidomimeticspeptidomimetics with an optimal balance of the overall charge and hydrophobicity.hydrophobicity. Some of these OAKs have a potent activity against many bacterial strainsstrains atat lowlow micromolar micromolar range. range. Interestingly, Interestingly, the the mechanism mechanism of actionof action is variable, is variable, from from cell membranecell membrane disruption disruption to inhibition to inhibition of DNA of DNA replication, replication, according according to environmental to environmental factors factors such as such pH, temperatureas pH, temperature and salt and concentration salt concentration [147]. [147].

Figure 6. AMP analogs; acyllysine oligomers (OAKs) with alternating acyl (A) and lysine (K) units.

Other useful peptidomimetics are peptoids, glycine oligomers where the “lateral chains” are attached to the amide nitrogen. The amphiphilic cyclic peptoids 41–43 shown in Scheme 9 present cationic chains, resembling the ornithine units, and hydrophobic chains containing aromatic groups.

Scheme 9. Peptoid analogs, alternating cationic and hydrophobic chains. Cycle 41 gave the best results. Reaction conditions (above): (a) R-NH2, base; (b) BrCH2COOH; (c) 20% HFIP, CH2Cl2; (d) PyBOP, DIEA, DMF. Conversion yields >95% were observed in the synthesis of cyclic peptoid analogues.

Molecules 2018, 23, x FOR PEER REVIEW 13 of 24 underwent 44% degradation in that time [124]. The ring size [143] and the flexibility [144] of the ring backbone also affect the bioactivity. In summary, the generation of peptide libraries and the subsequent SAR studies have shed light on the structural features required for antimicrobial activity. As a result, completely synthetic AMPs (SAMPs) and then peptidomimetics have been developed in an effort to improve selectivity, stability, bioavailability and also to reduce costs. The generation of SAMPs has been commented before. In the next section, the development of peptidomimetics will be discussed.

2.2.3. SAR Studies: Natural AMPs as Inspiration for Peptidomimetics In a further stage, chemical synthesis has allowed the preparation of peptide analogues where at least part of the α-amino acid units have been replaced by other structural elements. However, the principle that hydrophobic and cationic structures must be balanced is still applied. For instance, Mor et al. have designed acyllysine oligomers (OAKs) with alternating acyl chain (A) and cationic lysine (K) units (such as compounds 36 and 37, Figure 6) [145]. The design intended to avoid the formation of stable secondary structures, while allowing the generation of the active structures on interaction with cell membranes [146]. The studies on the variation of the A chain lenght, and the number of A and K units, finally afforded peptidomimetics with an optimal balance of the overall charge and hydrophobicity. Some of these OAKs have a potent activity against many bacterial strains at low micromolar range. Interestingly, the mechanism of action is variable, from cell membrane disruption to inhibition of DNA replication, according to environmental factors such as pH, temperature and salt concentration [147].

Molecules 2018, 23, 311 14 of 26 Figure 6. AMP analogs; acyllysine oligomers (OAKs) with alternating acyl (A) and lysine (K) units.

Other usefuluseful peptidomimeticspeptidomimetics areare peptoids,peptoids, glycine oligomers where the “lateral“lateral chains”chains” areare attached to thethe amideamide nitrogen.nitrogen. The amphiphilic cyclic peptoids 41–43 shown in SchemeScheme9 9 present present cationiccationic chains,chains, resemblingresembling thethe ornithineornithine units,units, andand hydrophobichydrophobic chainschains containingcontaining aromaticaromatic groups.groups.

Scheme 9. Peptoid analogs, alternating cationic and hydrophobic chains. Cycle 41 gave the best results. 2 2 2 2 ReactionReaction conditionsconditions (above):(above): ((a)) R-NHR-NH2, base; ( b) BrCH 2COOH;COOH; ( cc)) 20% 20% HFIP, CHCH2Cl2;;( (dd)) PyBOP, PyBOP, DIEA, DMF. ConversionConversion yields >95% were were observed observed in in the the synthesis synthesis of of cyclic cyclic peptoid peptoid analogues. analogues. Molecules 2018, 23, x FOR PEER REVIEW 14 of 24

These peptidomimetics are are readily synthesized on on a solid support through cycles of bromoacetylation and and substitution substitution of the of thehaloge halogenn group group by primary by primary amines. amines. The peptoids The disrupt peptoids S. aureusdisrupt membraneS. aureus membrane through throughthe formation the formation of pores, of pores, and anddisplay display a apotent potent activity activity against methicillin-resistant S.S. aureus aureus(MRSA). (MRSA). A goodA good selectivity selectivity is achieved, is achieved, and as anand additional as an advantage,additional advantage,the unnatural the backboneunnatural isbackbone more resistant is more to resistant proteases to [proteases148]. A related [148]. A strategy related is strategy carried is out carried with outthe αwith-peptide/peptoid the α-peptide/peptoid hybrids hybrids44 and 4544 (Figureand 45 7(Figure, left), where7, left), αwhere-aminoacids α-aminoacids with cationic with cationic chains chainsalternate alternate with N -benzylwith N glycines.-benzyl glycines. By introducing By introducing changes in thechan lateralges in chains the lateral and the chainsN-substituents, and the Na-substituents, decreased cytotoxicity a decreased and cytotoxicity a high activity and againsta high activity multidrug-resistant against multidrug-resistantE. coli are obtained E. coli [149 are]. obtainedOther interesting [149]. Other peptidomimetics interesting peptidomimetics are lipidated cyclic are lipidatedγ-AApeptides, cyclic γ where-AApeptides, the amino where groups the amino in the groupsmacrocyclic in the ring macrocyclic are attached ring to are cationic attached chains. to cationic These compounds chains. These disrupt compounds bacterial disrupt membranes bacterial but membranesalso induce thebut immunealso induce response the immune and display response an and anti-inflammatory display an anti-inflammatory effect [150]. effect [150].

Figure 7. AMPAMP analogs: analogs: ( (LeftLeft)) αα-- and and ββ-peptoids-peptoids 4444 andand 4545;;( (CenterCenter)) substituted substituted PrAMPs PrAMPs 4646––4848;; (Right) introduction of cationiccationic chains in analogues 49 and capped-termini derivativesderivatives 5050..

Unnatural Proline-Rich Peptides were also used to introduce cationic and hydrophobic “lateral chains” on the backbone, but in the case of compounds 46–48 (Figure 7, center), the substituents were attached to the 4-hydroxy group of the Hyp (hydroxyproline) units. It must be said that Proline-rich antimicrobial peptides (PrAMPs) represent promising agents against Gram-negative bacteria, and these new derivatives are active both against Gram-positive and Gram-negative bacteria. Library screening identified a derivative with antimicrobial activity in the low micromolar range but low cytotoxicity, which was superior to the reference compound mellitin [151]. Finally, two interesting (and opposite) strategies to introduce cationic and hydrophobic units are shown in Figure 7 (right, compounds 49 and 50). In the first example, cell-penetrating synthetic antimicrobial peptides (SAMPs) are prepared using an aspartic acid oligomer, which is amidated, transforming the anionic aspartic unit into a cationic residue. The compounds 49 display potent and selective bactericidal effect against Mycobacterium (M. smegmatis), by interacting with its DNA [152]. The opposite approach, “capping” lysine residues, decreases the cationic nature of the peptide. Thus, the activity of short synthetic antimicrobial peptides was modulated by lipidation of the C- or N-terminal lysine units (compound 50). Lipidation greatly increased the activity against Gram-positive and Gram-negative bacteria, and a derivative was isolated with activity against S. aureus, A. baumannii and P. aeruginosa in the low micromolar range [153]. The development of AMPs with aminoacid analogs, such as β-amino acids, has also received much attention. Thus, α,β-peptide hybrids and β-peptides often achieve well-defined conformations, and are more resistant to protease degradation. β-Peptides (such as compounds 51–53) usually form globally amphiphilic helices (Figure 8); the groups of Gellmann, Seebach, De Grado, Fülöp, and others have made seminal contributions to understand their folding patterns and their relationships with biological activity [154–161]. Although in the initial studies the β-peptides displayed undesirable haemolytic properties, a better understanding of these foldamers has resulted in the development of β-AMPs with potent antimicrobial activity and low cytotoxicity (hemolysis).

Molecules 2018, 23, 311 15 of 26

Unnatural Proline-Rich Peptides were also used to introduce cationic and hydrophobic “lateral chains” on the backbone, but in the case of compounds 46–48 (Figure7, center), the substituents were attached to the 4-hydroxy group of the Hyp (hydroxyproline) units. It must be said that Proline-rich antimicrobial peptides (PrAMPs) represent promising agents against Gram-negative bacteria, and these new derivatives are active both against Gram-positive and Gram-negative bacteria. Library screening identified a derivative with antimicrobial activity in the low micromolar range but low cytotoxicity, which was superior to the reference compound mellitin [151]. Finally, two interesting (and opposite) strategies to introduce cationic and hydrophobic units are shown in Figure7 (right, compounds 49 and 50). In the first example, cell-penetrating synthetic antimicrobial peptides (SAMPs) are prepared using an aspartic acid oligomer, which is amidated, transforming the anionic aspartic unit into a cationic residue. The compounds 49 display potent and selective bactericidal effect against Mycobacterium (M. smegmatis), by interacting with its DNA [152]. The opposite approach, “capping” lysine residues, decreases the cationic nature of the peptide. Thus, the activity of short synthetic antimicrobial peptides was modulated by lipidation of the C- or N-terminal lysine units (compound 50). Lipidation greatly increased the activity against Gram-positive and Gram-negative bacteria, and a derivative was isolated with activity against S. aureus, A. baumannii and P. aeruginosa in the low micromolar range [153]. The development of AMPs with aminoacid analogs, such as β-amino acids, has also received much attention. Thus, α,β-peptide hybrids and β-peptides often achieve well-defined conformations, and are more resistant to protease degradation. β-Peptides (such as compounds 51–53) usually form globally amphiphilic helices (Figure8); the groups of Gellmann, Seebach, De Grado, Fülöp, and others have made seminal contributions to understand their folding patterns and their relationships with biological activity [154–161]. Although in the initial studies the β-peptides displayed undesirable haemolytic properties, a better understanding of these foldamers has resulted in the development of βMolecules-AMPs 2018 with, 23 potent, x FOR PEER antimicrobial REVIEW activity and low cytotoxicity (hemolysis). 15 of 24

Figure 8. β-peptides-peptides with with alternating alternating hydrophobic hydrophobic and ca cationictionic units and different folding patterns.patterns.

Other amino acid analogues incorporate heteroatom functions, such as the silaamino acids Other amino acid analogues incorporate heteroatom functions, such as the silaamino acids [162]. [162]. Incorporation of silicon-containing amino acids in peptides increases their lipophilicity and Incorporation of silicon-containing amino acids in peptides increases their lipophilicity and their their proteolytic stability. Thus, when the antimicrobial peptide alamethicin was replaced by a proteolytic stability. Thus, when the antimicrobial peptide alamethicin was replaced by a derivative derivative containing β-silicon-β3-amino acids, a 20-fold increase in membrane permeabilization containing β-silicon-β3-amino acids, a 20-fold increase in membrane permeabilization was observed. was observed. In another example, helical sulfono-γ-AApeptide foldamers were described, which In another example, helical sulfono-γ-AApeptide foldamers were described, which were resistant to were resistant to proteolytic degradation. The hit compound presented potent activity against multi-drug-resistant Gram-positive and Gram-negative pathogens [163]. The synthesis of other peptidomimetics, with structures less related to peptides, but which mimic the action of membrane active proteins, has been recently reviewed [164].

3. Delivery and Bioavailability Issues Although beyond the scope of this review, it is worth mentioning that the stability and bioavailability of AMPs can be enhanced by loading them into different delivery systems, such as liposomes, lipic discs, polymer nanoparticles, inorganic nanoparticles, graphene and carbon nanotubes, quantum dots, etc. These systems have been recently reviewed by Piotrowska et al. [165].

4. Scale-Up: Overcoming Production Cost Problems The cost of production can be a limiting factor for the development of AMP drugs. However, biotechnological or chemical production of AMPs and their mimetics have undergone many advances, decreasing the synthetic costs and making large-scale production feasible. The methodologies that allowed the efficient generation of peptide libraries during the discovery process, are also useful for the industrial production of small peptides (4–10 residues) [166]. Thus, several lipopeptides and other small peptides (<6 units) have been prepared by solution synthesis or by chemoenzymatic methods [167]. The ongoing progress in the field has allowed the synthesis in a multi-tonne scale of the antiviral agent enfuvirtide (Fuzeon®, Genentech, USA), a 36-residue peptide [168]. The economical large-scale production of peptide mimics with simpler structures is also feasible, as commented before. However, the chemical synthesis of large peptides at reasonable costs is still challenging. A biotechnological approach is then preferred. Many large peptides have been produced in microorganisms [169]. However, the generation of AMPs has specific problems, since these peptides can be toxic for the producer cell and are also prone to proteolytic degradation. Therefore, several strategies have been developed, such as the use of fusion proteins, tandem polypeptides, secretion as inclusion bodies, etc. The large-scale production of plectasin, a fungal defensin from P. nigrella, has been achieved in high yield and

Molecules 2018, 23, 311 16 of 26 proteolytic degradation. The hit compound presented potent activity against multi-drug-resistant Gram-positive and Gram-negative pathogens [163]. The synthesis of other peptidomimetics, with structures less related to peptides, but which mimic the action of membrane active proteins, has been recently reviewed [164].

3. Delivery and Bioavailability Issues Although beyond the scope of this review, it is worth mentioning that the stability and bioavailability of AMPs can be enhanced by loading them into different delivery systems, such as liposomes, lipic discs, polymer nanoparticles, inorganic nanoparticles, graphene and carbon nanotubes, quantum dots, etc. These systems have been recently reviewed by Piotrowska et al. [165].

4. Scale-Up: Overcoming Production Cost Problems The cost of production can be a limiting factor for the development of AMP drugs. However, biotechnological or chemical production of AMPs and their mimetics have undergone many advances, decreasing the synthetic costs and making large-scale production feasible. The methodologies that allowed the efficient generation of peptide libraries during the discovery process, are also useful for the industrial production of small peptides (4–10 residues) [166]. Thus, several lipopeptides and other small peptides (<6 units) have been prepared by solution synthesis or by chemoenzymatic methods [167]. The ongoing progress in the field has allowed the synthesis in a multi-tonne scale of the antiviral agent enfuvirtide (Fuzeon®, Genentech, USA), a 36-residue peptide [168]. The economical large-scale production of peptide mimics with simpler structures is also feasible, as commented before. However, the chemical synthesis of large peptides at reasonable costs is still challenging. A biotechnological approach is then preferred. Many large peptides have been produced in microorganisms [169]. However, the generation of AMPs has specific problems, since these peptides can be toxic for the producer cell and are also prone to proteolytic degradation. Therefore, several strategies have been developed, such as the use of fusion proteins, tandem polypeptides, secretion as inclusion bodies, etc. The large-scale production of plectasin, a fungal defensin from P. nigrella, has been achieved in high yield and purity using recombinant techniques with the fungus Aspergillus oryzae [170]. Being an eukaryotic organism, Aspergillus is less sensitive to the antimicrobial peptide. However, for the expression in the bacteria Escherichia coli of several AMPs, Vogel used calmodulin (CaM) as a carrier protein [171]. CaM presents a structure with two binding domains which can accommodate different chains containing basic and hydrophobic units, avoiding the toxic effects of the amphipathic AMPs and at the same time, protecting them from degradation during the production process. With this technique, several AMPs were produced, such as melittin, fowlicidin-1, tritrpticin, indolicidin, puroindolide A peptide, magainin II F5W, lactoferrampin B, MIP3α51−70, and human β-defensin 3 (HBD-3). These peptides, in the absence of CaM, would be very toxic to E. coli and would kill the producer cells. In another example, Kalman described the expression in E. coli and the purification of seven recombinant AMPs, in industrial scale [172]. Fusions to sumoase protease (SUMO) produced high yields of the intact recombinant AMPs without toxicity to the bacteria; in addition, a cost-efficient, two-step method for the purification of the recombinant peptides was developed. The peptides IDR1, MX226, LL37, CRAMP, HHC-10, E5 and E6 were produced using this methodology, under the GMP conditions required for human-use drugs. On the other hand, Wink achieved recombinant production of the folded, disulfide-containing Snakin-2 in E. coli [173]. The use of microalgae to express recombinant peptides has been commented in Section 2.1. Other examples [124,174] and the promising use of plants as biofactories to obtain crop-protecting peptides has been recently reviewed by Montesinos and Bardají [124]. For instance, in a recent work from the group, the peptide cecropin A was expressed in rice seeds [175,176]. Some years ago, the biotechnological processes were limited to AMPs containing proteinogenic amino acids that could be synthesized in the bacterial ribosomes. Recently, different methods have Molecules 2018, 23, 311 17 of 26 been introduced to ‘reprogram the genetic code’ and allow the assembly of nonproteinogenic amino acids in the ribosomes. These methods have been recently reviewed by Suga [177], and include in vitro translation, engineered aminoacyl tRNA synthetases, and RNA ‘flexizymes’, offering extraordinary opportunities for the preparation of ‘tailored’ peptides with improved properties.

5. Perspectives and Conclusions Antimicrobial peptides (AMPs), also called host-defense peptides for their immunomodulatory properties, could be the next-generation of antimicrobial “superdrugs” if the problems related to their stability, bioavailability, and production at reasonable costs are solved. Their potent activity against pathogens which do not respond to current treatments, their multiple mechanism of action which results in very low or negligible induction of resistance, their synergy with other antimicrobials, and their ability to modulate our immune response are clear assets for the pharmaceutical companies. However, currently only a few antimicrobial peptides have reached the market, and most of the approved drugs are for topical use. But in recent years, a better understanding of their physical features and their complex mechanism of action has allowed the development of analogs and mimetics with improved pharmacological properties. On the other hand, the development of new biotechnological and chemical processes for their industrial production has considerably reduced their costs. Currently, there are promising products in pharmaceutical pipelines with excellent results in animal models or in early clinic trials. Interestingly, some of them are intended to fight systemic infections produced by the top-ten pathogen threats. Although not all of these drug candidates will reach the market, it is reasonable to expect that some AMPs will join the list of approved drugs in the coming years. If these expectations are met, a new generation of versatile, potent and long-lasting antimicrobial drugs will soon be available.

Acknowledgments: This work was supported by the Research Program SAF-2013-48399-R, Plan Estatal de I+D, Ministerio de Economía, Industria y Competitividad, Spain, and European Social Funds (FSE). Author Contributions: A.B. prepared the abstract artwork and scale-up sections, and together with C.C.G. wrote the introduction and the chemical sections; the work of their groups is described as part of Section 2.2.1. J.M.P.d.l.L. and A.B. wrote the biotechnological Section 2.1, where the work of J.M.P.d.l.L. et al. is used to explain several advances in this research area. Conflicts of Interest: The authors declare no conflict of interest.

References and Notes

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