Antimicrobial Host Defense Peptides

REVIEWS
Antimicrobial host defence peptides:

functions and clinical potential
Neeloffer Mookherjee 1 ✉, Marilyn A. Anderson 2
, Henk P. Haagsman 3
and Donald J. Davidson 4
Abstract | Cationic host defence peptides (CHDP), also known as antimicrobial peptides, are
naturally occurring peptides that can combat infections through their direct microbicidal
properties and/or by influencing the host’s immune responses. The unique ability of CHDP to
control infections as well as resolve harmful inflammation has generated interest in harnessing
the properties of these peptides to develop new therapies for infectious diseases, chronic
inflammatory disorders and wound healing. Various strategies have been used to design
synthetic optimized peptides, with negligible toxicity. Here, we focus on the progress made in
understanding the scope of functions of CHDP and the emerging potential clinical applications
of CHDP-based therapies.
The global increase in multidrug-resistant pathogens Initial research in the field was focused on the anti
along with a steady decline in the discovery of new microbial functions of this family of peptides, with a
antibiotics underscores the need for new therapies to drive to discover new antibiotic-like therapies based
control infections. In addition, common treatments on the naturally occurring peptides. However, stud-
used for many chronic inflammatory diseases such as ies published in the first decade of this century estab-
corticosteroids can increase susceptibility to infections1,2, lished that the microbicidal action of some of these
including antibiotic-recalcitrant pathogens3. There is peptides (for example, the human cathelicidin LL-37
therefore an urgent need for alternative strategies that and β-defensins) was severely impaired under phys-
can both kill pathogens and resolve harmful inflamma- iological salt concentrations (particularly by divalent
Manitoba Centre for tion. One promising approach has emerged from the cations) and in the presence of host factors such as
1
Proteomics and Systems

Biology, Department of
identification of cationic host defence peptides (CHDP), anionic polysaccharides, apolipoproteins, DNA, F-actin
Internal Medicine and which can control infections by their direct micro and glycosaminoglycans8,9. The relatively poor direct
Department of Immunology, bicidal properties and/or by modulating host immune microbicidal capabilities of these peptides under physio
University of Manitoba, responses, while also exhibiting the capacity to limit logical conditions, at the low, naturally occurring con-
Winnipeg, Manitoba, Canada.
enhanced inflammation. centrations observed in vivo, did not adequately explain
2
La Trobe Institute for CHDP, also known as antimicrobial peptides, were first their contribution in host defence against infections10,11.
Molecular Science, La Trobe
University, Melbourne,
described in the 1960s by Kiss and Michl in the speck- Subsequently, research over the last two decades has
Victoria, Australia. led frog4. Several seminal studies in the 1980s further demonstrated that CHDP exhibit a wide range of func-
3
Division of Molecular Host defined antimicrobial peptides, notably cecropin isolated tions beyond microbicidal activity, including the ability
Defence, Department of from moths by the Boman group5, defensins isolated from to influence immune responses, which contribute to their
Infectious Diseases and human neutrophils by Ganz and Lehrer6 and magainins antimicrobial impact (reviewed in refs12–15). This has led
Immunology, Faculty of isolated from amphibians by Zasloff et al.7. CHDP are to the adoption of the current name for this family of
Veterinary Medicine,
Utrecht University,
now known to be expressed across a diverse range of peptides, cationic host defence peptides (CHDP), which
Utrecht, Netherlands. species, from microorganisms, plants and invertebrates encompasses the wide range of functions described.
4
University of Edinburgh to more complex amphibians and mammals. Over the last three decades there has been substantial
Centre for Inflammation CHDP are typically amphipathic small peptides with interest in therapeutically harnessing CHDP, with more
Research, Queen’s fewer than 50 amino acids and a net positive charge of than 5,000 articles published in this area of research
Medical Research Institute, +2 to +9 at physiological pH. However, these natural since 2017 alone. These include the examination of
Edinburgh, UK.
peptides differ significantly in sequence and structure, potential clinical uses for CHDP ranging from infections
✉e-mail:
and are broadly classified into four structural groups on involving multidrug-resistant bacteria16–19 to chronic
neeloffer.mookherjee@
umanitoba.ca the basis of conformation; α-helical linear peptides, those inflammatory diseases such as arthritis20, asthma21 and
https://doi.org/10.1038/ with β-sheet with disulfide bridges, cyclic peptides and colitis22, as well as some cancers23. Peptide-based thera-
s41573-019-0058-8 peptides with extended flexible loop structures. peutics currently in clinical trials are primarily intended
NaTuRe RevIews | DRug DISCOVeRy volume 19 | May 2020 | 311

Reviews
Host defence peptides

for the treatment of infections such as respiratory tract, divergence by both positive and negative selection of
Naturally occurring typically oral and catheter-related infections, and for wound mammalian defensin genes following ancestral gene
amphipathic cationic peptides healing (see Dramp Database). duplication36–38.
that modulate host immune This Review will provide an overview of current Cathelicidins are produced as prepropeptides con-
responses to control infection
and regulate inflammation
understanding of the scope of functions of CHDP, pri- taining an amino-terminal (N-terminal) signal pep-
(commonly used as an marily from eukaryotes. Emerging therapeutic appli- tide, a cathelin-like domain and the carboxy-terminal
alternative name for cations of these peptides, current clinical trials and mature peptide. The pro-cathelin-like domain is cleaved
antimicrobial peptides to the associated clinical developmental challenges will off primarily by serine proteases once the peptide is
encompass all their functions).
be discussed. Although there is increasing interest in secreted39,40. These peptides are named after the con-
Antimicrobial peptides
the development of non-p eptide mimics of CHDP served cathelin-like domain in the propeptide which
Naturally occurring cationic for therapeutic application, such as peptoid analogues is the common denominator of this family of pep-
peptides, typically shorter (reviewed in ref.24), a comprehensive discussion of these tides, as the active mature cathelicidins do not share
than 50 amino acids in length, approaches is beyond the scope of this Review. sequence similarities. Most cathelicidins are α-helical
with the ability to kill
microorganisms.
(23–37 amino acids), amphipathic, cationic peptides
Naturally occurring CHDP with a hydrophobic surface allowing interaction and
Defensins The antimicrobial peptide database has catalogued more perturbation of membranes with anionic surfaces. The
A family of host defence than 2,600 natural antimicrobial peptides, including only human cathelicidin, LL-37, is the most well studied
peptides with a common
those annotated as immunomodulatory25. The major peptide in this family. LL-37 is an α-helical peptide and
β-sheet core stabilized with
three or four disulfide bridges
families of CHDP from eukaryotes that are of interest is one of several cleavage products of hCAP18, the pro
depending on the source. from a drug discovery perspective are summarized in duct of the only human cathelicidin gene CAMP26. Other
the following subsections. α-helical cathelicidins include the sole mouse catheli-
Cathelicidin cidin CRAMP41 and chicken CATH-2 (ref.29). Another
A member of a family of host
defence peptides named after
Vertebrate CHDP class of cathelicidins comprises β-hairpin peptides
the conserved cathelin-like CHDP from vertebrates have an essential role in the first (12–18 residues), such as the bovine bactenecin, that
domain in the propeptide line of defence against microbial pathogens. On infec- have one intramolecular disulfide bond. Cathelicidins
precursor. tion, CHDP can kill pathogens through diverse mecha- such as protegrins (16–18 residues) have two intra
nisms26–31 (discussed later), acting rapidly and directly on molecular disulfide bonds and adopt β-sheet structures.
Histatins
A family of histidine-rich host
the pathogen when present in high local concentrations, Finally, linear cathelicidins (13–39 residues) are enriched
defence peptides. or indirectly to modify components of host defence. in specific amino acids (for example, tryptophan-
These peptides exhibit immunomodulatory activities that enriched bovine indolicidin and proline-rich porcine
can be either pro-inflammatory or anti-inflammatory PR-39). Cathelicidins are immunomodulatory anti
depending on the phase of the infection12–14,29 (see below). microbials with an important role in the regulation of
CHDP from vertebrates are amphipathic peptides con- the inflammatory response29–31.
taining amino acids with hydrophilic and hydrophobic Several other vertebrate CHDP are also being explored
side chains at opposite sides of the molecules. These as potential alternatives to antibiotics. Among these are
CHDP can interact with the negatively charged mem- histatins, which are histidine-rich CHDP constituents
branes of bacteria. The two main classes of CHDP in ver- of saliva of mammals. Histatins are less amphipathic
tebrates, the defensins and the cathelicidins (Fig. 1a), are than cathelicidins or defensins and may have different
produced as prepropeptides which are cleaved to yield modes of action compared with other vertebrate CHDP
mature active peptides. as illustrated by their antifungal activity42. Finally, sev-
Defensins have a common β-sheet core stabilized with eral CHDP produced by amphibian skin granular glands,
three disulfide bridges between six conserved cysteine such as magainin 2, have been used as prototypes for the
residues, and are subdivided into α-, β- and θ-defensins development of novel antimicrobials43.
on the basis of the linkage of cysteine residues. The genes
encoding α- and β-defensins are adjacent on chromo Invertebrate, plant and fungal CHDP
some band 8p23.1 and probably have common ances- CHDP are a vital component of the innate immune
try, with α-defensins thought to have evolved by gene system of all eukaryotic organisms, including plants,
duplication and found only in some mammals, pri- fungi and invertebrates which lack an adaptive immune
marily primates and rodents32. Several of the human system. These CHDP, similarly to those from vertebrates,
α-defensins are highly expressed in neutrophils, and are also small amphipathic peptides with an overall posi-
other α-defensins are produced and secreted by Paneth tive net charge44,45. These peptides fall into several diverse
cells in the small intestine6,33,34. The β-defensins are ubiq- and distinct structural groups, including α-helical pep-
uitous and present in all vertebrates. The human genome tides, β-sheet peptides, peptides with mixed α-helical
has more than 30 genes coding for β-defensins, and and β-sheet structures, extended peptides and pep-
mice have even more genes coding for these peptides. tides enriched in specific amino acids (Fig. 1b). Their
β-defensins are produced mainly in epithelial cells33,35. classification is generally dependent on the number,
Finally, the cyclic θ-defensins arose from α-defensins spacing and connectivity of cysteine residues as well as
after the divergence of primates and have been purified structure, which is normally highly conserved within a
from the leukocytes of rhesus macaques and baboons. CHDP family.
These molecules are the only cyclic peptides found in In plants, gene duplication and rapid evolution has
mammals and exhibit antiviral activity. Thus, varying produced large families of antimicrobial peptides within
between different species, evidence exists for sequence the same organism46. Genome sequencing has revealed
312 | May 2020 | volume 19 www.nature.com/nrd

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a CATH-2 HBD2 HD5 ion channel perturbation and enzyme inhibition46,47.

Similarly to the mammalian defensins, these also exhibit
other functions, including roles in innate immunity, in
addition to antimicrobial activity33,48. Although once
considered an example of evolution of an ancient innate
immunity molecule, it is now evident that mammalian
and plant defensins evolved independently and are an
excellent example of convergent evolution46.
Synthetic peptides derived from CHDP

LL-37 Magainin 2
The vast repertoire of sequences and structures of natu-
ral antimicrobial peptides allows the design and devel-
opment of novel therapeutic peptide analogues, or
peptidomimetics. Various approaches are being applied in
b the design of new drug candidates based on CHDP, such
30 Fungal AFP Cystatin as single amino acid substitution, use of internal segments
of CHDP to derive smaller peptides and in silico meth-
ods to predict new synthetic peptides on the basis of the
understanding of structure–function relationships49–51.
Big defensin
Synthetic innate defence regulator peptides
20 Early approaches to the development of CHDP as thera-
β-Sheet content (a.a.)
C8 defensin
peutics focused heavily on optimizing the direct micro-
bicidal properties of these peptides. However, it has
Cyclotide
become clear that strategies adopted to enhance in vitro
microbicidal functions often resulted in peptides with
β-Thionin
increased levels of cytotoxicity. In addition, naturally
10 Hevein
Lipid transfer occurring CHDP can also exhibit other functions that are
protein undesirable for drug development, such as an ability to
α-Hairpinin Snakin induce mast cell degranulation, with release of histamine
and prostaglandin, as well as activation of complement
factors52,53. Therefore, in the last decade, effective strate-
SFTI gies have focused on designing synthetic peptides from
0
sequences of CHDP to optimize antimicrobial func-
0 20 40 60
α-Helical content (a.a.) tions in vivo, through a combination of some microbi
cidal activity along with beneficial immunomodulatory
Fig. 1 | Structures of CHDP. a | Examples of structures of cationic host defence peptides functions, in the absence of cytotoxic effects.
(CHDP) from vertebrates. Two cathelicidins are depicted: human LL-37 and chicken Innate defence regulator peptides (IDR peptides) are
CATH-2 (with a proline-induced kink). Two human defensins are shown: the human such small synthetic cationic peptides derived from
α-defensin 5 (HD5) and the human β-defensin 2 (HBD2). The pairing and positioning of natural CHDP, and have been screened and optimized
the six conserved cysteine residues is as follows: α-defensins, Cys1–Cys6, Cys2–Cys4, for immunomodulatory functions12,54. Libraries of IDR
Cys3–Cys5; β-defensins, Cys1–Cys5, Cys2–Cys4, Cys3–Cys6. Magainin 2 is a peptide peptides have been generated by screening of over-
from Xenopus laevis; its analogue, pexiganan, was developed as a topical agent. Amino lapping fragments representing internal sequences of
acid side chains are coloured as follows: red, hydrophobic; blue, basic; green, acidic.
CHDP, and by single amino acid substitutions49–51. Most
b | Examples of the diversity of CHDP structures. Tertiary structures of selected peptides
from plants, fungi and invertebrates, arranged by secondary structure content. β-Strands
of the IDR peptides reported to date are derivatives of
are shown in blue, α-helices are shown in red and disulfide bonds are shown in yellow. the bovine CHDP bactenecin21,49,54. IDR peptides are
Protein Data Bank IDs 1MR4, 1NB1, 2RNG, 1BHP, 1GD3, 2L2R, 1JBL, 1HEV, 2MAL, 5E5Q non-immunogenic and do not have the potentially
and 5OQS. a.a., amino acid; AFP, antifungal peptide; SFT1, sunflower trypsin inhibitor 1. adverse effects associated with some natural CHDP.
In general, IDR peptides have a modest direct effect
on the pathogen, yet can control infection in vivo and
that plants such as Arabidopsis and Medicago have up reduce inflammation, as shown in a variety of infection
Peptidomimetics to 300 defensin or defensin-like sequences. Apart from models54–56. The beneficial use of IDR peptides for the
Compounds that mimic a the cysteine residues and a glycine residue which are control of infections is now well established in pre
natural peptide and retain required for the defensin fold, these are highly diverse, clinical models of a wide range of pathogens, including
the biological function of the
wherein the sequence diversity is displayed on the sur- multidrug-resistant bacteria, viruses, parasites and
parent protein.
face loops presented on conserved scaffolds. This may antibiotic-recalcitrant bacterial biofilms54,56–60. IDR pep-
Innate defence regulator explain the diversity of biological functions defined for tides also exhibit adjuvant functions to enhance mucosal
peptides this family of proteins47. Plant defensins are generally immunity and antigen-specific humoral response61–64,
(IDR peptides). Small synthetic potent antifungal molecules. However, plant defensins and are effective in controlling inflammation and related
cationic peptides with
immunomodulatory functions,
with antibacterial and insecticidal activity have also been physiological outcomes in models of sterile inflamma-
derived from natural host described, along with those with roles in cell signalling, tion65 and chronic inflammatory diseases21. A distinct
defence peptides. self–non-self recognition during sexual reproduction, advantage of IDR peptide-based therapy, compared

Reviews
with current anti-inflammatory therapeutics, is the of CHDP for bacterial membranes compared with the
potential to resolve infections along with the ability to membranes of cells from plants, invertebrates and verte
control inflammation54,57. brates. The amphipathic nature of CHDP is important
for the membrane-destabilizing properties of these
Cryptic and synthetic peptides peptides. CHDP can bind to the bacterial inner mem-
Several mammalian cationic proteins, such as histones, brane, leading to penetration of the peptide, leakage
lactoferrin and thrombin, and their cleavage products of bacterial cell contents and cell death27,28 (Fig. 2). In
have antimicrobial activities. Histones and fragments Gram-negative bacteria, interaction of CHDP with
thereof have a wide range of antimicrobial activities66. LPS results in perturbation of the outer membrane, and
These are not restricted to nucleosomes but are also this has been defined as a primary mode of action for
found in the cytoplasm of cells and can be released on the antimicrobial activity of CHDP76. Four main mod-
activation. Up to 70% of the proteins of neutrophil extra els of (inner) membrane perturbation have been pro-
cellular traps (NETs) are histones and histone fragments67. posed: aggregate, toroidal pore, barrel-stave and carpet
Lactoferrin is another example of a protein that yields anti models76 (Fig. 2). It should be cautioned that these pro-
microbial peptides on proteolytic degradation in vivo. posed mechanisms of action stem largely from experi
This protein is not confined to milk; it is produced ments with model membranes. As the interaction of
by several tissues and has several biological activities CHDP with bacterial membranes does not involve a spe-
related to host defence, including antimicrobial acti cific target, it has been speculated that the development
vity. Its proteolysis in the gastrointestinal tract produces of microbial resistance is unlikely. Indeed, CHDP can
fragments that are more active than the native protein68. elicit transient bacterial adaptations, the mechanisms of
Proteolytic digestion with pepsin releases lactoferricin, a which are different from those involved in the develop-
peptide derived from the N-terminal region of lactoferrin. ment of bacterial resistance to conventional antibiotics.
Other active fragments described include lactoferricin For example, a study showed that removal of CHDP from
1–11 and lactoferrampin69. Similarly, synthetic molecules the culture medium resulted in the bacteria reverting to
based on human θ-defensin pseudogenes, termed ‘retro their original state: the adaptation to counter the effect
cyclins’, have been developed as potential therapeutics, of the CHDP was not maintained77. Therefore, microbial
for example as antivirals70. These observations that anti- ‘resistance’, as described for conventional antibiotics, is
microbial and/or immunomodulatory peptides can be unlikely to develop for CHDPs.
released from larger proteins during inflammation or In addition to inducing gross damage of bacterial
infection has led to the exciting concept of developing membranes, CHDP may also affect cell wall synthesis.
synthetic CHDP as prodrugs in which the N terminus For example, defensins such as HNP1 and HBD3 exert
of the peptide is modified by a linker and a negatively their antibacterial activity by docking on lipid II, the
charged promoiety. Such prodrugs can be selectively intermediate in peptidoglycan synthesis78–80. In addi-
cleaved by a disease-associated enzyme, thereby target- tion, CHDP such as the ribosome-binding proline-rich
ing peptide activity to pathologically affected parts of the peptides can cross the bacterial membrane and kill bac-
body71. In addition, these discoveries have led to bio teria from within by binding to intracellular targets such
informatics approaches to identify segments of secreted as nucleic acids or nascent proteins and subsequently
proteins that could be developed as novel antimicrobial affect cell processes such as replication, transcription,
peptides of human origin. Bioinformatic tools have been translation, protein folding and cell wall synthesis28,76,81,82
developed to find those cryptic sequences, and the pep- (Fig. 2). Simultaneous exposure of a pathogen to multi-
tides thus defined are known as cryptic peptides72. Among ple different CHDP, potentially using different modes
the identified and tested cryptic peptides are those from of action, may be a critical mechanism by which these
two apolipoproteins73. Other researchers have developed peptides are so effective in vivo. An exciting recent
entirely novel antimicrobial peptides by high-throughput study in Drosophila used CRISPR gene editing to delete
screening of libraries of peptides that were synthesized all known immune-inducible CHDP and demonstrated
semirandomly74,75 or by rational design51. both additive and synergistic antimicrobial functions of
the peptides, as well as highly specific CHDP–pathogen
Antimicrobial actions of CHDP interactions in vivo83.
The antimicrobial effects of CHDP on a wide range of It should be noted that the antibacterial effects of
pathogens have provided the impetus for the develop- CHDP are measured under non-physiological condi-
ment of these peptides as broad-spectrum antimicrobials. tions in most in vitro studies. This is a problem as the
The related mechanisms of action are diverse and appear direct microbicidal activities of many CHDP are antag-
to be dependent on the microbial pathogen. onized under physiological in vivo conditions, such as
higher ionic strength and presence of divalent cations
Antibacterial activity and host lipids and proteins. Thus, it may be argued that
The bacterial membrane is the main target for most CHDP may not function in vivo as antimicrobials with
cationic peptides. Bacterial membranes are negatively direct microbicidal properties11,84. However, some CHDP
Cryptic peptides charged because of the presence of anionic lipids, lipo (and derived synthetic peptides) with compromised
Segments of secreted proteins polysaccharides (LPS; in Gram-negative bacteria) or direct antimicrobial activity in vitro exhibit the capacity
identified by bioinformatics
screening to potentially
teichoic acids (in Gram-positive bacteria). These negatively to actively control infections in vivo, which may be due to
function as antimicrobial charged molecules initiate an electrostatic interaction local concentrations of CHDP released by neutrophil
peptides. with cationic compounds which explains the preference degranulation at the site of infection being higher than

Reviews
CHDP/AMP
Membrane translocation Membrane perturbation Bacterial lysis
Models
Barrel stave
Carpet
(detergent-like)
Intracellular targets
DNA/RNA Protein Protein Toroidal pore

synthesis synthesis folding
Bacterial killing
Fig. 2 | Models of antibacterial mechanisms of CHDP. Direct antimicrobial mechanisms of cationic host defence
peptides (CHDP) can be mediated by membrane translocation of the peptides followed by binding to intracellular targets
such as nucleic acids and/or proteins to kill bacteria. The mechanisms of translocation are not clear and may depend on
the peptide and bacterial species. Proline-rich antimicrobial peptides (AMP) use inner membrane transporters as Trojan
horses to gain entry and subsequently bind within the ribosomal exit tunnel. Other CHDP may use transient pores for
translocation. Interaction of CHDP with negatively charged bacterial membrane resulting in membrane perturbation
has been defined as a primary mode of direct antimicrobial action. The models of membrane perturbation proposed
are the barrel-stave, carpet and toroidal pore models. The barrel-stave model was the first permeabilization mechanism
proposed and is considered to be the prototype in peptide-mediated transmembrane pore formation. In this model,
peptides act as staves and vertically insert themselves into the lipid bilayer, forming barrels. Peptides which act according
to the carpet model cover the negatively charged membrane based on electrostatic attraction. Above a certain peptide
threshold concentration, the membrane ruptures in a detergent-like manner, resulting in micelle formation of peptide with
membrane lipids. The toroidal pore model is a variation of the aggregate model, where after parallel binding of the
peptide to the membrane, the peptide distorts the alignment of the polar head groups of the lipids. This results in
perturbation of the acyl chain interactions of the lipids, changes in membrane curvature and destabilization of membrane
surface integrity. At certain peptide-to-lipid ratios, the peptides orient themselves perpendicularly to the membrane and
induce the formation of transient toroidal channels.
in vitro minimum inhibitory concentrations. A more syncytial virus (RSV), Zika virus, vaccinia virus and
plausible explanation is that CHDP-mediated immuno Kaposi’s sarcoma-associated herpesvirus) in vitro is
modulatory functions and/or the concerted action of the capacity of CHDP (including defensins and cathel-
CHDP with other immunity-related factors is critical icidins) to destabilize the viral envelope on contact,
in the resolution of infections in vivo (discussed later). damaging the virions and inhibiting infectivity86–94.
However, the relationship between antibacterial potency This may happen on contact in solution or on viral
of CHDP in vitro and the immunomodulatory functions exposure to plasma membrane-associated CHDP dur-
is not understood. There is no evidence of an inverse ing cell entry88. However, CHDP have also been shown
correlation between antibacterial potency and the ability to have antiviral activity against non-enveloped viruses
of CHDP to induce an immune response84. (such as rhinoviruses, human papillomavirus 16 and
adenoviruses) by decreasing viral replication95,96 and/or
Antiviral activity by binding viral capsid, thereby preventing uncoating
In addition to the antibacterial properties of CHDP, early and nuclear entry of the viral genome97–101. An addi-
observations of the antiviral potential of CHDP85 have tional mechanism of action against a number of viruses
been expanded to demonstrate activity against a range (such as herpes simplex virus (HSV) and HIV) relates
of viruses. Most of these studies in recent years were to specific CHDP binding to cellular receptors involved
performed in vitro and described a variety of mecha- in viral infection, which is dependent on the lectin-
nisms that underpin the antiviral effects, differing in the like properties of some peptides102–110. Further antiviral
context of specific viruses (Table 1). effects may also result from CHDP-mediated aggrega-
A common mechanism of action against many envel- tion of viral particles111, inhibition of protein kinase C
oped viruses (such as influenza viruses, respiratory activity112,113 and immunomodulatory effects (discussed

Reviews
Table 1 | Antiviral activities of cationic host defence peptides

Virus Peptide Proposed mechanism of action in vitro Refs
Influenza virus HNP Virus aggregation; inhibition of PKC disrupts IAV endosomal 85,111,112
trafficking; enhanced neutrophil phagocytosis of IAV

Retrocyclins Virus aggregation; blockade of increased virus uptake by 113,274,275
professional phagocytes; RC2: haemagglutinin-mediated

fusion of viral and endosomal membranes
β-Defensins Inhibition of IAV infectivity at higher concentrations applied 114,115,218
before viral entry

LL-37 Disruption of viral envelope 116,217,276
Urumin Virion destruction, targeting H1 haemagglutinin 87
RSV HBD2 Viral envelope destabilization in solution or on exposure 88,277
to plasma membrane-associated HBD2

LL-37 Virion binding and destruction; prevention of infection 89,90
and spread; function retained by core 22-mer

Rhinovirus Cathelicidins Decreased infectivity and replication 91,95
Adenovirus α-Defensins Peptide binding to adenoviral capsid prevents uncoating 97–99,278
and nuclear entry of the viral genome; dependent on

optimal peptide hydrophobicity and charge
HPV-16 α-Defensins Inhibition of uncoating and nuclear entry of the viral genome 101
HSV α-Defensins, HSV binding to cellular receptors glycoprotein B and heparin 100,102,103,105
HBD3, retrocyclins sulfate inhibited; dependent on lectin-like properties rather

than charge
HIV HNP Disruption of cellular entry; inhibition of PKC activity, 106,279–281
interfering with HIV replication

Retrocyclins Blockade of viral entry into cells by peptide binding to 107–110,282–284
gp120 and CD4; dependent on lectin-like properties

β-Defensins Direct effects on virions; intracellular, postviral entry 285–287
inhibitory functions
LL-37 Suppression of HIV reverse transcriptase activity 288,289
Vaccinia virus Cathelicidins Damage to integrity of the double-layered viral envelope 92,290
Zika virus Cathelicidins Direct inactivation of Zika virus; protective modulation 93
of interferon signalling pathways

Kaposi’s sarcoma- Cathelicidin- Disruption of viral envelope 291
associated herpesvirus derived peptide

HBD, human β-defensin; HNP, human neutrophil peptide; HPV, human papillomavirus; HSV herpes simplex virus; IAV, influenza A virus;
PKC, protein kinase C; RC2, retrocyclin 2; RSV, respiratory syncytial virus.
in detail later) of the peptides on host immune cells such life-threatening fungal infections, and the development
as enhancing phagocyte function111,114 or by modifica- of antifungal drug resistance continues to increase118.
tion of cytokine responses115,116. These antiviral mecha- This is particularly evident for azole resistance because
nisms highlight the possibility that baseline expression of the widespread use of azoles in agriculture119. In addi-
of CHDP could create an ‘antiviral shield’ at mucosal tion, the worldwide number of immunocompromised
surfaces and prevent replication and spread of the virus patients is increasing, which leads to more fungal infec-
if upregulated after initial infection. Therapies aimed at tions. Both trends are very disturbing and demand the
inducing host CHDP expression, or the application of development of new potent antifungals that do not easily
synthetic CHDP-derived peptides with defined, selective elicit resistance. Furthermore, it is important that novel
properties, could therefore have both preventative and antifungal compounds be used only in humans to pre-
therapeutic potential. vent rapid resistance development in the environment.
Because of their diverse modes of action, CHDP-derived
Antifungal activity molecules may be used as paradigms for the develop-
Fungal infections in humans are a growing problem world- ment of potent antifungals. Antifungal CHDP from
wide. Many patients experience non-life-threatening plants and vertebrates have been described48. The activity
fungal infections of the skin and oral cavity. However, of plant defensins has been described primarily against
invasive infections by fungal species such as Aspergillus fungi120. Various killing mechanisms of CHDP with
fumigatus, Cryptococcus neoformans, Candida albicans regard to yeasts and fungi have been reported, ranging
and Histoplasma capsulatum are responsible for the from effects on mitochondrial functions of C. albicans
death of 1.5 million people annually117. Only a lim- by histatin 5 to membrane effects on this yeast by cathe
ited set of antifungals is available for the treatment of licidins and CHDP mimics121–123. It should be noted that

Reviews
Immune homeostasis
fungal biofilms are highly resistant to antifungals124. It is on factors such as the peptide concentration, the kinet-
Balanced and tightly regulated thus important to screen newly developed CHDP-based ics of response and the environmental stimuli (reviewed
immune response under antifungals for antibiofilm activity124. in refs12,15,128,129). The pleiotropic immunomodulatory
physiologically normal functions of CHDP raise questions regarding the primary
conditions.
Immunomodulatory actions of CHDP biological role of these peptides. In the following sub
The earliest studies of the non-microbicidal properties sections, we summarize the activities of CHDP on modu
of CHDP were on their effects on immune cells, related lation of immunity and inflammation (Fig. 3), focusing
primarily to the ability of these peptides to recruit leuko primarily on cathelicidins and defensins. Understanding
cytes125–127. Following this, research on immunity-related the mechanisms that underpin the ability of CHDP to
functions of CHDP increased exponentially over the modulate immunity to protect against infection, resolve
next two decades, defining a diverse range of functions. inflammation and contribute to immune homeostasis
Immunity-related functions of CHDP seem to be depen is critical to the development of novel therapeutic
dent on the environmental stimuli, cell and tissue type, approaches based on CHDP-derived peptides.
interaction with different cellular receptors and the
concentration of the peptides. Studies to date indicate Immune activation
that the molecular mechanism underpinning the ability Protective activation of the innate immune system
of CHDP to selectively modulate immune responses by CHDP has emerged as one of the key mechanisms
is highly complex, involving intracellular uptake of underpinning the ability of these peptides to promote
the peptides, which may or may not be mediated by early clearance of infections. The actions of CHDP
membrane-associated G protein-coupled receptors include recruiting leukocytes, modulating neutrophil
(GPCRs), interaction with several intracellular interact- responses and influencing antigen-specific adaptive
ing protein partners or receptors (for example, GAPDH immunity (discussed later). In addition, the interaction
and p62), alteration of several signalling pathways of CHDP and the microbiota in mucosal immunity is
(nuclear factor-κB (NF-κB), p38 and JNK mitogen- a rapidly emerging area of research. Although beyond
activated protein kinase (MAPK), MKP1 and phospho- the scope of this Review, this exciting area of research
inositide 3-kinase (PI3K)) and engagement of different suggests the potential for CHDP-mediated selectivity
transcription factors, all of which seem to be dependent in control of the microbiota130–133, but also the potential
Promotion
of NETs
Microbicidal/
microbiome modulation Wound healing
Recruitment
and polarization
Microbes Neutralization of LPS of T cells
Differentiation
LPS of dendritic cells
CHDP
Enhanced
Gram-negative Induction of phagocytosis
bacteria chemokines
Induction of Enhanced
anti-inflammatory pro-inflammatory
Altering cytokines response to
inflammatory DNA/RNA
cytokine
milieu Suppression of Altering endotoxin-induced LPS
pro-inflammatory cellular signalling Recruitment
cytokines of monocytes/
macrophages
Fig. 3 | Summary of immunomodulatory mechanisms of CHDP. Immunomodulatory functions exhibited by cationic

host defence peptides (CHDP) include but are not limited to: recruitment of antigen-presenting cells to the site of
infection either directly or indirectly by induction of chemokines to enhance antimicrobial effects, facilitating the
activation of neutrophil extracellular traps (NETs), altering endotoxin-mediated signalling pathways, suppression of
pro-inflammatory cytokines, enhancing phagocytosis and pro-inflammatory responses to nucleic acids, induction
of anti-inflammatory cytokines, influencing differentiation of dendritic cells and polarization of T cells, and wound
healing. LPS, lipopolysaccharide. Adapted from ref.15, Springer Nature Limited.

Reviews
for components of the microbiota to contribute to the antimicrobial functions. The ability of CHDP to mod-
regulation of CHDP expression. Further research on ulate the host cellular response to infections is not
CHDP–microbiota interaction may enhance our under- restricted to effects on neutrophils; these peptides can
standing of the consequences of microbial dysbiosis also modify other innate and adaptive cellular responses
in disease states, ageing and following treatment with (reviewed in refs12,14,29,33).
broad-spectrum antibiotics.
Antigen presentation and adaptive immunity. CHDP
Leukocyte recruitment. Immune cells (for example, serve as a link between innate and adaptive immunity,
neutrophils, macrophages, mast cells and T cells) exhibit as a consequence of their capacity to recruit antigen-
direct chemotactic activity towards CHDP and their presenting cells such as monocytes/macrophages and
IDR derivatives54,57,127,134–136. CHDP also indirectly pro- dendritic cells (DCs) to the site of infections (as dis-
mote recruitment of leukocytes by inducing the release cussed earlier). These peptides can also enhance phago
of chemokines10,128,136–139. The ability of these peptides cytosis in macrophages, facilitating clearance of bacteria
to induce chemokine release and enhance recruitment by boosting immune activation155–158. CHDP can not
of leukocytes has been defined as a primary immuno- only activate antigen-presenting cells but can also influ-
modulatory mechanism related to their ability to protect ence the generation and polarization of lymphocyte
against infections54,57,140. The underlying mechanisms responses, thus shaping the adaptive immune response.
involve several cellular receptors, such as chemokine For example, the human defensins HBD2 and HBD3 can
receptors (for example, CCR6 and CCR2), GPCRs, both induce production of interferon-α from plasma
including the formyl peptide receptors (reviewed in cytoid DCs and consequently influence the initiation of
refs12,141), and Toll-like receptors (TLRs)135,142,143, as well as T cell responses159. The expression of the co-stimulatory
interaction with intracellular proteins such as GAPDH molecules CD80, CD86 and CD40 on myeloid cells
and p62 (refs144,145). However, these functions appear to is also upregulated by exposure to HBD3, facilitating
be dependent on the phase of infection and inflamma- the promotion of adaptive immune response via the
tion, as it has been shown that LL-37 can mediate the engagement of TLRs160. LL-37 can modulate the adaptive
internalization of chemokine receptor CXCR2 on mono- immune response by multiple effector functions; LL-37
cytes and neutrophils, consequently dampening chemo- facilitates enhanced expression of co-stimulatory molec
taxis146. Molecular processes that control the dichotomy ules and promotion of a modified adaptive response
of CHDP to selectively induce chemokine secretion and via modulation of DC differentiation and function
enhance leukocyte recruitment, without altering the in vitro158,161 and in vivo162, promotes DC activation163,164
anti-inflammatory functions of the peptide (discussed and enhances the activation/proliferation of B cells via
in the next section), are not completely understood. activating follicular DCs165. The influence of CHDP on
adaptive immunity has led to the examination of appli-
Neutrophil function. Neutrophils are the major innate cation of these peptides, primarily cathelicidins, as adju-
immune effector cells of the early-phase response to vants to enhance systemic and mucosal antigen-specific
infections, and are a primary source of both defensins immune responses143,159,165.
and cathelicidins, which are stored preformed in the
secondary and primary granules, respectively, and are Regulation of inflammation
released on neutrophil degranulation. CHDP can influ- As discussed already, CHDP exhibit multiple functions
ence the function of neutrophils to modify infection to activate the immune system, which can be classified
outcomes. For example, these peptides can enhance as pro-inflammatory responses that aid in the clear-
influx of neutrophils both by direct chemotactic func- ance of pathogens. In contrast, potent CHDP-mediated
tion127,136,143 and indirectly by promoting the secretion of anti-inflammatory effects have been demonstrated in
neutrophil chemokines (for example, IL-8 and GRO-α) the presence of an inflammatory and/or pathogenic
in an MAPK-dependent manner137,147, to promote con- challenge, thus suggesting that CHDP are also regu-
trol of infections. Similarly, a recent study showed that latory molecules that limit enhanced inflammation.
the CHDP hepcidin induced in the skin during bacterial Therefore, rather than describing them as either pro-
infections can enhance chemokine production and pro- inflammatory or anti-inflammatory molecules, it may
mote neutrophil responses148. CHDP are also located on be appropriate to define CHDP as molecules that can
NETs, potentially contributing to NET-mediated anti- balance inflammation to promote immune homeosta-
bacterial effects149. LL-37 can facilitate NET formation sis. The anti-inflammatory function of CHDP is rein-
and contribute to antiviral activity, as reported for influ- forced by several studies that have demonstrated that
enza A virus (IAV)86. Recent studies have shown that deficiency of these peptides results in increased inflam-
post-translational modifications of CHDP, in particular matory responses; cathelicidin-deficient mice exhibit a
citrullination, may alter the functions of NET-associated more severe inflammatory phenotype compared with
CHDP such as cathelicidins150–152. LL-37 can also pro- wild-type mice154,166,167. Similarly, reduced expression of
mote bacterial clearance in vivo by enhancing early β-defensin in enterocytes of humans has been associated
neutrophil responses153. In addition, LL-37 can induce with Crohn’s disease168. Indeed the critical role of defen-
intracellular calcium mobilization and the generation of sins in maintaining the integrity of intestinal mucosa
reactive oxygen species (ROS)146, as well as enhance ROS and immune homeostasis is well established (reviewed
production mediated by other inflammatory stimuli154, in ref.34). Notably, exogenous application of CHDP (for
indicating the capacity to prime and enhance neutrophil example, LL-37, CATH-2, BMAP-28 and HBD2) and

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synthetic peptides (for example, IDR-1 and IDR-1002) via TLR2 and TLR4 (ref.182). This is one of the mecha-
has been shown to control inflammation in various ani- nisms described for cathelicidin-mediated dampening
mal models of infection and sepsis55,57,59,60,169–172. Similarly,
of inflammatory responses induced by bacterial pro
an LL-37-derived peptide controlled the disease products, which may be especially important in pulmonary
cess in a mouse model of inflammatory arthritis20, infections to protect the respiratory epithelium172.
and IDR-1002 effectively alleviated airway inflamma- Similarly, human defensins (for example, HBD3)
tion in vivo21. In contrast, cathelicidin-deficient mice exhibit antiendotoxic properties in vitro and in vivo, via
demonstrate increased survival in a caecal ligation and modulation of TLR signalling pathways184,185, but also
puncture model of sepsis, despite increased expression induce the production of pro-inflammatory cytokines
of pro-inflammatory genes166. Therefore, the outcome of by monocytes160, increase TLR9-dependent responses to
CHDP-mediated immunomodulatory responses seems bacterial DNA186 and modify melanoma differentiation-
to be context dependent and reliant on the cellular associated protein 5 (MDA5)-mediated and TLR-
environment. This has been demonstrated in studies mediated responses to poly(I:C)187. In addition, HBD3
examining the crosstalk between TLRs and CHDP, can induce the maturation of DCs160, while HBD2 and
as discussed next. HBD3 can induce TLR9-dependent plasmacytoid DC
interferon-α release, secondary to interaction between
Crosstalk of CHDP with TLRs. Several studies have the peptides and otherwise non-inflammatory DNA to
demonstrated that cathelicidins exhibit potent anti- mediate in vivo adjuvant properties188. This is similar
endotoxin properties in vitro and in vivo10,138,169,173,174, to the capacity to mediate TLR7-, TLR8- and TLR9-
both by binding bacterial LPS173,174 and by intervening dependent immunogenic responses to self-DNA/RNA,
in TLR signalling mechanisms138. The ability of cathe which was first described for LL-37 (refs 163,164,189) .
licidins such as LL-37 to modulate TLR-to-NF-κB sig- However, LL-37 enhances TLR3-mediated responses190,191
nalling is not restricted to the effect on TLR4, as LL-37 and promotes release of IL-1β192. These observations
also dampens responses to TLR2/TLR1 agonists and demonstrate the potential of CHDP to modulate innate
whole bacteria10,175. Downstream outcomes of CHDP- and downstream adaptive responses via their impact on
mediated modulation of the TLR-to-NF-κB pathway in pattern recognition signalling pathways.
the presence of inflammatory stimuli result in the selec-
tive suppression of specific pro-inflammatory responses, Modulation of cytokine-mediated responses. Limited
such as production of tumour necrosis factor (TNF) studies have demonstrated the ability of CHDP, espe-
and ROS, without the neutralization of innate immune cially LL-37, to modulate cytokine-mediated responses
functions such as chemokine production, and concur- in various cell types. LL-37 induces the expression of
rent enhancement of anti-inflammatory mediators such the IL-1 family of genes, including T helper 17 cell/
as IL-10, IL-1RA, A20 and NF-κB inhibtor-α138,176–179. T helper 1 cell-related genes such as IL6 and IL23A
A caveat is that the effect of endogenous CHDP to limit in keratinocytes 193. Consistent with this, another
inflammation may be impeded by peptide modifications study demonstrated that LL-37 can synergize with the
that occur under inflammatory conditions. A recent cytokines IL-1β and granulocyte–macrophage colony-
study demonstrated that the enzyme peptidyl arginine stimulating factor to enhance chemokine production,
deaminase released by inflammatory cells can mediate but not with cytokines such as IL-4 or IL-12, in human
citrullination of LL-37, which compromises the ability peripheral blood mononuclear cells194. In contrast, LL-37
of the peptide to intervene in TLR signalling to dampen suppresses cytokine IL-32-induced pro-inflammatory
pro-inflammatory responses in macrophages and to con- cytokines such as TNF, IL-6 and IL-1β by activating
trol sepsis in vivo150. Therefore, it is essential to consider the dual phosphatase MKP1 which is a known negative
the composition of the inflammatory milieu in studies regulator of inflammation, without altering its ability
that aim to define or optimize the anti-inflammatory to induce chemokine production176. The mechanisms
functions of cationic peptides. that control the ability of CHDP such as LL-37 to limit
The modulation of TLR-mediated signalling by inflammation without altering chemokine production
CHDP is not necessarily ‘anti-inflammatory’, as these are not completely understood. To that end, a recent
peptides can also induce chemokines such as IL-8 and study demonstrated that LL-37-induced chemokine
MCP1 (pro-inflammatory response) to attract leuko release and subsequent leukocyte recruitment are
cytes at the same time as inhibiting LPS-i nduced selectively mediated by GPCRs via the CDC42 RHO
TNF10,138,180. Similarly, cathelicidins can also exhibit GTPase pathway, without impacting LL-37-induced
both pro-inflammatory and anti-inflammatory activi- anti-inflammatory cytokine release137.
ties depending on the phase of the infection. For exam-
ple, CATH-2 enhances the sensing of bacterial DNA181 Cell death
and is not anti-inflammatory if the bacterial infection Although CHDP can rapidly permeabilize prokaryo
has not been completely resolved181,182, while LL-37 tic membranes, most natural peptides are relatively
promotes acute protective pro-inflammatory responses less toxic to eukaryotic cells. CHDP such as LL-37 and
primarily in the context of an infection, including via CATH-2 can enter eukaryotic cells181,195 and facilitate
activation of the inflammasome, but not in the absence the entry of nucleic acids and DNA dyes181,192,196 with-
of pathogens153,183. However, in instances where the out inducing cell lysis, suggesting a temporary mem-
bacteria are killed, both LL-37 and CATH-2 exhibit brane disruption or pore opening. However, exposure
anti-inflammatory activity and can prevent activation to high concentrations of LL-37 induces eukaryotic cell

Reviews
apoptosis in vitro and in vivo197–200. This phenomenon is Several factors have limited the success of oral or
cell type dependent, with the viability of primary human systemic use of CHDP in preclinical studies, including
lymphocytes and monocytes relatively unaffected by (1) high local concentrations of CHDP, (2) concerted
exposure to high concentrations of the peptide158,201. actions of different CHDP and (3) synergism with other
The impact of CHDP-mediated induction of apo molecules at the site of infection. However, many in vivo
ptosis on innate or adaptive responses in vivo remains studies have shown efficacy of CHDP-derived peptides
unknown. However, high concentrations of LL-37 have when these are administered topically. For example, a
been found to preferentially induce death in infected recent study showed that topical application of a pep-
epithelial cells and represent a potential additional tide designed using LL-37 and tachyplesin 1 as chemi-
host defence mechanism183,198. Furthermore, different cal benchmarks was protective in a methicillin-resistant
modes of cell death have important roles in maintain- Staphylococcus aureus mouse model212. Similarly, local-
ing immune homeostasis and in amplifying or damp- ized administration of a CHDP-derived peptide was
ening, and later resolving, inflammatory responses. shown to be better in lowering bacterial load compared
In this context, the control of neutrophil death and with rifampicin treatment for tuberculosis213. Moreover,
anti-inflammatory properties of apoptotic neutrophils nanostructure-based technology has recently shown
is particularly important202. LL-37 can induce rapid promise as an effective delivery system for slow release
secondary necrosis of apoptotic neutrophils177,203, with of peptides for infection control in vivo214.
anti-inflammatory effects on activated macrophages A growing area of interest is the potential use of
being associated with LL-37-mediated release of gran- CHDP with indwelling medical devices, prosthetic
ule contents from the apoptotic cells, perhaps result- joints and other implants for the prevention of noso-
ing from release of LL-37 and α-defensins from these comial infections215,216. Bacterial biofilm formation on
apoptotic neutrophils204. Thus, CHDP-mediated regu- medical devices includes pathogens recalcitrant to anti-
lation of cell death has the potential to affect the mag- biotics, which results in biomaterial-associated infec-
nitude and the resolution of inflammatory responses tions, a major problem in clinical practice. CHDP may
to infection. be immobilized on the surfaces of biomaterials to pre-
vent adhesion of bacteria. For example, synthetic pep-
Development of CHDP-based therapies tides designed from LL-37 and a trombocidin-derived
On the basis of the properties discussed so far, many peptide were shown to be effective in inhibiting biofilm
CHDP from either prokaryotes or eukaryotes and their formation by a biomaterial-associated clinical isolate
derivatives are currently being investigated for a vari- of S. aureus215. In addition, depending on the chemical
ety of indications, including their use as antimicrobials tethering procedure, it is possible to retain antibacte-
and anti-inflammatories, as well as their application in rial activity of CHDP after the surface has been coared.
wound healing. The disadvantage of this approach is that only bacteria
in the immediate vicinity of the surface are killed. Appli
Antimicrobial therapies cation of CHDP-releasing biomaterials may be a better
Preclinical studies. Studies in murine models have approach to prevent infections from implants. It has
demonstrated the critical role of CHDP in the control been reported that several hydrogels and also nanotubes
of infections. For example, cathelicidin-deficient mice205 and microporous calcium phosphate coatings inhibit
exhibit less effective host defence against streptococcal bacterial growth in vivo216.
skin infections205, impaired clearance of Pseudomonas Another potential application of CHDP is their devel-
aeruginosa infections from the lung206 and the cornea207, opment as potential first-line antiviral treatments for
develop severer pox skin lesions on infection with vac- use during pandemics, where there is insufficient time
cinia virus92, develop severer infection with RSV on to produce vaccines (such as new influenza (IAV) pan-
challenge90 and exhibit increased susceptibility to infec- demics), or for viral infections for which vaccines are not
tion with Citrobacter rodentium in the intestinal tract208, available (such as for RSV infections), and more broadly
Escherichia coli in the urinary tract209 and Klebsiella for other viral pathogens. An early proof-of-concept
pneumoniae in the lung210. The impaired antimicro- study in mice demonstrated that gel-based application of
bial activities observed in these cathelicidin-deficient HD5 protected against HSV infection102. More recently,
mice may be explained by the absence of direct effects intravaginal instillation of a synthetic defensin, identi-
of the mouse cathelicidin CRAMP on these pathogens, fied by HD5-mutant screening, showed prophylactic
although the observed effects may also be the result of and/or therapeutic efficacy in a lethal HSV-2 infection
altered immunomodulation in the knockout mice. model103. In another example, prophylactic application
Exogenous administration of many CHDP has been of retrocyclin 2 in a murine HSV-mediated ocular ker-
found to be effective in various animal infection mod- atitis model modestly reduced viral titres and reduced
els for bacterial, viral and fungal infections (reviewed disease105 but had no effect on disease pathology when
in refs12,211). However, it remains to be determined if applied after infection. Intranasally administered human
the observed efficacy is primarily due to the direct anti- and murine cathelicidins have shown in vivo antiviral
microbial activities of the administered CHDP on the activity equivalent to that of current first-line neuramin
pathogen or is due to the effect of the peptides on host idase inhibitors in a lethal IAV mouse model when
Immunomodulation
The process of activating,
immunity, or a combination of both. Therefore, the applied concomitantly with the virus and daily thereafter,
suppressing or altering the mode of action of the peptides needs to be carefully dramatically increasing survival despite modest effects
nature of immune function. examined in vivo. on viral load116. Similarly, intranasal delivery of urumin87,

Reviews
intranasal delivery of mouse BD2 (mBD2; optimal trial demonstrated that supplementation with vitamin D
when premixed with the virus before infection217), was beneficial to control exacerbations in COPD, espe-
intravenous delivery of recombinant mBD3 (ref.218) cially for patients deficient in vitamin D224. Vitamin D3
and intramuscular delivery of mBD1–mBD3 fusion causes the induction of LL-37 in macrophages, which
genes219 were all protective in murine lethal IAV infection has also been associated with intracellular killing of
models. These studies all demonstrate the therapeutic Mycobacterium tuberculosis in a human trial225. Similarly,
potential of CHDP as antivirals in vivo, although further orally administered phenylbutyrate, with or without
investigation is needed. Many in vivo studies suggest that vitamin D supplementation, leads to the induction of
while early direct contact of the virus and CHDP may be LL-37 in macrophages and lymphocytes, and has been
protective due to direct damage to the virions, later-stage evaluated in the treatment of adults with active pulmo-
modulation of host immune/inflammatory responses by nary tuberculosis225. Therefore, strategies to enhance
the peptides may also be critical in the antiviral activ- endogenous CHDP production may be valuable for
ity of a CHDP115,220. Such observations highlight the antimicrobial therapies to counter challenges associated
importance of studies that address both the direct micro with peptide delivery, stability and bioavailability.
bicidal activities and the immunomodulatory properties In summary, only a handful of peptide-derived treat-
of these peptides. ments have made it to market. They include PAC-113, a
histatin analogue that is being sold in Taiwan as a topical
Clinical trials. Most CHDP in clinical trials so far have treatment for oral candidiasis, and dalbavancin, a semi-
been formulated for topical applications or as inhalants synthetic lipoglycopeptide that has been approved in the
for the treatment of infections221 (see Dramp Database USA for intravenous treatment of acute skin infections.
and Table 2). One of the most advanced of these was Although the failure rate has so far been high, the number
pexiganan, an analogue of the magainin peptide, which of CHDP in clinical trials has grown rapidly and is likely
was tested as a topical cream for the treatment of infected to lead to success in the future.
diabetic foot ulcers in phase III clinical trials. However,
development was terminated because it did not perform Immunomodulatory therapies
better than current treatments. There are several trials in Preclinical studies. Early clinical trials using synthetic
progress using omiganan, a CHDP-derived antimicrobial analogues of CHDP that had been designed to maximize
compound (detailed in Table 2). For example, a phase III microbicidal activity achieved only moderate efficacy226,
trial evaluating the long-term safety for topical applica- perhaps due to failure to recognize the importance of
tion of omiganan for treatment of rosacea is ongoing. In immunity-related functions of these peptides. Despite
addition, localized application of a human lactoferricin- issues of concentration at mucosal surfaces and antago-
derived peptide PXL01-containing hydrogel was shown nizing factors at sites of inflammation, CHDP are clearly
to be safe, well tolerated and effective as antiadhesion essential for the control of infections in vivo90,92,205–210.
treatment postoperatively after tendon repair surgery in Application of LL-37 is protective against infection with
an inpatient phase II clinical trial222. Also, clinical trials P. aeruginosa, influenza viruses and RSV in vivo89,116,227,
are ongoing for topical application of LL-37 for treatment with the mode of action involving enhanced, protective
of venous leg ulcers223 (Table 2). early neutrophil responses, rather than direct micro
A small number of clinical trials investigating the toxi bicidal activity against the pathogen153. Comparable
city and efficacy of CHDP using oral and intravenous results were also obtained after in ovo application of
administration routes have also been conducted or are CATH-2 in chickens to induce a long-lasting protection
ongoing (Table 2). For example, iseganan, an analogue of against respiratory E. coli infections228. Similarly, CHDP-
the peptide protegrin, was used as an oral solution for derived synthetic peptides such as IDR peptides were
treatment of oral mucositis in a phase III clinical trial also shown to be protective in various infection models
but did not show significant efficacy (Table 2). Similarly, wherein IDR peptides protect the host from the patho
surotomycin and iseganan completed phase III trials but gen by modulating the host immune response and, in
were rejected for ongoing development due to either parallel, suppress the release of inflammatory cytokines
poor efficacy or efficacy that was not superior to that of such as TNF and IL-6, suppress ROS and dampen
current drugs (Table 2). Phase III clinical studies using neutrophil degranulation54,57–59. These studies demon-
intravenously administered brilacidin, a synthetic defen- strate that CHDP or related synthetic peptides can pro-
sin mimetic, for treatment of skin infections are start- vide protection against infections by modulating host
ing soon. Recently, phase III trials of murepavadin as immune response rather than by directly targeting the
an intravenous treatment for bacterial pneumonia were pathogen.
terminated due to increased serum creatinine levels in Synthetic IDR peptides are being developed primar-
patients, indicative of acute kidney injury. This is rem- ily as immunomodulatory therapies to control infec-
iniscent of nephrotoxicity issues with polymyxins, the tions, with particular focus on antibiotic resistant
cationic nonribosomal peptides which were used for infections. IDR-1 was the first such peptide shown to
treatment of Gram-negative bacterial infections and are be protective in vivo against both Gram-positive and
currently used as the antibiotics of last resort. Gram-negative bacteria, including antibiotic-resistant
Another strategy being evaluated and tested in clinical infections such as methicillin-resistant S. aureus and
trials is to enhance the expression/production of endog- vancomycin-resistant Enterococcus and Salmonella
enous CHDP for chronic inflammatory and infectious enterica serovar Typhimurium54. Subsequently, an ana-
disease. A multicentre, double-blind, randomized clinical logue of IDR-1, peptide SGX94, showed broad-spectrum

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Table 2 | Selected antimicrobial peptides that are in clinical trials or approved

Peptide Origin Indication Status Company Clinical trial identifiers
Topical
Pexiganan Analogue of Infected diabetic foot Phase III complete; PLx Pharma Inc. NCT00563394, NCT00563433,
(Locilex, MSI-78) magainin, isolated ulcers rejected, efficacy (formerly Dipexium NCT01590758, NCT01594762
from African not superior Pharmaceuticals Inc.)
clawed frog
Xenopus laevis
D2A21, Demegel Synthetic cecropin Burn wound Phase III Demegen Not listed
peptide infections
CLS001 Omiganan Local catheter site Phase III complete Mallinckrodt, NCT00231153, NCT00027248,
(omiganan, pentahydrochloride, infections (discontinued) BioWest 2005-003194-24
MBI-226) synthetic cationic Therapeutics
indolicidin derivative Inc., Cadence
Pharmaceuticals Inc.
Topical skin antisepsis Phase III complete Mallinckrodt NCT00608959
Papulopustular Phase III current Cutanea Life NCT02576860, NCT02547441,
rosacea Sciences Inc. NCT02576847, 2015-002921-20,
2015-002919-15, 2015-002920-23
Acne vulgaris Phase II complete Cutanea Life NCT02571998, NCT02066545,
Sciences Inc., NCT00211497, NCT00211523
BioWest
Therapeutics Inc.
Atopic dermatitis Phase II complete Cutanea Life NCT03091426, NCT02456480,
Sciences Inc. 2016-003849-28, 2014-003689-26
Vulvar intraepithelial Phase II complete Cutanea Life NCT02596074, 2015-002724-16
neoplasia Sciences Inc.
Condylomata Phase II complete Cutanea Life NCT02849262, 2015-005553-13
acuminata (external Sciences Inc.
genital warts)
Facial seborrhoeic Phase II current Cutanea Life NCT03688971, 2017-003106-41
dermatitis Sciences Inc.,
Maruho Co. Ltd
Iseganan (IB-367) Analogue of Ventilator-associated Phase II/III; IntraBiotics NCT00118781
protegrin 1 pneumonia rejected, no Pharmaceuticals
efficacy
PXL01 Synthetic Prevention of Phase IIb complete; Promore Pharma NCT01022242, 2009-012703-25
macrocyclic postsurgical phase III trials (formerly
25-amino acid adhesions and scar planned Pergamum AB)
peptide derived prevention
from human
lactoferricin
NVXT (Novexatin Cyclic arginine- Fungal nail infection Phase IIb complete NovaBiotics NCT02933879, NCT02343627
NP213) based heptamer (onychomycosis)
PAC-113, P-113 Histatin 5 derivative Oral candidiasis Phase IIb complete Demegen, Pacgen NCT00659971
(12 amino acids) Biopharmaceuticals
Corporation; sold
over the counter
in Taiwan by
General Biologicals
Corporation
LL-37 Human cathelicidin Venous leg ulcers Phase IIb current Promore Pharma 2018-000536-10
subunit (formerly
Pergamum AB)
HXP124 Plant defensin Fungal nail infection Phase IIa complete Hexima ACTRN12618000131257
(onychomycosis)
Brilacidin Synthetic defensin Ulcerative Phase II complete; Alfasigma S.p.A. Not listed
(PMX-30063) mimetic proctitis/ulcerative phase III planned
proctosigmoiditis
Oral mucositis in Phase II complete; Innovation NCT02324335, NCT01211470
patients with head FDA fast track Pharmaceuticals
and neck cancer designation (formerly Cellceutix)

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Table 2 (cont.) | Selected antimicrobial peptides that are in clinical trials or approved
Topical (cont.)
LTX-109 (Lytixar) Synthetic cationic Atopic dermatitis, Phase II complete Lytix Biopharma NCT01223222, 2010-021438-68
tripeptide skin infection
Impetigo Phase II complete Lytix Biopharma NCT01803035
Nasal infections by Phase I/II complete Lytix Biopharma NCT01158235, 2010-019254-40
methicillin-resistant/
methicillin-sensitive
Staphylococcus aureus
(CKPV)2, Derivative of Vulvovaginal Phase II complete Zengen, Abiogen 2005-001360-31
CZEN-002 α-melanocyte candidiasis Pharma
stimulating hormone
OP-145 Cathelicidin family Chronic suppurative Phase II complete OctoPlus BV, ISRCTN12149720
(AMP60.4Ac) (LL-37 derivative) otitis media (middle Dr Reddy’s
ear infections) Research and
Development BV
C16G2 Synthetic peptide Prevention of Phase II complete Armata NCT03052842, NCT03004365,
dental caries due to Pharmaceuticals NCT02594254, NCT02509845,
Streptococcus mutans NCT02254993, NCT02044081,
NCT03196219
DPK 060 Derived from Acute external otitis Phase II complete DermaGen AB NCT01447017, 2011-004356-20
kininogen, cationic and Promore
random-coil peptide Pharma (formerly
Pergamum AB)
Atopic dermatitis Phase I/II complete DermaGen AB NCT01522391
and Promore
Pharma (formerly
Pergamum AB)
Lotilibcin Lipodepsipeptide Methicillin-resistant Phase I complete aRigen Not listed
(WAP-8294A2) S. aureus Pharmaceuticals,
Green Cross
Corporation
PL-5 α-Helical peptide Bacterial skin Approval by State Changchun Not listed
infections Food and Drug ProteLight
Administration of Pharmaceutical &
China for clinical Biotechnology Co.
trial
Oral
Surotomycin Cyclic lipopeptide, Diarrhoea caused by Phase III complete; Cubist NCT01597505, NCT01598311,
(CB-183, 315) analogue of Clostridioides difficile rejected, efficacy Pharmaceuticals, 2012-000252-3
daptamycin not superior Merck & Co. Inc.
Iseganan (IB-367) Analogue of Oral mucositis in Phase III complete; National Cancer NCT00022373
protegrin 1 patients with head no efficacy Institute, IntraBiotics
and neck cancer Pharmaceuticals
RDP58, Delmitide d-Amino acid Ulcerative colitis Phase II complete Genzyme, Procter 2004-004077-29
acetate, allotrap decapeptide & Gamble
1258
NVB-302 Synthetic type B C. difficile infection Phase I complete Novacta ISRCTN40071144
lantibiotic
Intravenous
Dalbavancin Semisynthetic Acute bacterial skin Approved Allergan (formerly NCT03233438
(BI397, Dalvance, lipoglycopeptide infections Actavis and Durata
Xydalba) Therapeutics)
Osteomyelitis and Phase IV current NCT03426761
septic arthritis
AB103 (p2TA) Synthetic anionic Necrotizing soft Phase III current Atox Bio Ltd NCT02469857, 2018-001125-15
CD28 dimer mimetic tissue infections
peptide
Dusquetide Synthetic 5 amino Oral mucositis in Phase III current; Soligenix NCT03237325, 2017-003702-41
(SGX942) acid peptide derived patients with head FDA fast track
from indolizidine, and neck cancer designation
immunomodulator

Reviews
Table 2 (cont.) | Selected antimicrobial peptides that are in clinical trials or approved
Intravenous (cont.)
Murepavadin Synthetic Ventilator- Phase III; Polyphor Ltd NCT03409679, NCT03582007
(POL7080) cyclic β-hairpin associated bacterial suspended, adverse
peptidomimetic pneumonia caused events
based on the cationic by Pseudomonas
antimicrobial aeruginosa
peptide protegrin 1
Neuprex, BPI-derived peptide Burns Phase II complete University of Texas NCT00462904
opebacan, Southwestern
BPI rBPI21 Medical Center
Myeloablative Phase I/II; Xoma LLC NCT00454155
allogeneic terminated, lack
haematopoietic stem of enrolment
cell transplantation
Brilacidin Synthetic defensin Acute bacterial skin Phase II complete; Innovation NCT02052388
(PMX-30063) mimetic and skin structure phase III planned; Pharmaceuticals
infections FDA fast track (formerly Cellceutix)
designation
EA-360 Linear tetrapeptide, Systemic Phase IIa/b current Exponential NCT03145220, 2014-002481-78
derived from inflammatory Biotherapies
human chorionic response and renal
gonadotropin function
hLF1-11 First cationic domain Infections during Phase I/II complete; AM-Pharma NCT00509938, NCT00430469
of human lactoferrin haematopoietic stem withdrawn
(11 residues) cell transplantations
Candidaemia Phase I/II; AM-Pharma NCT00509834
withdrawn
Bacteraemia due Phase I/II; AM-Pharma NCT00509847
to Staphylococcus withdrawn
epidermidis
Friulimicin B Cyclic lipopeptide Pneumonia, Phase I; rejected, MerLion NCT00492271
staphylococcal skin unfavourable Pharmaceuticals
infections pharmacokinetics
activity against bacterial infections solely by modulat- colitis murine model232,233. Similarly, therapeutic admin-
ing the host immune response to control infections istration of exogenous LL-37 or mouse CRAMP can
in preclinical studies229. Other IDR peptides, such as modulate Clostridioides difficile colitis by inhibiting
IDR-1002 and IDR-1018, have also been shown to toxin A-associated intestinal inflammation234. These
be beneficial in preclinical models of a wide range of studies suggest that CHDP and/or derived synthetic
pathogens, including multidrug-resistant bacteria, peptides have the potential to control chronic inflam-
viruses, parasites and antibiotic-recalcitrant bacte- matory diseases. Consistent with this, limited preclin-
rial biofilms56–59. In general, the mechanism of action ical studies have demonstrated the beneficial effects
of IDR peptides is primarily mediated by influencing of CHDP-derived synthetic peptides for treatment of
host innate immune responses to enhance immune cell chronic inflammatory diseases such as in inflammatory
recruitment to the site of infection to promote bacterial arthritis20, asthma21, and colitis22 models. For example,
clearance rather than by directly targeting the patho- exogenous administration of an LL-37-derived synthetic
gen. IDR peptides also influence neutrophil functions peptide prevented the development of arthritis, sup-
to augment neutrophil-mediated killing of bacteria and pressed autoantibodies and prevented cartilage degra-
the release of natural CHDP from neutrophils230. dation of the joints in a collagen-induced arthritis mouse
In addition to the potential application of CHDP in model20. Elafin delivered by adenovirus ameliorated coli-
infections, the immunomodulatory functions of these tis and suppressed inflammatory cytokines and related
peptides are being examined for use in other indications. NF-κB activation in a non-infectious DSS-induced colitis
Cathelicidin-deficient mice develop severer colitis in a murine model22. In addition, the peptide IDR-1002, a
non-infectious dextran sulfate sodium (DSS)-induced bactenecin derivative, improved allergen-induced airway
model with greater pro-inflammatory cytokine expres- hyper-responsiveness, controlled airway inflammation
sion and cell death compared with wild-type controls, and suppressed inflammatory cytokine IL-33 produc-
and with impaired mucus production231. In contrast, tion in an allergen-challenged animal model of asthma21.
therapeutic intrarectal administration of the mouse These preclinical studies suggest that CHDP and their
cathelicidin CRAMP (or mouse CRAMP-expressing derivative peptides can be developed for treatment of
Lactococcus lactis) is protective in the DSS-induced non-infectious inflammatory diseases.

Reviews
Due to the ability of CHDP to regulate inflamma- with no local or systemic adverse reactions, and to
tion, many studies have also explored the effects of these enhance healing of hard-to-heal venous leg ulcers223
peptides on cancers. However, the therapeutic use of (Table 2). A recent notable study in current clinical devel-
CHDP-based peptides for cancers remains controver- opment is the use of silicone hydrogel contact lenses
sial, as the effect of these peptides on cancer pathology coated with a synthetic immunomodulatory peptide,
seems to be dependent on the specific type of cancer235. Mel4, to reduce contact lens-associated infections and
Discussion of the nuances of the pharmaceutical rele- inflammation248. Notably, the potential therapeutic
vance of CHDP in cancer pathology is beyond the scope and prophylactic success of immunomodulatory CHDP-
of this Review236,237. Nevertheless, a gene therapy study derived peptides as alternatives to antibiotics in veteri
using HBD2 showed that application of this peptide nary medicine may pave the way for clinical trials in
resulted in enhanced local antitumour effects in pre- humans (One Health approach)249.
clinical murine models using CT26, MethA and LL/2
tumour cells, the mechanism of which was associated Considerations in CHDP-based drug development
with immunomodulatory functions of the peptide, The use of natural CHDP as effective therapeutics is
namely its ability to activate endogenous DCs23. not particularly viable as the concentrations that exhibit
Another promising avenue for harnessing the direct antimicrobial effects are relatively high, and in
immunomodulatory functions of IDR peptides is their that concentration range these peptides exhibit cyto-
application as adjuvants for new vaccine formulations, as toxic effects such as mast cell degranulation, comple-
IDR peptides enhance mucosal immunity and antigen- ment activation and apoptosis of mammalian cells, and
specific humoral responses61–64. Adjuvant formulations induce pro-inflammatory cytokine production11,12,15,250.
with IDR peptides have shown promise in various pre- Thus, synthetic peptides derived from natural CHDP,
clinical studies for vaccines against mycoplasma63, IAV synthetic designed peptides and peptides found by semi
HINI strain238 and pertussis239. In these studies, IDR pep-random high-throughput screening are now emerging
tides with modest antimicrobial activity used in different as putative lead compounds. Recent studies have also
vaccine formulations showed profound immunomod- focused on non-peptide CHDP mimics such as peptoid
ulatory effects. For example, a bactenecin-derived IDR analogues and developing compounds using CHDP
peptide used in a vaccine formulation with Mycoplasma on small abiotic scaffolds for therapeutic applications
bovis proteins elicited a balanced humoral IgG1/IgG2 (reviewed in ref.24).
response for use in cattle63. Intranasal administration of Major challenges facing peptide-based drug develop-
a nanoparticle-based vaccine formulation containing an ment include formulation and delivery, as well as high
immunomodulatory IDR peptide as an adjuvant resulted production cost. Biological factors that affect peptide
in a strong cellular and humoral response against IAV stability and bioavailability must be taken into consider-
H1N1 strain238. The potential use of IDR peptides as an ation; for example, mucosal pH and the presence of host
adjuvant in vaccines is strongly supported by studies or microbial proteases that can degrade candidate pep-
demonstrating that the presence of IDR peptides in these tides251–253, as well as several other factors that can impair
formulations is effective in eliciting a balanced type 1 peptide activity, such as physiological salt concentration,
T helper cell/type 2 T helper cell immune response along mucus, DNA and microbial saccharides251,253. Several
with facilitating the reduction of antigen dose required approaches are therefore being explored to enhance
for immunity240. peptide stability, such as using d-amino acid peptides
The application of immunomodulatory CHDP in and modification of peptides by amidation or acetylation
wound healing is also being explored, driven by studies of the terminal regions or by targeted replacements of
showing that growth factors associated with stimulation tryptophan or histidine with a non-natural amino acid
of regeneration of tissues also induce the production of (reviewed in ref.211).
endogenous CHDP such as LL-37, β-defensins and The antimicrobial activity of CHDP is generally less
lipocalins in keratinocytes241. Aligned with this, in vitrothan that of conventional antibiotics. Regulatory author-
studies have demonstrated that CHDP such as defensins ities require new antimicrobials to be non-inferior to
and LL-37 promote angiogenesis and wound healing242–245, existing antibiotics, even if the new compounds do not
prevent protease-mediated skin barrier damage246 and elicit antimicrobial resistance. CHDP may be unsuit-
promote re-epithelialization of wounds247. These pheno able for standard in vitro antimicrobial susceptibility
typic changes are driven by peptide-mediated activation test methods to predict in vivo efficacy, even for topi-
of signalling intermediates and transcription factors cal application, and thus more physiologically relevant
that activate EGFR through the induction of a GPCR, modified approaches may be vital254. Taking all this into
in particular for the peptide LL-37 (ref.243). Accordingly,consideration, the oral or systemic use of CHDP, with
application of LL-37 was shown to be effective in a mouse the aim of directly killing microorganisms, will likely
model of excisional wound243. be difficult to achieve. However, discoveries of CHDP
demonstrating effects against antibiotic-resistant patho
Clinical trials. Despite many preclinical studies describ- gens do hold promise216, as does the new direction of
ing the immunomodulatory therapeutic potential of applying immunomodulatory CHDP as an adjunct to
CHDP-derived peptides, there have been very few suc- antibiotics, due to the observed synergy of CHDP with
cessful clinical trials. The human cathelicidin LL-37 has conventional antibiotics255–258.
been applied in a first-in-human randomized placebo- Additional approaches that are being considered to
controlled clinical trial, and was shown both to be safe, counter the challenges associated with CHDP-based

Reviews
therapy include the use of nutritional supplements study of in vivo survival and pathogenicity of a CHDP-
such as vitamin D or phenylbutyrate or other products resistant S. aureus (evolved in vitro in the presence of
based on short-chain fatty acids to enhance the levels cationic peptides) showed that resistance to the pep-
of endogenous CHDP225. In addition, topical applica- tides provided no survival advantage to the bacteria in
tion of analogues of vitamin D was shown to enhance an insect host environment that is dominated by anti-
local expression of cathelicidin in psoriatic skin, which microbial peptides, and bacterial clearance was at least
would circumvent any challenges that may be related to as efficient as for the sensitive strains269. Nevertheless,
high systemic levels of vitamin D259,260. Also, formula- harnessing the immunomodulatory actions of CHDP
tions using nanoparticles or liposomes for slow release to selectively boost the host immune response rather
and targeted delivery of CHDP261–263 as well as the use than directly targeting the pathogen may be the
of shorter synthetic peptides such as IDR or cryptic path forward in the development of CHDP-b ased
peptides are being examined (discussed earlier). In anti-infective therapies.
preclinical studies56–59, shorter synthetic peptides such
as IDR peptides have demonstrated negligible toxicity Outlook
and no immunogenicity, and cost considerably less to The repertoire of functions exhibited by CHDP ranges
produce than most CHDP, thus making these valua- from direct antimicrobial activity to a wide range of
ble candidates to investigate for clinical application. In effects on host defence mechanisms, highlighting
addition, considerably less peptide is required if CHDP the critical role of these molecules in infection and
are used as immunomodulatory agents compared with immunity. Although, research in this field was ini-
their use as direct antimicrobial agents. Furthermore, tially focused on the development of new ‘antibiotics’
new production methods such as the efficient use of based on cationic antimicrobial peptides, it is now well
expression systems rather than chemical synthesis appreciated that CHDP have a critical role in immunity,
reduce the costs of production216,264,265. from activation of innate immunity, enhancement of
Although CHDP offer a promising approach to antigen presentation and phagocytosis, to influencing
treat infections, a key challenge for the use of CHDP adaptive immunity and memory functions, along with
or related synthetic analogues as antimicrobials is the potent anti-inflammatory functions. It is thus not sur-
development of pathogen-associated resistance mecha- prising that research in this field has intensified in the
nisms. Indeed, resistance of pathogens to human CHDP context of drug development for a variety of clinical
has been demonstrated, with multiple mechanisms applications ranging from the control of antibiotic-
adapted by bacteria to evade the direct antimicrobial resistant pathogens, alleviation of inflammation in
effects of the peptides266–268 (Box 1; Fig. 4). However, chronic disease and their use as antibiotic adjuvants,
bacteria have a limited number of ways to resist CHDP, to the targeting of specific cancers. However, there are
and this resistance is often costly. An important recent many challenges associated with CHDP-based drug
Box 1 | Development of antimicrobial resistance to CHDP

A major consideration for the potential application of cationic host defence peptides (CHDP) as new-generation
antibiotics will have to include a thorough understanding of the frequency of resistance development of pathogens
to peptide-based therapies. CHDP have promise over small-molecule antibiotics because the surfaces with which they
interact with targets in the pathogen are larger270 and hence single amino acid substitutions are unlikely to lead to
adaptations of bacteria to mitigate CHDP activity. Furthermore, CHDP have complicated mechanisms of action, often
interacting with more than one target in microorganisms, such that multiple mutations within the pathogen are needed
for ‘resistance’ to the peptides. Indeed, a recent study showed that bacterial adaptations to resist CHDP action do not
develop easily271. The additional, indirect CHDP-mediated effects of enhancing host immune responses to control
infections provide an important complementation to the direct microbicidal activities, providing a multifaceted attack on
pathogens during infection. Nevertheless, recent studies have revealed that bacterial and fungal pathogens are capable
of developing mechanisms to resist the effects of CHDP. The mechanisms of adaptation of pathogens to human CHDP have
been studied extensively and reviewed266–268. Common mechanisms in bacteria to counter the effects of CHDP include
repulsion, sequestration, removal and degradation (Fig. 4). Additional mechanisms of bacterial adaptations include
modification of the pentapeptide on lipid II, a prominent CHDP target, and altering the rigidity of the membrane by
acylation of lipid A. Mechanisms that fungi use to enhance tolerance of CHDP have focused mainly on Candida species,
which also use repulsion, sequestration, removal by efflux pumps and proteolytic degradation against peptides such as
LL-37, histatin 5, HNP1, HBD3 and lactoferrin272. Commensal bacteria of the host microbiota must be able to survive
the CHDP presented by epithelial and mucosal surfaces. This may in part be due to the relatively low concentrations of
CHDP at mucosal surfaces (except for specific niches such as the intestinal crypts) and somewhat inhibitory environments
in which secreted CHDP are present normally in the absence of inflammation. In addition, proteases that cleave
endogenous peptides to generate the active form of mature CHDP are also either absent or inactive in the absence of an
inflammatory response. In contrast, CHDP in the phagolysosome of a neutrophil, for example, are at a high concentration
in a controlled environment optimized for pathogen killing. Relative bacterial resistance to host CHDP is a prerequisite for
effective commensal colonization131, a property which may be most critical for stability through periods of inflammation,
where increased levels of CHDP may be capable of preferentially removing pathogens without totally decimating the
healthy microbiota. Harnessing this sort of selectivity would have clear therapeutic advantages over broad-spectrum
antibiotics and is indeed the focus of an exciting new approach to treating atopic dermatitis273 that is now being developed
in human clinical trials.

Reviews
a b
Capsule/EPS/biofilm Capsule/EPS/biofilm LPS
++
1 Protease digestion 2 Sequestration 2 Sequestration
++ ++ 1 Protease O antigen x40
+ ++ ++
+ ++ – – – – – – digestion ++
++ – – – – – –
++ + ++ Core
+
Peptidoglycan
8 Decrease negative P N N P Lipid A
++
charge (amination) 9 ↑OM rigidity
+Ala +Ala 5 Lipid II modification
WTA ++ LTA Outer (acylation)
3 Repulsion ++
membrane
G
+
M Peptidoglycan
PG P ++
+Lys P ++ ++
Membrane
+Ala
+
4 Phospholipid modification
PG
Inner +Lys
Cytoplasm membrane +Ala
++ 6 Efflux
7 Signal transduction pathways pumps 4 Phospholipid modification
Sigma Two-component
factors systems Cytoplasm
6 Efflux
Expression of cell wall biosynthesis, 1 Protease digestion + ++ ++ pumps
DNA damage, detoxification proteins +
c ++
2 Sequestration
++
++
Msb2
Mannoprotein
++
++ ++
++
β-Glucan/ 1 Protease
chitin
Msb2
digestion ++ ++
+
+ ++ ++
Membrane
Msb2
Msb2
P Phosphate
Cytoplasm
6 Efflux ++
pumps Cationic AMP
++ ++
G GlcNAc
10 MAPK signalling pathways
M MurNAc
Hog1 CWI
N Glutamine
ROS
Oxidative/osmotic Cell wall stress
stress response response Protein glycosylation
Fig. 4 | Common resistance mechanisms to CHDP in bacterial and fungal pathogens. a | Gram-positive bacteria.
b | Gram-negative bacteria. c | Yeasts/fungi. Mechanism 1, degradation by secreted proteases, outer membrane (OM)
proteases or cytosolic proteases. Mechanism 2, sequestration by secreted proteins, anionic polysaccharides or
mannosylphosphate side chains on glycoproteins (fungi) or O antigen (Gram-negative bacteria). Mechanism 3,
electrostatic repulsion by alanylated lipoteichoic acid (LTA) or wall teichoic acid (WTA). Mechanism 4, electrostatic
repulsion by aminoacylated phospatidylglycerol (PG). Mechanism 5, blocking of cationic host defence peptides (CHDP)
binding by altering the pentapeptide on lipid II. Mechanism 6, export of CHDP by efflux pumps. Mechanism 7, activation
of signal transduction pathways that induce expression of genes that reinforce the wall or detoxify products of CHDP
activity. Mechanism 8, lipid A modification by amine compounds. Mechanism 9, enhanced membrane rigidity by lipid A
acylation. Mechanism 10, activation of mitogen-activated protein kinase (MAPK) signalling pathways in fungi for protection
against oxidative, osmotic or cell wall stress. AMP, antimicrobial peptide; CWI, cell wall integrity pathway; EPS, extracellular
polysaccharide; GlcNAc, N-acetylglucosamine; LPS, lipopolysaccharide; MurNAc, N-acetylmuramic acid; ROS, reactive
oxygen species. Adapted with permission from ref.267, Royal Society of Chemistry, and from Swidergall and Ernst, Eurkaryot.
Cell 13, 950–957 (2014) (ref.272), with permission from the American Society of Microbiology.

Reviews
development, notably those associated with formu associated challenges and the limited understand-
lation and delivery, the potential for drug resistance and ing of structure–function relationships, the potential
the lack of solid pharmacokinetic data. Nevertheless, of CHDP-based therapies remains a promising new
the wide range of CHDP functions defined to date clinical direction.
provide a diverse range of natural molecules for the
design and optimization of new drugs. Despite many Published online 27 February 2020
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antimicrobial peptides. (2006). © Springer Nature Limited 2020

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REVIEWS

Antimicrobial host defence peptides:

Proteomics and Systems

NaTuRe RevIews | DRug DISCOVeRy volume 19 | May 2020 | 311

Host defence peptides

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a CATH-2 HBD2 HD5 ion channel perturbation and enzyme inhibition46,47.

Synthetic peptides derived from CHDP

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Membrane translocation Membrane perturbation Bacterial lysis

DNA/RNA Protein Protein Toroidal pore

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Table 1 | Antiviral activities of cationic host defence peptides

trafficking; enhanced neutrophil phagocytosis of IAV

professional phagocytes; RC2: haemagglutinin-​mediated

before viral entry

Urumin Virion destruction, targeting H1 haemagglutinin 87

RSV HBD2 Viral envelope destabilization in solution or on exposure 88,277

to plasma membrane-​associated HBD2

and spread; function retained by core 22-mer

Adenovirus α-​Defensins Peptide binding to adenoviral capsid prevents uncoating 97–99,278

and nuclear entry of the viral genome; dependent on

HBD3, retrocyclins sulfate inhibited; dependent on lectin-​like properties rather

interfering with HIV replication

gp120 and CD4; dependent on lectin-​like properties

Zika virus Cathelicidins Direct inactivation of Zika virus; protective modulation 93

of interferon signalling pathways

associated herpesvirus derived peptide

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Fig. 3 | Summary of immunomodulatory mechanisms of CHDP. Immunomodulatory functions exhibited by cationic

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Table 2 | Selected antimicrobial peptides that are in clinical trials or approved

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Box 1 | Development of antimicrobial resistance to CHDP

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professional phagocytes; RC2: haemagglutinin-mediated

to plasma membrane-associated HBD2

Adenovirus α-Defensins Peptide binding to adenoviral capsid prevents uncoating 97–99,278

HBD3, retrocyclins sulfate inhibited; dependent on lectin-like properties rather

gp120 and CD4; dependent on lectin-like properties