Received: 30 April 2018 | Revised: 11 September 2018 | Accepted: 12 September 2018

DOI: 10.1002/med.21542

REVIEW ARTICLE

Antimicrobial peptides: Promising alternatives

in the post feeding antibiotic era

Jiajun Wang | Xiujing Dou | Jing Song | Yinfeng Lyu | Xin Zhu |
Lin Xu | Weizhong Li | Anshan Shan

Institute of Animal Nutrition, Department of

Animal Nutrition, Northeast Agricultural
University, Harbin, China
Correspondence
Anshan Shan, Institute of Animal Nutrition,
Department of Animal Nutrition, Northeast
Agricultural University, No. 59 Mucai Street,
Xiangfang District, 150030 Harbin, China.
Email: asshan@neau.edu.cn
Funding information
National Natural Science Foundation of
China, Grant/Award Numbers: 31272453,
31472104, 31672434; Program of Ministry of
Education of China, Grant/Award Number:
20092325110009; Program for Universities
in Heilongjiang Province, Grant/Award
Number: 1254CGZH22; China Agriculture
Research System, Grant/Award Number:
CARS‐35
Abstract
Antimicrobial peptides (AMPs), critical components of the
innate immune system, are widely distributed throughout
the animal and plant kingdoms. They can protect against a
broad array of infection‐causing agents, such as bacteria,
fungi, parasites, viruses, and tumor cells, and also exhibit
immunomodulatory activity. AMPs exert antimicrobial ac-
tivities primarily through mechanisms involving membrane
disruption, so they have a lower likelihood of inducing drug
resistance. Extensive studies on the structure‐activity
relationship have revealed that net charge, hydrophobicity,
and amphipathicity are the most important physicochemical
and structural determinants endowing AMPs with antimi-
crobial potency and cell selectivity. This review summarizes
the recent advances in AMPs development with respect to
characteristics, structure‐activity relationships, functions,
antimicrobial mechanisms, expression regulation, and appli-
cations in food, medicine, and animals.

KEYWORDS
antimicrobial peptides, application, mechanism, structure‐activity
relationship

Abbreviations: ADG, average daily gain; AMP, antimicrobial peptide; CAMP, cathelicidin antimicrobial peptide; DON, deoxynivalenol; HDP, host‐defense
peptide; HP‐CPP, tumor‐homing cell‐penetrating peptide; IKK, IκB kinase; LPS, lipopolysaccharides; MAPK, mitogen‐activated protein kinase;
MDT, maggot debridement therapy; MIC, minimum inhibitory concentration; NF‐κB, nuclear factor κB; PAMP, pathogen‐associated molecular pattern;
PG‐1, protegrin 1; TLR, Toll‐like receptor; VDR, vitamin D receptor; YB‐1, Y‐box protein 1.

[Correction added after online publication on 13 November 2018: “postfeeding” has been changed to post feeding]

Med Res Rev. 2019;39:831–859. wileyonlinelibrary.com/journal/med © 2018 Wiley Periodicals, Inc., | 831
832 | WANG ET AL.

1 | INTRODUCTION

In recent decades, antibiotics have been used to improve feed efficiency in livestock production, disease treatment,
and infection prevention in the conventional medical industry.1 Alarmingly, antibiotic‐resistant bacteria have been
increasingly isolated from patients and animals treated with antibiotics, and this resistance to the most
conventional antibiotics has become one of the most pressing global public health concerns worldwide. Therefore,
it is imperative to find an alternative antimicrobial strategy.2
Antimicrobial peptides (AMPs), also known as host‐defense peptides (HDPs),3 have the potential to be
developed as a new generation of antimicrobials. AMPs are an abundant and diverse group of molecules that are an
important component of the innate immune system and are expressed in many tissues and cell types in response to
activation of the Toll‐like receptor (TLR) signaling pathway.4 In addition to rapid and broad‐spectrum activities
against Gram‐negative and ‐positive bacteria, fungi, parasites, viruses, and tumor cells, AMPs also mediate
chemotaxis, apoptosis, immunomodulatory effects, and wound healing.5 The antimicrobial mechanism of AMPs
varies from membrane permeabilization to interaction with an array of intracellular target molecules, and differs
from that of antibiotics that target specific molecular receptors of pathogens (Figure 1). The membrane interactions
between AMPs and bacteria make it both theoretically and clinically difficult for bacteria to acquire resistance to
AMPs, which supports the wide application of AMPs in food, medicine, and animals for avoiding the overuse of
antibiotics.

2 | C L A S SI F I C A T I O N A N D S T RU C T U R A L PR O P E R T I E S
OF ANTIMICROBIAL P EPTIDES

2.1 | Primary structures

2.1.1 | Sequence length
Most AMPs exhibit variable sequence lengths, ranging from 10 to 60 amino acid residues. However, shorter AMPs
are preferred to reduce production costs and many short AMPs exhibit antibacterial potency against clinical isolates
similar to those of longer AMPs; for example, the hexapeptide MP196 (RWRWRW‐NH2) shows robust activity
against Escherichia coli and Staphylococcus aureus with an minimum inhibitory concentration (MIC) value of 5 µg/mL.6
Moreover, long‐chain linear peptides are often more hemolytic and cytotoxic but their N‐ or C‐terminal–truncated
sequences usually have a lower cytotoxicity but retain robust activity.7 However, peptides with excessively short
lengths show a decreased tendency to form amphipathic secondary structures, which leads to compromised

FIGURE 1 Targets of antibiotics [Color figure can be viewed at wileyonlinelibrary.com]

WANG ET AL. | 833

membrane‐disruption capacity and is associated with decreased antimicrobial potency. This indicates that AMPs with
a certain length threshold bind membranes with high affinity and form helical structures.8

2.1.2 | Amino acid composition

Despite considerable structural variation, AMPs mainly include two types of amino acid residues: cationic and
hydrophobic residues.9 In naturally occurring AMPs, the cationic resides are Arg, Lys, and His,10 and the
hydrophobic resides are mainly aliphatic and aromatic amino acids. In addition, Cys and Pro residues are conserved
in natural AMPs. It is generally accepted that positively charged residues of AMPs directly interact with the
negatively charged components of bacterial cells. Then, the hydrophobic residues get incorporated into lipid
bilayers to mediate membrane permeabilization and disruption, which lead to rapid cell death.

2.1.3 | AMPs rich in specific amino acids

Several approaches have been applied to AMPs to improve their bactericidal activity and reduce their hemolytic
activity, including modifications to the sequence, cyclization, and synthesis of multivalent constructs. A number of
amino acids have been selected to allow for the expansion of the pharmacological applications of AMPs; some
amino acids have special functions.

Proline‐rich peptides
In general, Pro amino acids form exceptional structures, which is a vital parameter for increasing the therapeutic
index of peptides.11 Pro‐rich AMPs can penetrate the bacterial cytosol through outer membrane protein
channels, which can modulate the immune system via cytokine activity or angiogenesis.12 Therefore, Pro‐rich
peptides are considered to be a potential type of cell‐penetrating peptides with the capability of internaliz-
ing membranes.

Cysteine‐rich peptides
One important role of Cys residues is to form disulfide bonds to stabilize β‐hairpin or sheet structures.13 The
presence of disulfide bridges is a prerequisite for pore formation in a membrane, and subsequent studies have
confirmed that antimicrobial activity is highly associated with the stability of β‐hairpin conformations, whereas the
removal of the disulfide bridges causes marked reductions in antimicrobial activity.14

Glycine‐rich peptides
The rational use of a small amino acid, such as Gly, can improve the activities of AMPs.15 Additionally, Gly‐rich
peptides have increased selectivity while retaining high antimicrobial activity.16 In addition, Gly‐rich peptides
usually potently destroy fungi, Gram‐negative bacteria, and cancer cells.17,18

Aromatic amino acid‐rich peptides

Trp residues are usually chosen for their tendency to form indole rings, and pairwise Trp‐Trp interactions lead to a
distinctive cross‐strand contact and stable tertiary structure.19,20 AMPs containing Trp penetrate a microbial cell
membrane and efficiently disrupt it.21–23 Many studies have shown that Trp‐rich peptide analogs are effective
against several isolated antibiotic‐resistant bacteria.24,25 Phe is usually chosen for its high hydrophobicity, and
Phe‐rich AMPs exhibit potent antimicrobial activity against Gram‐positive and negative bacteria as well as yeast
without hemolytic activity.26–29
834 | WANG ET AL.

2.2 | Characteristics of secondary structures

2.2.1 | α‐Helixes
According to the updated database (APD: http://aps.unmc.edu/AP/main.php), the proportion of natural α‐
helical peptides is the highest among AMPs (Figure 2),9 and they are mainly derived from different species,
including insects, fish, amphibians, mammalians, and plants. Moreover, many studies have shown that most of
these peptides exhibiting membrane‐like properties or interacting with membranes are converted to α‐helical
structures. This transformation induces the segregation of hydrophilic/charged amino acids in space from the
hydrophobic residues, which results in an amphipathic structure that is recognized as a prerequisite for AMPs
to act on membranes.9,31 Electrostatic interactions between the positively charged residues of AMPs and
negatively charged components of bacterial membranes have been widely reported to be the primary
antimicrobial mechanism of most AMPs, including melittin, magainin, and cathelicidins, and the nonpolar face
of those peptides can penetrate further into the membrane.34–37
In the porcine cathelicidin family, PMAP‐36 has received a great deal of attention. Sequence analysis
showed that PMAP‐36 has the highest proportion of cationic amino acids (36%), which are most frequent at the
N‐terminus.38 Structural analyses of PMAP‐36 further demonstrate that this highly cationic sequence adopts a
typical amphipathic α‐helical conformation and random hydrophobic tail.39 Recent studies have indicated that
the N‐terminal region (α‐helical domain) of PMAP‐36 is the active region.40–42 However, perfect
amphipathicity often results in increased cytotoxicity.9 To investigate the relationship between the structure
and bio‐activities of amphipathic α‐helical AMPs, a series of short α‐helical AMPs were designed with imperfect
amphipathicity by replacing the paired Lys residues on the polar face with Trp along the N‐terminal
amphipathic region of PMAP‐36. The researchers found that the disruption of amphipathicity increased
antimicrobial activity with very little hemolysis.30
Many studies have focused on the de novo design of short α‐helical peptides with potent activity.43–47
According to the helical‐wheel projection, Ma et al48,49 designed a small combinatorial library of Val/Arg‐rich
peptides, and peptide G6 was found to have optimal cell selectivity. G6 has been further demonstrated to
reduce peritoneal bacterial counts and increase survival after Salmonella typhimurium infection.50 Further

F I G U R E 2 Common structural classes of antimicrobial peptides (AMPs): A, α‐helix; B, β‐sheet; and C, extended
structures. Reproduced with permission from Takahashi et al,9 Copyright (2010) Elsevier Masson SAS. Helical‐wheel
projections of the peptides: D, PMAP‐36. By default, the output presents the hydrophilic residues as circles,
hydrophobic residues as diamonds, potentially negatively charged as triangles, and potentially positively charged as
pentagons. Hydrophobicity is color coded as well: the most hydrophobic residue is green, and the amount of green
decreases proportionally to the hydrophobicity, with zero hydrophobicity coded as yellow. Hydrophilic residues are
coded red with pure red being the most hydrophilic (uncharged) residue, and the amount of red decreases
proportionally to the hydrophilicity. The potentially charged residues are light blue. Reproduced with permission from
Zhu et al,30 Copyright (2014) Elsevier Ltd [Color figure can be viewed at wileyonlinelibrary.com]
WANG ET AL. | 835

studies were conducted to assess the effect of net charge and Pro on the activity of Val/Arg‐rich peptides. It
was found that an appropriate increase in net charge improved antimicrobial activity and decreased
hemolysis, and the substitution of Gly with Pro significantly reduced toxicity.51 Sources of some AMPs along
with their chemical and biochemical properties are summarized in Table 1.

2.2.2 | β‐Sheets
Besides α‐helixes, β‐sheets are another principal secondary structure of AMPs that can be induced by a membrane‐
mimetic environment. Moreover, in an aqueous solution, most β‐sheet peptides change their conformation from
unstructured ones to β‐sheet structures52,53 in a membrane‐mimetic environment. Generally, β‐sheet peptides
consist of 2 to 10 Cys residues that form 1 to 5 disulfide bridges as conformational constraints for stabilizing their
bioactive conformation. Additionally, peptides that contain disulfide bonds often adopt a cyclic β‐hairpin
conformation, such as tachyplesin and protegrin 1 (PG‐1).14,54,55
Many studies have reported that most natural peptides with potent antimicrobial activity exhibit a β‐sheet
structure, such as horseshoe crab polyphemusin‐1 and pig PG‐1.56 Linked by two disulfide bridges, they adopt an
antiparallel β‐sheet structure and robust β-hairpin conformation. The turn region of PG‐1 contains three positively
charged Arg residues, and this is considered to be the active center of PG‐1.57 The majority of natural β-sheet
AMPs play important roles in the innate immune system, and are categorized as defensins that can kill invading
pathogens or regulate immune responses. Defensins are well‐known for their characteristic six to eight Cys
residues in defined positions that are evolutionarily conserved across most multicellular organisms. Additionally,
the disulfide bridge between conserved Cys residues is a crucial structure stabilizer for defensins, although these
covalent bonds are not directly associated with antimicrobial activity.
Peptides that typically adopt α‐helix and β‐sheet structures have been designed to improve the prospects for
applying AMPs therapeutically and the relationship between their structures and bio‐activities has been
investigated.43,45,58 The β‐sheet structure possesses similar antimicrobial activities to and greater cell selectivity
than that of α‐helical peptides with equal hydrophobicity and charge.59 However, it is difficult for short peptides to
form robust β‐structures, so systematic studies of β‐sheet peptides are somewhat under‐represented. Recently, the
simplification and repeated‐stack segments of amino acids have been shown to be an effective strategy for the
design or optimization of AMPs. Several rational designs of synthetic β‐sheet folding peptide amphiphiles with
broad‐spectrum and highly selective antimicrobial activities have been reported.57,58,60 Therefore, the protein
folding theory and the common features of natural AMPs provide a basis for the design of β‐sheet AMPs, including:
(1) the intrinsic β‐sheet propensities of amino acids in strands and cross‐strand interactions across strands; (2) a net
positive charge mediating peptide interactions with negatively charged membranes of bacteria; (3) hydrophobic

T A B L E 1 Classification of antimicrobial peptides (AMPs)

Classes Representatives Sequences Hosts

α‐Helix Melittin GIGAVLKVLTTGLPALISWIKRKRQQ Honey bee
Magainin‐1 GIGKFLHSAGKFGKAFVGEIMKS Frog
LL‐37 LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES Human
β‐Sheet HNP‐1 AC[1]YC[2]RIPAC[3]IAGGRRYGTC[2]IYGGRKWAFC[3]C[1] Human
HBD‐1 DHYNC[1]VSSGGQC[2]LYSAC[3]PIFTKIQGTC[2]YRGKAKC[1]C[3]K Human
Protegrin 1 RGGRLC[1]YC[2]RRRFC[2]VC[1]VGR Pig
Extended Indolicidin ILPWKWPWWPWRR Cow
Tritrpticin VRRFPWWWPFLRR Pig
PR‐39 RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPPRFPPRFP Pig

Abbreviations: HBD‐1, human α‐defensin 1; HNP‐1, human α‐defensin 1.

[1], [2], and [3] are disulfide bonds intramolecularly formed by Cys residues in one peptide.
836 | WANG ET AL.

residues providing lipophilic anchors and ultimately inducing membrane disruption, and (4) the reasonable
arrangement of amino acid residues to form a structure with amphipathic characteristics, which segregates cationic
and hydrophobic residues to opposite faces of the folded molecule.52,60 Based on previous reports, the rational
design of short synthetic β‐hairpin AMPs consisting of VR recurring Ac‐C(VR)nDPG (RV)nC‐NH2 (n = 1, 2, 3, 4, or 5)
aimed at investigating the potency of the designed peptides against pathogenic bacteria and the relationship
between strand length and the activity of β‐hairpin peptides. It was found that longer peptides exhibited
significantly greater toxicity to mammalian cells. Notably, the antimicrobial activity of the peptide initially increased
and then decreased as the chain length was increased. Furthermore, a synthetic β‐hairpin peptide effectively
provided resistance to challenge by a bacterial infection with a lethal dose of a S. typhimurium strain.52
Subsequently, a series of symmetric‐end β‐sheet peptides showed the effect of the type of amino acid on AMP
activity. Different subtypes of hydrophobic amino acids significantly influenced the secondary structure and
antimicrobial activity of synthetic AMPs. Peptides that contained the aliphatic residue Ile were more likely to form
a β‐sheet conformation and exhibit greater selectivity for microbial membranes.57

2.2.3 | Extended structures

Most of the extended peptides with a high proportion of Pro and Gly residues usually exhibit a linear structure,
rather than typical secondary structures. Therefore, extended peptides can be divided into Pro‐ and Gly‐rich
peptides.61–63 Pro‐rich extended peptides are isolated from mammals, such as indolicidin and tritrpticin, and from
insects such as apidaecins.12,64–67 The size of the Pro‐rich peptides varies from 15 to 39 residues. The Pro residue is
often associated in doublets and triplets with basic residues (Arg and Lys). Generally, most short‐chain Pro‐rich
peptides exhibit potent activity against Gram‐negative bacteria, while maintaining low antimicrobial activity against
Gram‐positive bacteria, which are suggested to contain extracellular proteases that can degrade Pro‐rich
peptides.12 Several antimicrobial Gly‐rich peptides have been isolated from various insect species. The size of
Gly‐rich peptides varies from 8 kDa (holotricin) to 30 kDa (sarcotoxin II).68,69

3 | MAJOR P ARAMETE RS THAT D ETERMINE THE ACT IVIT Y

OF ANTIMICROBIAL P EPTIDES

3.1 | Charge
The net positive charge is one of the main characteristics of AMPs. The importance of a positive charge and its
impact on antimicrobial potency are evident. Most cationic amphipathic AMPs present a net positive charge
ranging from +2 to +9 because the interaction between AMPs and cell membranes mainly relies on electrostatic
attraction.70 It is generally assumed that cationic AMPs initially interact with negatively charged lipid head groups
on the outer surface of the cytoplasmic membrane. The peptide then penetrates the outer leaflet of the cytoplasmic
membrane lipid bilayer in an approximately parallel orientation to the bilayer, which leads to the displacement of
lipids.30 The enhancement of the total positive charge often results in increased affinity for the microbial
membrane and greater antimicrobial activity.
Nevertheless, an excessive charge can have a negative effect on activity, and a highly cationic peptide may even
be devoid of antimicrobial activity. As a cationic peptide continuously increases the number of positive charges,
antimicrobial activity may no longer increase.71,72
For negative charges, the introduction of negative charges generally results in decreased antibacterial activity
of AMPs. However, there are some anionic AMPs (rich in Gln and Asp) that have been shown to participate in the
eukaryotic innate immune response.73 These peptides have net charges ranging from −1 to −2, and they generally
require cations, for example Zn2+, as cofactors for biocidal activity.74
WANG ET AL. | 837

3.2 | Hydrophobicity
Hydrophobicity is another important parameter that can define antimicrobial potency and cell selectivity. AMPs
with high hydrophobicity can damage the membrane structure, which results in cell lysis or the formation of
transient pores and the transport of peptides inside the cell; this property enables them to interact with
intracellular targets.75
Studies have shown that hydrophobicity is strongly correlated with antimicrobial and hemolytic activities.76 At
a relatively lower level of hydrophobicity, increasing the hydrophobicity of the amphipathic α‐helical AMPs
improves antimicrobial activity until an optimal hydrophobicity threshold is reached. A reasonable explanation for
this finding is that the self‐association of peptides prevents a peptide from passing through the cell wall.77
However, higher hydrophobicity has also been correlated with greater hemolytic activity. In red blood cell
membranes, increased hydrophobicity induces peptides to penetrate deeper into the hydrophobic core.78

3.3 | Amphipathicity
Amphipathicity that results from the segregation of hydrophobic and polar residues on the opposite face of the
molecular framework has been recognized as the most important physicochemical and structural parameter for the
activity of AMPs. This is especially so for the ability of an AMP to form an α‐helix as without this arrangement
broad‐spectrum antimicrobial activity is not observed (Figure 2D). Many reports have shown that membrane lytic
peptides tend to cluster in regions of high hydrophobicity,79,80 which has been used as a preferred strategy to
design synthetic peptides. However, other studies suggest instead that perfect amphipathicity often results in a
simultaneous increase in both bactericidal activity and cytotoxicity.81 But most of these studies focused on the
effect of perfect/imperfect amphipathicity via an amino acid substitution approach and did not sufficiently consider
whether such a modification in a peptide sequence generally alters more than one structural characteristic that
might modulate antimicrobial activity. Therefore, it would be impossible to only assign an observed effect to
changes in perfect/imperfect amphipathicity. To address this issue, Wang et al used a minimal sequence
modification approach that allowed for the modification of one structural characteristic, while other characteristics
were kept largely constant. They found that imperfectly amphiphilic peptides showed better antimicrobial activity
than the corresponding perfectly amphipathic peptides; however, the toxicity of the engineered peptides was
independent of the type of amphipathicity, which was a greatly different result from those of previous studies.82

4 | FUNC T I O NS O F A N TI M I CRO BI A L P EP T I D ES

4.1 | Antibacterial activity

Antibacterial activity is one of the most basic functions of AMPs. Many peptides show activity against Gram‐
positive and ‐negative bacteria, such as melittin,83 magainins,84 cecropins,85 and LL‐37.86 Despite these differences
in membrane composition, phospholipids, the lipid A core of lipopolysaccharides (LPS) of Gram‐negative bacteria,
and the teichoic and teichuronic acids of Gram‐positive bacteria are anionic, which support electrostatic
interactions with cationic AMPs. A net positive charge and hydrophobicity are essential for peptides to act as
antibacterial agents. As mentioned above, a net positive charge enhances interactions with anionic bacterial targets
and hydrophobicity promotes the insertion of peptides into bacterial membranes. Additionally, flexibility has a
great influence on the antibacterial activity of AMPs as it permits peptides to transform conformations from an
aqueous solution to a membrane‐mimicking environment. In response to environment stimuli, changes in the
peptide secondary structure can cause more efficient membrane leakage.87
838 | WANG ET AL.

4.2 | Antifungal activity

To date, more than 70 000 fungi have been found and 399 of them are known to threaten human health. Immune‐
compromised individuals undergoing chemotherapy and organ transplant recipients taking immune inhibitors are
extremely susceptible to fungal infections. While there are drugs that are active against fungi, they act on few
specific molecular targets and drug‐resistant strains can lead to reduced efficacy of these drugs in certain cases.88
Recently, the number of studies on antifungal peptides has increased.89 PAF26 is a synthetic de novo designed
tryptophan‐rich peptide, and PAF26 and PAF26 derivatives have been tested against many kinds of fungi including
the model fungus Neurospora crassa90 and several dermatophytes.91 PAF26 exerts antifungal activity via energy‐
dependent endocytic internalization before killing fungal cells. Astacidin 1, a novel antibacterial peptide, was
isolated from hemocyanin of the freshwater crayfish Pacifastacus leniusculus and exhibited antifungal activity
against Candida albicans, Trichosporon beigelii, Malassezia furfur and Trichophyton rubrum.92 Moreover, 14‐helical
hydrophobic β‐peptides are able to prevent the formation of C. albicans , Candida glabrata , Candida parapsilosis , and
Candida tropicalis biofilms.93
Generally, membrane permeabilization is the first process whereby most antifungal peptides kill fungi.94
Subsequently, some antifungal peptides can also interact with C. albicans genomic DNA and significantly induce cell
cycle arrest during the S‐phase.95 Additionally, the induction of apoptosis in yeast via ROS production is also an
important mechanism for some antifungal peptides.96 Moreover, some peptides bind to a receptor on the fungal
cell membrane, interfere with cell wall synthesis or the biosynthesis of essential cellular components, and even
enter the cytoplasm where they cause mitochondrial depletion, eventually killing the fungi.92
Peptides that primarily exhibit antifungal activity, such as many of those isolated from plants, tend to be
relatively rich in polar and neutral amino acids, suggesting a unique structure‐activity relationship.97 Evidence
suggests that no conserved sequences are apparent for antifungal peptides.97

4.3 | Antiviral activity

Many AMPs have shown the ability to inhibit a wide spectrum of virus infections, including those caused by
enveloped RNA and DNA viruses, feline calicivirus, and echovirus.98–101 Some α‐helical peptides, such magainins,
dermaseptin, and melittin, have shown potent anti‐HSV activity.102–104 Additionally, some β‐sheet peptides, such as
defensins, tachyplesin, protegrins and the β‐turn peptide lactoferricin, have all shown high activity against
HSV.104–109

4.4 | Antitumor activity

Currently, cancer is the third leading cause of human death. Therefore, the development of novel antitumor drugs is
needed. Fortunately, some cationic AMPs exhibit a broad spectrum of cytotoxic activity against tumor cells, which
could provide a new class of anticancer drugs. Notably, AMPs present significant advantages over traditional
antitumor agents, such as higher specificity and circumvention of drug resistance because they act via a distinct
mechanism.110,111 Most tumor cells carry a net negative charge on the membrane as a consequence of the
overexpression of various anionic molecules, such as phosphatidylserine.112 AMPs mainly act on target cell
membranes via a non‐receptor–mediated pathway, for which it is more difficult for tumor cells to develop
resistance compared to conventional chemotherapeutic agents.113,114 For example, the antitumor mechanism of B1
and its analogs involves three steps: cell membrane disruption resulting from changes in membrane permeability;
penetration of the cytoplasm after membrane disruption; disruption of mitochondrial membranes and release of
cytochrome C.115 In another case, the high cell selectivity of peptide (G(IIKK)3I‐NH2) could stem from its
preferential binding to outer cell membranes, and it can also induce the programmed cell death of tumor cells via
both the mitochondrial and death receptor pathways without inducing nonspecific immunogenic responses.116
WANG ET AL. | 839

Moreover, many cell‐penetrating peptides have been synthesized for showing improved tumor‐targeting ability. For
example, several tumor‐homing cell‐penetrating peptides (HP‐CPPs) have been successfully developed by means of
conjugating HPs with CPPs,117 and several tumor‐targeting drug delivery systems have been established by
conjugating epidermal growth factor receptor family targeting domains with nanoparticles or CPPs.118
AMPs with ideal antitumor activity should meet at least three conditions: (1) high net positive charge. The
existence of the high net positive charge of AMPs contributes to electrostatic attraction between the negatively
charged components (such as phosphatidylserine) of tumor cells and the positively charged AMPs.119 (2) High
structural flexibility. The flexibly of AMPs allows for changes in conformation in different environments (aqueous
and membrane‐mimic environments), which allows them to traverse the phospholipid layer of tumor cells. (3) High
oligomerization. AMPs should be easily clustered on the membrane surface of a tumor cell so that they can form a
pore on the tumor cell membrane.120

4.5 | Antiparasitic activity

Protozoan parasites cause millions of deaths annually worldwide. More than three billion residents in tropical
regions are threatened by malaria infection. AMPs that show good activity against parasites include melittin,
cecropin, magainin, bovine myeloid antimicrobial peptide 18 (BMAP‐18), BMAP‐27, and PG‐1, and the parasitic
resistance activity of some AMPs does not depend upon the developmental stage of the parasite.121 In addition,
ParaPep is a repository of antiparasitic peptides; most of the ParaPep peptides have been evaluated for growth
inhibition activity against various species of Plasmodium, Leishmania, and Trypanosoma.122

4.6 | Immunomodulatory activity

Many AMPs show strong antibacterial activity in vitro under the appropriate conditions, while their activity is often
inhibited at modest concentrations of peptides in the presence of physiological concentrations of monovalent and
divalent cations, serum and anionic macromolecules, such as glycosaminoglycans, in vivo.123 In vivo, AMPs can
selectively increase or regulate host immune mechanism to promote resistance of microbial infection rather than
direct killing of bacteria.123 More recently, it has become evident that AMPs have a diverse range of functions in
modulating immunity.124 The immunomodulatory properties of AMPs include reduction in the levels of
proinflammatory cytokines produced in response to microbial signature molecules; modulation of the expression
of chemokines; stimulation of angiogenesis; enhanced wound healing; and macrophage and leukocyte
differentiation.125,126 The functions of AMPs in immune modulation are presented in Figure 3.32,33 In addition,
some AMPs exert immune activities by binding protein molecules. For example, muramyl dipeptides MDP and
GMDP are potent adjuvants and immune stimulators released from the cell wall of invading bacteria that can exert
immune activity by specifically binding YB‐1 (Y‐box protein 1), a member of a specialized class of proteins that can
cycle between the nucleus, cytosol, membrane, and extracellular milieu.127 Moreover, LFP‐20 attenuated the LPS‐
induced release of proinflammatory factors that were likely associated with the MyD88/nuclear factor‐κB (NF‐κB)
and MyD88/mitogen‐activated protein kinase (MAPK) signaling pathways.128

5 | ANT I MI C R O B I A L M E C H A N I S M O F A N T I M I C R O B I A L PEP T I D ES

The antimicrobial mechanism of AMPs is extremely complex. It is difficult to characterize the mode of action of
each AMP, and one AMP may act on multiple targets to protect against pathogens.129 It is widely accepted that the
main antimicrobial mechanism of AMPs against pathogens involves dissipation of the electrochemical potential,
induction of lipid asymmetry, and loss of important metabolites and cellular components, which ultimately leads to
cell shrinkage and cell death (Figure 4). Many studies have provided clear evidence about the antimicrobial
840 | WANG ET AL.

F I G U R E 3 Overview of the immunomodulatory properties of antimicrobial peptides (AMPs). (1) anti‐endotoxin

activity; (2) vaccine adjuvant; (3) mast cell degranulation; (4) wound repair; (5) induce immune cell differentiation;
(6) inhibit proinflammatory mediators such as tumor necrosis factor (TNF‐α); (7) Activate and recruit macrophages,
monocytes, dendritic cells, and T cells; (8) Promote angiogenesis; (9) Neutralize proinflammatory cytokines
released from macrophages and monocytes. Refer the Figure 2 of Afacan et al32 and the Figure 2 of Lai and Gallo33
[Color figure can be viewed at wileyonlinelibrary.com]

F I G U R E 4 The interactions between antimicrobial peptides and bacterial membranes [Color figure can be
viewed at wileyonlinelibrary.com]
WANG ET AL. | 841

mechanism of the given AMPs, for which increased membrane permeability may not be sufficient alone to cause
cell death.56 In addition to membrane permeabilization, AMPs cause cell death by targeting not only intracellular
contents for inhibiting transcriptional130,131 translational132 or other processes,133,134 but also precursors and/or
essential intermediates in peptidoglycan, LPS or other biosynthetic pathways for interfering with the functional
synthesis of the cell wall and impairing subsequent bacterial replication.135

5.1 | Peptide‐membrane interactions

5.1.1 | Adsorption and binding to membranes
AMPs are thought to be unstructured in an aqueous environment and fold into their final amphipathic
conformation upon interaction with an anionic membrane environment.30,136 These AMPs must be attracted to the
pathogen cell wall by electrostatic interactions between the positive charge of a peptide and the anionic component
of a membrane.137,138 Some studies have argued that the composition and structure of Gram‐negative and ‐positive
cell walls is the basis for the susceptibility of different pathogens to AMPs, such as anionic phospholipids, the lipid A
core of LPS of Gram‐negative bacteria and the teichoic and teichuronic acids of Gram‐positive bacteria.139 Once
bound to a microbial surface, peptides cross capsular polysaccharides and other components of the cell wall before
they interact with the cytoplasmic membrane in Gram‐positive bacteria.140,141 For Gram‐negative bacteria, the
initial action of peptides involves the competitive displacement of LPS‐associated divalent cations, for example
Mg2+ and Ca2+. In this way, peptides destabilize this supramolecular assembly and gain access to both outer and
inner membranes.30

5.1.2 | Threshold concentration and conformational transition

Peptides exert their antimicrobial activity when they reach a certain concentration at the target surface that is
described as the minimum threshold concentration.51 Below this threshold concentration, peptides tend to interact
and concentrate at the level of the lipid headgroup, fold simultaneously, and remain adsorbed parallel to the lipid
bilayer.56 As the concentration increases, the peptides begin to orient themselves perpendicular to the membrane,
insert and partition into the hydrophobic core of the bilayer.34,46 Above the threshold concentration, peptides
promote alterations in conformation, both in‐depth membrane localization and association state, as well as indirect
changes in bilayer topology, for example, pore formation or disintegration.129
Once bound to the membrane, AMPs can change their conformations, which is considered to be one of the most
important actions of AMPs. Subsequently, AMPs alter the conformation observed at the water‐lipid interface.
Although the precise conformational change mechanism is the subject of debate, fundamental thermodynamics
now appear to contribute to such conformational changes, especially for α‐helical AMPs. It has been reported that
the interaction between peptides and the lipid membrane consists of four thermodynamic steps: partitioning,
folding, insertion, and association.142 After the electrostatic adsorption and correct orientation according to the
plane of binding, the key stage of this process is the partitioning action of the membrane and the conformational
transition. It has been shown that the partitioning action to membranes could be described by a partitioning‐folding
coupling process, as the formation of a secondary structure makes the partition of a structured peptide less
energetically costly. Some studies have proposed that the formation of peptides bonds upon folding to a structured
conformation reduced the Gibbs energy for peptide partitioning, which contributes to the partitioning pathway.143
It has been shown that α‐helix formation is driven by negative enthalpy and opposed by entropy based on
calorimetric measurements and noncalorimetric estimates.143 The enthalpy of helix formation has been found to
be largely independent of the peptide sequence and can be mainly attributed to the formation of intramolecular
CO–NH hydrogen bonds.144 When peptides insert into the hydrophobic core of a membrane, the peptides shield
bonding into intramolecular H‐bonds in secondary structures, which is another contribution to the overall
reduction in the Gibbs energy.145 Besides reductions in the Gibbs energy, there are other contributions, such as
842 | WANG ET AL.

side‐chain packing, the effect of folding/assembly entropy, relative exposure of side chains to membranes and
water and the depth of membrane penetration of secondary structure units. By contrast, β‐sheet AMPs are
typically much more ordered in aqueous solution and membrane environments because of the constraints imposed
by disulfide bonds and the intrinsic rigid structure of this type of secondary structure as compared to α‐helixes.57

5.1.3 | Peptide insertion and membrane permeability

Following the initial attachment, peptides insert into the bacterial membrane to form transmembrane pores (membrane
permeabilization and lysis).146 There are several widely accepted models accounting for the antimicrobial mechanism of
AMPs, including the barrel‐stave model, carpet model and toroidal model.9 In brief: (1) in the toroidal model, the
hydrophilic portion of the amphipathic conformation of peptides is associated with the lipid headgroup; (2) in the barrel‐
stave model, the peptides penetrate the membrane and form pores in the hydrophilic portion; and (3) in the carpet
model, peptides disrupt the membrane structure by a detergent‐like action.

5.2 | Targeting the intracellular receptors

In some cases, AMPs may kill bacteria by other antimicrobial mechanisms rather than by inducing membrane
damage (Figure 5).98 Inhibition of cellular nucleic acids by cationic AMPs is possible because of the polyanionic

F I G U R E 5 The mechanism of antimicrobial peptides (AMPs) targeting the intracellular receptor.

A, Dermaseptin, buforin‐II and pleurocidin have been shown to inhibit both DNA and RNA synthesis.147 B, PR‐39
and indolicidin have been shown to inhibit the rate of protein synthesis.129 C, Pyrrhocoricin and drosocin have
been shown to reduce enzymatic activity via inhibition of ATPase activity of the heat‐shock protein DnaK, an
enzyme involved in chaperone‐assisted protein folding.148 D, AMPs may also inhibit resistance mechanisms linked
to bacterial pathogenesis, for example, enzymes with anionic binding site pockets linked to the modification of
aminoglycoside antibiotics.149 E, Lantibiotics such as mersacidin and nisin target the formation of structural
components of the cell wall, specifically the transglycosylation of lipid II, necessary for the synthesis of
peptidoglycan.150 Adapted with permission from Jenssen et al98 Copyright (2006) American Society for
Microbiology [Color figure can be viewed at wileyonlinelibrary.com]
WANG ET AL. | 843

charge present in nucleic acids and some intracellular enzymes.71 Some AMPs have been shown to penetrate
bacterial cell membranes rendering them relatively undamaged but with antimicrobial activity by means of
targeting intracellular receptors.151 For example, in mammals, AMPs LL‐37 and PR‐39 have multiple membrane and
intracellular mechanisms, but they also mitigate the immune response to foreign pathogens.152 Indeed,
dermaseptin, buforin‐II, and pleurocidin have been shown to inhibit both DNA and RNA synthesis.147 Pyrrhocoricin
and drosocin can bind the bacterial chaperone DnaK, an enzyme involved in chaperone‐assisted protein folding, and
prevent its multi‐helical lid from functioning.148 Lantibiotics, such as mersacidin and nisin, can form a tight complex
with lipid II and block the transglycosylation of its substrate, thereby inhibiting peptidoglycan synthesis.150

6 | REGULATING THE EXPRESSIO N O F M A M M A L I A N A N T I M I C R O B I A L

PE PTIDES

The immune system provides protection against a wide variety of pathogens, and it consists of innate and adaptive
immune responses. The innate immune system is phylogenetically ancient and confers broad protection against
pathogens without previous exposure; most multicellular organisms depend upon it to combat microbial
infections.153 The innate immune system utilizes TLRs to recognize and bind pathogen‐associated molecular
patterns (PAMPs). In studies of the regulation of the expression of mammalian AMPs, PAMPs have been shown to
play a key role and act via various pathways.

6.1 | NF‐κB signaling pathway

TLRs recognize distinct PAMPs, such as LPS of Gram‐negative bacteria. After ligand binding, the intracellular
adaptor molecule MyD88 is recruited to the receptor complex and then MyD88‐dependent or ‐independent signal
transduction pathways are activated. TAK1 is activated, which in turn activates the IκB kinase (IKK) complex and
then activates the nuclear localization domain of NF‐κB, which ultimately induces the expression of AMPs. For
example, TLR3 is involved in increased hBD‐2 and hBD‐3 expression in mammalian bronchial epithelial cells in the
common cold caused by rhinovirus‐16 (RV16), an inflammatory virus.154

6.2 | MAPK signaling pathway

The MAPK pathway transduces a large variety of external signals, leading to a wide range of cellular responses,
including growth, differentiation, inflammation, and apoptosis. In mammals, three major MAPK pathways have been
identified: MAPK/ERK, MAPK/JNK, and MAPK/p38.155 Similar to the activation of the NF‐κB pathway, TAK1
activates the MAPK pathways, also leading to the expression of multiple proinflammatory genes and the induction
of AMP expression. In human intestinal cells, TLR4 dependent and TLR2 dependent increased hBD‐2 expression
occur in response to LPS, while PGN156 expression is mediated by the MAPK/JNK pathway. However, increased
hBD‐2 expression by Salmonella enteritidis flagellin (FliC) (a TLR5 ligand) together with gangliosides on intestinal
cells leads to the activation of the MAPK/p38 and MAPK/ERK pathways.157

6.3 | Vitamin D receptor signaling pathway

Circulating monocytes are activated by TLR2/1 agonists present on specific microbes. The genes encoding vitamin
D receptor (VDR) and CYP27B1 are induced. CYP27B1 induces the production of 1ɑ, 25‐dihydroxyvitamin D3
(1,25‐D3). The combination of 1,25‐D3 with the VDR leads to increased expression of AMPs. For example,
expression of the cathelicidin AMP (CAMP) gene is strongly induced in macrophages and epithelia by VDR and its
ligand 1,25‐D3 in humans and primates, but not in other mammals.158 Unlike other AMP genes that are directly
844 | WANG ET AL.

induced during infection and inflammation by NF‐κB, the CAMP gene is induced by the VDR and its ligand 1,25 ‐ D3,
which are upregulated via TLR‐signaling.

7 | AMP M IMIC S

AMPs represent the most promising alternatives to antibiotics, but some disadvantages still limit the practical
application of AMPs, such as high cytotoxicity, low resistance to proteolytic degradation resulting in short half‐lives,
and high production cost. To solve these problems, peptide mimics (peptidomimetics) have been designed to mimic
the amphiphilic structure, function, and mode of action of AMPs but whose backbone is not based on the regular
amino acid linked in a chain.147,159 Compared to AMPs, peptidomimetics often exhibit high bioavailability and long
half‐lives in vivo, while maintaining a similar function activity and selectivity. Furthermore, most peptidomimetics also
kill various bacteria through mechanisms involving membrane disruption, and some peptidomimetics can neutralize
the proinflammatory activities of LPS and lipoteichoic acid,160 while inducing the production of the chemokines
interleukin 8, monocyte chemotactic protein 1 (MCP‐1), and MCP‐3,6 thereby boosting the innate immune response,
specifically the recruitment of immune cells such as neutrophils required for the resolution of infections.161 To date,
many approaches have been used to explore the potency of peptidomimetics—from the use of unnatural amino acid
residues to alternative backbone structures, mainly including N‐substituted glycines (peptoids), β‐peptides, peptide‐
peptoid hybrids, ceragenin‐based mimetics, α/γ N‐acylated N‐aminoethyl peptides (AA peptides) and oligoacylly-
sines.162 Among these strategies, peptoids are particularly well suited to mimic AMPs because of the convenient
synthesis strategy and relatively low synthesis cost. Peptoids are poly‐N‐substituted glycines in which side chains are
attached to the backbone Nα‐amide nitrogen instead of the Cα‐atom,163 making them protease‐resistant. Peptoids
often preclude both backbone chirality and intrachain hydrogen bonding; however, they can be induced to form
stable polyproline type‐I‐like helices by the incorporation of bulky, α‐chiral side chains.164 Based on the trimer repeat
(X‐Y‐Z)n, Chongsiriwatana et al164 designed 13 amphiphilic helical peptoids derived from H‐(NLys‐Nspe‐Nspe)4‐NH2
and H‐(NLys‐Nssb‐Nspe)4‐NH2 to study structure‐activity relationships. They observed that highly charged (≥3),
moderately hydrophobic, and helical peptoids exhibit high cell selectivity. Checkerboard assays demonstrated that
peptoids and AMPs can interact synergistically, with fractional inhibitory concentration indices as low as 0.16.165
Czyzewski et al also established a new QSAR model based on 27 diverse peptoids, which accurately correlates
antimicrobial peptoid structures with antimicrobial activity. They found that the peptoid 1 exhibited optimal cell
selectivity against a range of Gram‐positive and negative strains including superbugs. In a murine model with invasive
S. aureus challenge, peptoid 1 significantly reduced colony forming units and mortality compared to the control group
without causing any medium‐term toxicity at 4 mg/kg, demonstrating the promising therapeutic potential of peptoid 1
as an antimicrobial agent.166
The hybrid structure of α‐amino acids and peptoids has also received wide attention for improving the cell
selectivity of peptidomimetics.167 By integrating chemometrics/cheminformatics techniques and in vitro susceptibility
assays, a combinatorial library based on 18 natural amino acids and peptoid residues was established, and two valid 8‐
mer peptide‐peptoid hybrids (IK‐Nssb‐NLys‐VRK‐Nssb‐NH2 and NLys‐L‐NLys‐W‐Nsmb‐IKRW‐NH2) with high
antibacterial activity against S. aureus and Pseudomonas aeruginosa were successfully screened out. Molecular
dynamics simulations revealed that IK‐Nssb‐NLys‐VRK‐Nssb‐NH2 can form a typical amphipathic helix that is
beneficial to membrane disruption, while another is unstructured in aqueous environments.168 Recently, a lysine‐
based α‐peptide/β‐peptoid hybrid LBP‐3 with potent activity against P. aeruginosa was designed and encapsulated into
a nanogel to improve its cell selectivity. Nanogel formulation F10 (size, 175 nm; ZP, −16 mV) was identified as the
most promising formulation with a high degree (88%) of LPB‐3 encapsulation and demonstrated a 3‐fold reduction in
cytotoxicity towards hepatocytes along with improved bacterial killing kinetics.169 Aminoglycoside antibiotic (eg,
tobramycin)‐peptoid conjugates as a novel hybrid structure have also been recently reported.170 Tobramycin was
conjugated with the lysine‐based peptoid at the C‐5 position with various alkyl tethers. The tobramycin‐lysine hybrid
WANG ET AL. | 845

3 with aliphatic C12 chain showed better activity against Gram‐positive bacteria than Gram‐negative bacteria, and it
exhibited lower hemolytic activities (<20%) than the lysine‐based peptoid (87%) at 512 μg/ml and showed no
cytotoxicity to human epithelial prostate (DU145) and breast (JIMT‐1) cancer cell lines at 25.2 μg/ml. Furthermore,
compound 3 displayed strong synergistic interactions with minocycline and rifampicin against MDR and extensively
drug‐resistant (XDR) P. aeruginosa isolated and enhanced the efficacy of both antibiotics in the Galleria mellonella
larvae in vivo infection model. Mode of action studies indicated that amphiphilic tobramycin‐lysine adjuvants enhance
outer membrane cell penetration and affect the proton motive force, ultimately inhibiting efflux pumps.171
Lipidation is a well‐known approach for increasing the antimicrobial activity of a peptide/peptoid by facilitating
interactions between an AMP/peptoid and the microbial envelope,172 even in some cases endowing inactive
cationic peptides/peptoids with antimicrobial activity.173 Lipid tail length has a great influence on the antimicrobial
activity and hemolysis of peptides/peptoids, mainly because the lipid tail length is closely related to the molecular
hydrophobicity.173,174 Furthermore, some lipopeptides/lipopeptoids also have strong synergistic activities with
conventional antibiotics,175 possess anticancer activity,176 induce cytokine production in macrophages and exert
immunomodulating properties.177 The mechanism of action of lipopeptides/lipopeptoids has been extensively and
deeply studied in recent years, such as that of Tridecaptin A1 (TriA1), which is a nonribosomal lipopeptide with
selective antimicrobial activity against Gram‐negative bacteria. It exerts bactericidal effects by crossing the outer
membrane through an interaction with LPS, then binding to lipid II on the inner membrane, followed by disrupting
the proton motive force.178

8 | C O MB I N AT IO N TH E RA PY

8.1 | Resistance to AMPs and peptidomimetics

With unique physical membrane disruption and diverse intracellular targets, resistance to AMPs is not readily
inducible for most organisms. However, in recent years, resistance to AMPs has been confirmed to be an inherent
component of some resistant bacteria and has been achieved in laboratory evolutionary experiments.179 Mechanisms
of resistance to AMPs mainly include cell surface modification,180 efflux pumps, uptake transporters, and proteolytic
degradation.181 These AMP resistances have involved chromosomal mutations, but have never been reported to
occur via horizontal gene transfer, until a mobilized colistin resistance 1 (MCR‐1) plasmid was isolated.182 MCR‐1 is a
member of the phosphoethanolamine transferase enzyme family, with expression in E. coli resulting in the addition of
positively charged amino groups phosphoethanolamine to lipid A, thereby blocking the binding of AMPs to lipid A.
Importantly, this plasmid can spread between strains.182 Thus, once AMPs have been put into clinical use, the
development of AMP‐resistant strains will be inevitable.183 In addition, peptidomimetic resistance also has been
reported by Hein‐Kristensen et al,184 but these resistant isolates showed no cross‐resistance against a panel of
membrane active AMPs and recovered highly susceptibility to peptidomimetics in the presence of blood plasma;
therefore, the current study does not imply additional concerns for peptidomimetics as future therapeutics.184

8.2 | Combination therapy as a strategy to combat resistance

Many previous studies have shown that the use of multiple antibiotic agents in a therapeutic cocktail can reduce
the dose of each drug in the combination. Such combination therapies may limit the development of resistance in
vitro compared to monotherapy. The impermeability of Gram‐negative bacterial membranes is the biggest
limitation for most antibiotics with cytosolic targets and a high molecular weight.170 Because of their
membranolytic characteristics, AMPs/peptidomimetics have become well suited for use in synergic combinations
with conventional antibiotics; in particular, they can significantly increase the activity of antibiotics, which act on
intracellular targets,185 such as deoxynybomycin, tetracycline,186 kanamycin, and rifampicin,187 against multidrug‐
resistant EKAPE (Enterococcus faecium, S. Aureus, Klebsiella pneumoniae,188 Acinetobacter baumannii, P. aeruginosa,
846 | WANG ET AL.

Enterobacter spp.).189 Furthermore, Garbacz et al190 demonstrated a synergistic activity of peptide IB‐367 in
combination with the antibiotics fusidic acid and cotrimoxazole against S. aureus isolated from the airways of cystic
fibrosis patients. Today, the synergistic effect between AMPs/peptidomimetics and antibiotics is well
appreciated but remains the subject of debate. He et al used a novel assay to measure interactions between
four conventional antibiotics and four membrane permeabilizing AMPs against Gram‐negative and positive
bacteria, yet none of the AMPs exhibited synergistic effects with any of the conventional antibiotic drugs in any
organism. Thus, they considered that large‐scale membrane disruption and permeabilization by AMPs is not
sufficient to drive synergistic activities with chemical antibiotics in either Gram‐negative or positive microbes.191
Accordingly, as an emerging field, the use of AMPs/peptidomimetics as potential antimicrobial adjuvants or
potentiators has been explored. These adjuvants may have weak activity on their own, but can either impede
antibiotic resistance mechanisms or potentiate antibiotic actions.170 An adjuvant may be an efflux pump inhibitor
(EPI), a membrane permeabilizer or an enzyme inhibitor.170 The above‐mentioned tobramycin‐lysine hybrid 3 can
be considered as an EPI and membrane permeabilizer, as it has weak activity against Gram‐negative bacteria on its
own, but can significantly increase the activity of minocycline and rifampicin against P. aeruginosa via enhancing
membrane penetration and inhibiting efflux pumps.171 A polymyxin‐based AMP SPR741 is currently being
developed as an adjuvant that can permeabilize the outer membrane to facilitate the entry of other antibiotics into
the bacterial cell, ultimately potentiating the activity of antibiotics against Gram‐negative pathogens.170

9 | A P P L I C A T I O N O F AN T I M IC RO B IA L P E P T ID E S

9.1 | In food
Preservatives used in the food industry are required to be natural antimicrobial compounds without toxicity to
humans or the environment. In recent years, with assured effectiveness for food preservation to satisfy increasing
consumer demands for natural and healthy food, much attention has been focused on the application of AMPs as an
alternative approach to control undesirable micro‐organisms in foodstuffs, while maintaining the sensory qualities
and nutritional properties of food. Many studies have demonstrated the potential of AMPs as food preservatives.
For example, Nisin, a polycyclic peptide with 34 amino acid residues derived from Lactococcus lactis, is usually used
in processed cheese, meats, and beverages.192 For further application of AMPs in food preservation, new methods
have been explored to preserve the bioactivity of AMPs in real foodstuffs. For example, Ple/PVA fiber, an AMP Ple
(Pleurocidin, a novel AMP with 25 amino acids, derived from the skin‐secreted mucous of the winter flounder)
incorporated into ultrafine PVA fiber mats via electrospinning technology, was demonstrated to be successfully
applied in apple cider, with efficient inhibition activity against E. coli.193

9.2 | In medicine
Considerable progress on AMPs as adjuvant treatment and/or drug therapy in the clinic has been made as the
result of many studies.56,194 For example, lucifensin and lucifensin II, two insect defensins secreted and excreted by
sterile larvae of the flies Lucilia sericata or Lucilia cuprina , can contribute to wound healing, especially in patients
with impaired healing due to underlying disorders (eg, diabetes and cardiovascular disease) during a procedure
known as maggot debridement therapy, which is routinely used at hospitals worldwide.195 Pexiganan, a 22‐amino‐
acid membrane disruptor analog of the Xenopus peptide magainin, has been clinically proven to replace ofloxacin in
the treatment of diabetic foot ulcers as early as 1996, and may avoid the selection of resistant bacteria that can
develop after oral systemic antibiotic therapy; however, the peptide was not approved by the Food and Drug
Administration in 1999 even after completion of a phase III trial. Currently the phase III clinical trials of this peptide
are being conducted again to treat mildly diabetic foot infection by Dipexium Pharmaceuticals.194 Omiganan is an
indolicidin analog isolated from bovine neutrophils and has a broad‐spectrum antibacterial activity. Currently, it has
T A B L E 2 Selected antimicrobial peptides (AMPs) in clinical phase of development
WANG

Clinical trial
ET AL.

AMPs Description Condition or disease Administration Phase Status company identifier if available
Pexiganan (MSI‐78) Analog of magainin Diabetic foot infection Topical Phase 3‐C MacroChem Corporation NCT00563433
NCT00563394
Dipexium NCT01594762
Pharmaceuticals, Inc. NCT01590758
Omiganan Derived from indolicidin Catheter infections Topical Phase 3‐C Mallinckrodt NCT00231153
Atopic dermatitis Phase 2‐C Cutanea Life NCT03091426
Rosacea Phase 3‐C Sciences, Inc. NCT02576847
Vulvar intraepithelial neoplasia Phase 2‐C NCT02596074
Acne vulgaris Phase 2‐C NCT02571998
Lytixar (LTX‐109) Synthetic antimicrobial Gram‐positive, skin infections Mild Topical Phase 2‐C Lytix Biopharma AS NCT01223222
peptidomimetic Eczema/Dermatoses Atopic Dermatitis
Nasal carriers MRSA Phase 1/2‐C NCT01158235
Non‐bullous Impetigo Phase 2‐C NCT01803035
Surotomycin Cyclic lipopeptide Clostridium difficile‐associated oral Phase 1‐C Merck Sharp & NCT02835118
diarrhea (CDAD) Dohme Corp. NCT02835105
197
Novexatin Cyclic peptide Onychomycosis Topical Phase 2‐C NovaBiotics
(NP‐213)
LL‐37 Host‐defense peptide Melanoma Intratumorally in Phase 1/2‐A M.D. Anderson Cancer NCT02225366
cutaneous or Center
subcutaneous
tumors
PXL01 Derived from lactoferricin Surgical adhesions Hyaluronic acid‐ Phase 2‐C Pergamum AB NCT01022242
based hydrogel
Iseganan (IB‐367) Derived from protegrin 1 Oral mucositis in head and neck cancer. Oral rinse Phase 3‐U National Cancer NCT00022373
Institute (NCI)
PAC‐113 Derived from histatin 3 Oral candidiasis Mouth rinse Phase 2‐C Pacgen NCT00659971
Biopharmaceuticals
Corporation
|

(Continues)
847
 (Continued)
848

TABLE 2
|

Clinical trial
AMPs Description Condition or disease Administration Phase Status company identifier if available

Dalbavancin Lipoglycopeptide Bone infection Osteomyelitis Septic Intravenously Phase 4‐R Infectious Diseases NCT03426761
arthritis Joint infection Prosthetic joint Physicians, Inc
infection
Infectious Peritonitis Intravenously Phase 4‐A University of Colorado, NCT02940730
Denver
Methicillin‐resistant Staphylococcus Intravenous Phase 3‐R Durata Therapeutics Inc NCT02814916
Aureus skin infections administration in
children
SGX942 5‐amino acid peptide Oral mucositis in head and neck cancer Intravenously Phase 3‐R Soligenix NCT03237325
OP‐145 Derived from LL‐37 Chronic otitis media Eardrops Phase 2‐C Leiden University, The ISRCTN84220089
Netherlands
Brilacidin (PMX‐ Defensin mimetic Bacterial skin infection Intravenously Phase 2‐C Cellceutix Corporation NCT02052388
30063) Mucositis in head and neck Neoplasms Oral Rinse Phase 2‐C Innovation NCT02324335
Pharmaceuticals, Inc
POL7080 Peptidomimetic Renal impairment Intravenously Phase 1‐C Polyphor Ltd NCT02110459
Ventilator‐associated pneumonia Phase 2‐C NCT02096328
aeruginosa
Healthy; Synergism with amikacin Phase 1‐C NCT02897869
AP‐214 Derivative from HDP Prevention of (acute) kidney injury after Intravenously Phase 2‐C Action Pharma A/S NCT01256372
cardiac surgery
Prevention of kidney injury after Phase 2‐C NCT00903604
thoracic aortic aneurysm repair
CD‐NP Chimeric 37‐mer derived Acute decompensated heart failure Infusions Phase 2‐C Nile Therapeutics NCT00839007
from combination of two
natriuretic peptides
Ghrelin Endogenous host‐defense Chronic respiratory infection Intravenously Phase 2‐C University of Miyazaki, JPRN‐
peptide Japan; UMIN000002599
Airway inflammation Phase 2‐C JPRN‐
UMIN000001598
WANG

Abbreviations: A, active, not recruiting; C, completed; HDP, host‐defense peptide; R, recruiting; U, unknown.
ET AL.
WANG ET AL.
T A B L E 3 Reports on application of antimicrobial peptides (AMPs) in animals
Animals AMPs Treatments/Doses
Weanling piglets CAPa Basal diet with 4 ppm
deoxynivalenol and
4% CAP
Weanling piglets SyntheticAMP‐ Basal diet with 60 mg/kg
A3band AMP‐P5c AMP‐A3 and basal diet
with 60 mg/kg AMP‐P5
Basal diet with 40 and
60 mg AMP‐P5/kg diet
Basal diet with 0, 60 and
90 mg AMP‐A3/kg diet
Weaned piglets Cecropin ADd Basal diet with 400 mg/kg
cecropin AD and piglets
were orally challenged
with E. coli K88
Weanling piglets Recombinant Basal diet with 0.1 g
Lactoferrampin‐ Lactoferrampin‐
lactoferricin lactoferricin and 0.1 g
chlortetracycline/kg diet
Weanling piglets cipB‐LFC‐LFA Basal diet with no
addition, 100 mg cipB,
100 mg cipB‐LFC‐LFA/
kg diet
Weanling piglets Colicin E1 Basal diets with0, 11, or
16.5 mg Colicin E1/kg
and piglets were orally
inoculated with E. coli
Weaned female Lactoferrin Basal diet with 1.0 g/kg
piglets lactoferrin
Indigenous male Cecropin AD‐ Basal diets with a CADN
chickens Asn (CADN) liquid sample at 0, 2, 4, 6,
and 8 ml/kg
| 849
Effects References
Attenuating the metabolic Xiao et al,201
disturbances in amino Xiao
acid, lipid, and energy et al,202Xiao
metabolism induced by et al204
DON; Improving
intestinal morphology,
intestinal epithelial cell
proliferation and protein
synthesis; Improving feed
efficiency, immune
function, and
antioxidation capacity,
alleviating organ damage
Improving the Yoon et al200
performance, nutrient
digestibility, intestinal
morphology and reducing Yoon et al199
pathogenic bacteria
Yoon et al205
Increasing immune status Wu et al206
and nitrogen and energy
retention as well as
reducing intestinal
pathogens
Improving performance Tang et al207
and affecting serum
parameters
Improving performance, Tang et al208
the regulation of immune
function and the
absorption of Fe, reducing
the incidence of diarrhea
Improving the Cutler et al209
performance and
reducing the incidence of
postweaning diarrhea
Increasing ADG, efficiency Wang et al210
of gain, intestinal villus
height and relative
abundance of mRNA for
PR‐39 and protegrin 1
Increasing nutrient Wen and He211
utilization, enhancing
intestinal villus heights,
decreasing aerobic
bacterial counts
(Continues)
850 | WANG ET AL.

TABLE 3 (Continued)

Animals AMPs Treatments/Doses Effects References

Arbor Acre male Pig AMP (PMAP)e Basal diet with PAMP at Improving the Bao et al212
broiler 150 and 200 mg/kg performance, the
chickens intestinal mucosal
immunity, and increasing
the intestinal ability to
absorb nutrients

Abbreviations: AD, atopic dermatitis; CAP, composite antimicrobial peptides; cipB‐LFC‐LFA, cipB‐lactoferricin‐lactofer-
rampin; DON, deoxynivalenol.
a
CAP consists mainly of antibacterial lactoferrin peptides, along with plant defensins and active yeast.
b
AMP‐A3 (amino acid sequence: AKKVFKRLEKLFSKIWNWK‐NH2) is an analog of antimicrobial peptide HP 2‐20 (amino
acid sequence: AKKVFKRLEKLFSKIQNDK‐NH2).
c
AMP‐P5 (amino acid sequence: KWKKLLKKPLLKKLLKKL‐NH2) is an analog of the hybrid antimicrobial peptide CA‐MA
[Cecropin A (1–8)‐Magainin 2 (1–12); KWKLFKK IGIGKFLHSAKKF‐NH2].
d
Cecropin AD is expressed in Bacillus subtilis and the amino acid sequence of Cecropin AD is KWKLFKKIEKVGQRVR-
DAVISAGPAVAT‐VAQATALAK‐NH2.
e
PAMP is isolated from pig small intestine.

completed phase III clinical trials for catheter infections and rosacea. It also has completed phase II for the
evaluation of the pharmacodynamics, safety and efficacy of topically applied Omiganan in patients with vulvar
intraepithelial neoplasia, atopic dermatitis and acne vulgaris. POL7080 as an antimicrobial peptidomimetic
specifically targets P. aeruginosa at the nanomolar level via non‐membrane‐disrupting activity.196 Currently, Phase I
clinical studies of the peptidomimetic POL7080 have been completed in the evaluation of the pharmacokinetics
(PK) of POL7080 in subjects with renal function impairment. Furthermore, phase II clinical trials for patients with
ventilator‐associated P. aeruginosa pneumonia and phase I clinical trials for synergism with amikacin also have been
completed. In addition to antibacterial activity, some AMPs have also been shown to have immune‐promoting
effects, such as PXL01, which is derived from human lactoferricin and been assessed for its efficacy, safety and
handling in patients with flexor tendon injuries in phase II trials.197 The selected AMPs in the clinical phase of
development are summarized in Table 2.

9.3 | In animals
It has been reported that several AMPs added in the diet have beneficial effects, including body weight, the average
daily gain, nutrient digestibility and intestinal morphology as well as effects on intestinal and fecal microflora.198
Additionally, antimicrobial activity is regarded as the basis of AMPs to improve animal performance, improve
nutrient digestibility and support normal intestinal morphology and function.199,200 Some reports also
demonstrated that AMPs can protect piglets from challenge with the mycotoxin deoxynivalenol (DON)201 and
repair the intestinal injury induced by DON.202 Moreover, AMPs as an immune molecule can attenuate
inflammation, enhance intestinal barrier function and improve microbiota composition in the intestines of weaned
piglets, ultimately resulting in significantly reduced rates of diarrhea in weaned piglets.203 Reports on the functions
of AMPs in animal health are summarized in Table 3.

10 | PERSPEC TIV E A ND C O NC LU SION S

Over the past several decades, AMPs have been developed as a promising alternative to antibiotics. However,
low in vivo stability is still considered to be the key factor limiting their clinical application. Indeed, the systemic
administration of peptide, for example oral or intravenous injection, results in rapid degradation by digestive
WANG ET AL. | 851

enzymes or rapid removal by the metabolic system, even upon local administration, as peptides are prone to
degradation by tissue proteolytic enzymes. To extend the half‐life of AMPs, AMP mimics with an amphiphilic
scaffold have been widely developed. Peptidomimetic studies have made great advances in recent decades, but
the systematic structure‐function relationships of peptidomimetics warrant further studies to gain insights into
the development of the molecular design of peptidomimetics and for exploring their medical and pharmacological
applications. For example, the introduction of different additional hydrophobic building blocks and more cationic
charges may strongly influence the biological activity of peptidomimetics. Furthermore, bacterial resistance to
peptidomimetics has been reported, but the resistance mechanisms need to be better characterized.
Combination therapy is considered to be an effective solution to drug resistance but the strategy remains
fallible as several important pharmacological questions remain unanswered. For instance, PK variances and
distribution rates of different drugs would certainly impose a challenge in fine‐tuning the dosages of
administered drugs to replicate their observed in vitro synergies.170 Thus, a hybrid molecule that combines two
or more pharmacophores has been reported to eliminate the problem of noncomplementary pharmacodynamics.
Additionally, these hybrid compounds could even alter the pharmacological spectrum of the hybrids and impart a
new mechanism of antibacterial action to the resulting hybrid agent, which could provide a new means of over-
coming drug resistance. Currently, it is unrealistic to completely abandon the use of antibiotics; however, the
advantages of AMPs/peptidomimetics and antibiotics could be combined to complement each other and play a
greater role in reducing drug resistance. To date, hybrids of AMP/peptidomimetic and antibiotic molecules are
just emerging, but in the future AMPs/peptidomimetics‐antibiotics hybrids may represent the next generation of
antimicrobial agents. In this review, we have described recent work on the characteristics, structure‐activity
relationships, functions, antimicrobial mechanisms, expression regulation, and application of AMPs. Additionally,
the development of peptidomimetics was briefly introduced. The broad‐spectrum antimicrobial activity and
nonspecific physical membrane disruption mechanism of AMPs highlight their potential to be developed as
potential alternatives to antibiotics. We hope that this review will be helpful to demonstrate the behavior of
AMPs and contribute to the further design and clinical application of AMPs as alternatives to antibiotics.

A C K N O W L E D GE M E N TS

The authors thank all the members and laboratory technicians involved in the studies based in the Institute of
Animal Nutrition, Northeast Agricultural University. This study was supported by financial support from the
National Natural Science Foundation of China (31672434, 31472104, and 31272453), the China Agriculture
Research System (CARS‐35), the Program of Ministry of Education of China (20092325110009), and the Program
for Universities in Heilongjiang Province (1254CGZH22).

CO NFLICTS OF INTE RES T

The authors declare that there are no conflicts of interest.

A UT HO R C ONT RI BU TIO NS

JW wrote the manuscript under the guidance of AS and both authors read and approved the final version of the
manuscript.

OR CID

Jiajun Wang http://orcid.org/0000-0002-2086-2105

852 | WANG ET AL.

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How to cite this article: Wang J, Dou X, Song J, et al. Antimicrobial peptides: Promising alternatives in the
post feeding antibiotic era. Med Res Rev. 2019;39:831–859. https://doi.org/10.1002/med.21542