Critical Reviews in Clinical Laboratory Sciences

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Designing and optimizing new antimicrobial

peptides: all targets are not the same

Adriana Barreto-Santamaría, Manuel E. Patarroyo & Hernando Curtidor

To cite this article: Adriana Barreto-Santamaría, Manuel E. Patarroyo & Hernando Curtidor (2019):
Designing and optimizing new antimicrobial peptides: all targets are not the same, Critical Reviews
in Clinical Laboratory Sciences, DOI: 10.1080/10408363.2019.1631249

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Published online: 09 Aug 2019.

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CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES
https://doi.org/10.1080/10408363.2019.1631249

REVIEW ARTICLE

Designing and optimizing new antimicrobial peptides: all targets are not
the same
Adriana Barreto-Santamarıaa,b , Manuel E. Patarroyoa,c and Hernando Curtidora,b
a
Fundacion Instituto de Inmunologıa de Colombia - FIDIC, Receptor-Ligand Department, Bogota D.C, Colombia; bUniversidad del
Rosario, School of Medicine and Health Sciences, Bogota D.C., Colombia; cUniversidad Nacional de Colombia - Bogota, Faculty of
Medicine, Bogota D.C., Colombia

ABSTRACT ARTICLE HISTORY

Because the resistance of microorganisms to the available antibiotics is a growing healthcare Received 10 December 2018
problem worldwide, the search for new antimicrobial peptides (AMPs) that provide useful thera- Revised 5 May 2019
peutic options has been increasing in importance. Many initial candidates have had to be dis- Accepted 10 June 2019
carded after having advanced to the preclinical and clinical stages. This has led to substantial Published online 9 August
2019
losses in terms of time and money. For that reason, the essential characteristics of AMPs (i.e.
their activity, selectivity, stability in physiological conditions and low production cost) must be KEYWORDS
considered in their design. In addition, peptides could be active against several kinds of cells Antimicrobial peptides;
with activity and selectivity resulting from interaction with multiple target cell components, minimum inhibitory
which sometimes are present in mammalian cells as well. Thus, the cellular composition is concentration (MIC);
important in the AMP-target cell interaction and must be considered in the design of AMPs, too. antimicrobial activity;
This review describes general aspects of AMP design, limitations concerning their therapeutic hemolytic activity;
application, and optimization strategies for overcoming such limitations. selectivity; SAR study

Abbreviations: Aa: amino acid; AMP: antimicrobial peptide; CAMPs: cationic antimicrobial pepti-
des; CL: cardiolipin; LPC: lysophosphatidylcholine; LPS: lipopolysaccharide; LTA: lipoteichoic acid;
MIC: minimal inhibitory concentration; MOA: mechanism of action; PA: phosphatidic acid; PC:
phosphatidylcholine; PE: phosphatidylethanolamine; PG: phosphatidylglycerol; PI: phosphatidyli-
nositol; PIP2: phosphatidylinositol 4,5-bisphosphate; PS: phosphatidylserine; RBC: red blood cell;
SAR: structure-activity relationship; SPPS: solid-phase peptide synthesis; STAMPs: specifically tar-
geted antimicrobial peptides; TA: teichoic acid; TCS: two-component regulatory systems; GlcCer:
glucosylceramide.

Introduction therapeutic agents with different mechanisms of action

The resistance of microorganisms to most currently
available antimicrobials is a worldwide public health
problem [1]. This resistance represents a large expend-
iture in terms of cost-effectiveness and health because
hospital-acquired infections sometimes induce other
injuries or diseases such as sepsis that require add-
itional treatment or that can lead to death [2].
Around 31.5 million cases of sepsis, 19.4 million
cases of severe sepsis and 5.3 million deaths occur
annually worldwide as a result of infections caused by
antibiotic-resistant pathogens [2]. It has been estimated
that if effective strategies are not introduced to coun-
teract such phenomena, such infections will cause
around ten million deaths annually by 2050 [3]. Hence
there is urgent need to develop regulatory strategies
concerning antibiotic use and to search for new
(MOA) that act effectively against the pathogens
responsible for these infections [4].
Peptides that have antimicrobial activity have been
isolated from plant and animal species since the 1980s;
they are considered natural antibiotics and are called
antimicrobial peptides (AMPs) [5]. Many natural AMPs
have been used as models to make synthetic AMPs,
thereby showing these molecules’ versatility in terms of
being improved through design. Although resistance to
some AMPs has been reported (i.e. staphylococci’s pro-
teolytic defense mechanisms induced by the anionic
AMP, dermicidin) [6], AMPs are of great interest in clin-
ical research because of their broad spectrum of activ-
ity, the ability to combine multiple MOAs, and their
rapid activity that minimizes resistance developing to
these molecules [7,8].

CONTACT Hernando Curtidor hernando.curtidor@urosario.edu.co Fundacion Instituto de Inmunologıa de Colombia - FIDIC, Carrera 50 No. 26-20,
Bogota D.C., Colombia.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group
2 A. BARRETO-SANTAMARIA ET AL.
However, despite the large number of studies on the
development of AMPs, few AMPs have advanced to the
stage of clinical trial and/or therapeutic use, partly due
to the pharmaceutical industry’s interests and the sup-
posedly disadvantageous characteristics of these types
of molecules [9,10], e.g. high production costs, suscepti-
bility to modification by enzymes, or lack of superiority
regarding currently available agents [9,11,12]. For a
peptide to be considered as a therapeutic option, it
must have powerful antimicrobial properties and low or
zero hemolytic and cytotoxic activities (high selectivity),
which is why obtaining AMPs that have high selectivity
has gained great importance [13,14,15]. It has been
argued that a major difficulty concerning AMP develop-
ment lies in the fact that candidates have been pro-
moted to preclinical and clinical trials too quickly,
without having been fully optimized, thereby leading
to them being discarded [9].
This paper reviews relevant aspects of AMP design,
limitations regarding their use as therapeutic agents,
and some optimization strategies that have been sug-
gested for overcoming such limitations early on, high-
lighting the importance of optimization during the
early stages of AMP development, i.e. in the laboratory.
Finally, the diverse composition of the potential target
cells of AMPs and their relevance in the design process
are reviewed.
Natural AMPs
Natural AMPs have been widely studied because they
represent an alternative to the antibiotic resistance
Figure 1. Biological activities described to date for AMPs. These peptides are active against different types of microorganism and
against cancer cells. Such peptides are sometimes characterized by having an immunomodulatory or anti-inflammatory effect due
to binding to certain pathogen components such as LPS from Gram-negative bacteria.
mechanisms of microorganisms [13]. They consist of
amino acid (aa) sequences having activity against a var-
iety of microorganisms; many AMPs are broad spectrum,
while some have narrow spectrum activity, meaning that
they have been classified as antibacterial, antifungal,
antiparasitic or antiviral peptides (Figure 1) [16].
Although AMPs are found naturally in almost all life
forms, from bacteria (e.g. bacteriocins produced by
Lactobacillus [17]) to mammals, most have been isolated
from different animal species; e.g. cecropins come from
insects [18,19] and magainins and dermaseptins have
been isolated from frog skin [20–22]. A considerable
number of AMPs, such as the puroindolines and some
defensins, have been obtained from plants [23,24].
Unfortunately, many naturally occurring AMPs have
certain limitations, e.g. low selectivity, which results in
toxic effects on host cells, or the requirement for a high
concentration for their antimicrobial activity. Thus, sev-
eral approaches to AMP design have been gaining
increasing relevance as a strategy for enhancing AMP
development [25,26].
Synthetic AMPs
Limitations of naturally occurring AMPs have hampered
their therapeutic use. The search for a new generation
of AMPs has focused on computational design of pepti-
des or on the selection of new candidates from large
peptide libraries [27–31]. Such designed or selected
sequences can be produced by chemical synthesis or
recombinant expression [32,33] for testing in antimicro-
bial, hemolytic and cytotoxic activity assays, thereby

enabling their selectivity for microorganisms and their
therapeutic potential to be ascertained [34,35].
Although AMP design is complex due to the consid-
erable number of natural aa and possible combinations
depending on peptide length (e.g. 4
1015 possible
combinations for a 12 aa sequence), this could be used
for overcoming natural limitations of AMPs [10,34,36].
Table 1 shows how candidates having potent activity
can be obtained through effective design. These mole-
cules’ scope goes beyond their use in clinical therapy as
some AMPs have been studied for alternative applica-
tions, e.g. the amidated antifungal peptide H-Orn-Orn-
Trp-Trp-NH2 has been evaluated regarding its use as a
preservative against food contamination [37].
Antimicrobial activity of AMPs
Evaluating a peptide’s antimicrobial activity during the
experimental stage consists of determining its activity
on the microorganism of interest (bacteria, fungi, para-
sites and/or viruses), the minimal inhibitory concentra-
tion (MIC) assay being the standard method [38].
Different culture conditions may be required to ensure
optimum microorganism growth and to obtain reliable
results [39–42]. Certain methodological conditions
are also required, because of the implications of work-
ing with cationic peptides; e.g. the need to use
Mueller–Hinton broth without cation adjustment for
the MIC assay against bacteria, since these ions (Caþþ
or Mgþþ) can cause substantial inhibition of cationic
AMP activity [38].
The MIC value obtained is a crucial factor in the ini-
tial selection of candidate peptides, because the US
Food and Drug Administration (FDA) approves a new
antimicrobial for use in humans only if it has thera-
peutic advantages over already available agents. AMPs
must thus be effective at concentrations equal to or
less than that of available antibiotics to justifying the
enormous investment in time and money required to
fully develop a new therapeutic agent [43].
Higher concentrations of AMPs are usually required
to inhibit pathogen growth than those for already avail-
able antibiotics. Their design thus requires an approach
which involves constantly seeking to improve these
molecules’ antimicrobial activity and to understand the
impact of certain modifications on such activity in order
to optimize new candidates in the early stages of
laboratory development [9,34,44–46].
An overview of MIC values for peptides that have
reached advanced stages of development is useful for
establishing MIC ranges, to decide whether a peptide
enters the clinical study phase and becomes profiled
CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES 3
within the therapeutic industry [47]. Such results can be
presented as MIC (concentration inhibiting 100% of the
growth of a particular strain), or MIC100, MIC90 or
MIC50 values (concentrations inhibiting 100% of the
growth of 100%, 90% or 50% of a considerable number
of evaluated strains) (Table 1).
The MIC values for AMPs being developed range
from <0.03 lg/mL to 32 lg/mL (Table 1), supporting
their potential as a therapeutic alternative to currently
available antibiotics whose MIC varies between 0.5 lg/
mL and 4 lg/mL [48]. For example, the lipoglycopep-
tide, dalbavancin, has a  0.03 mg/mL MIC against
Staphylococcus aureus (Gram-positive strain) whilst the
glycopeptide antibiotic, vancomycin, has a 0.5–2 lg/
mL MIC against the same type of bacteria [49]. It is
important to note that as AMPs are generally molecu-
larly larger than antibiotics, their molecular weights,
and consequently, their MIC values in mg/mL, are usu-
ally higher too. MIC values in lmol/L should thus be
used for a more apt comparison between AMPs and
antibiotics.
Although a  16 lg/mL or  16 lmol/L MIC has been
posed as a requirement for a candidate to be evaluated
in clinical studies [10], this may vary depending on the
pathogen against which it acts and the advantages it
may offer compared to the available agents against the
same pathogen. For example, pexiganan, a synthetic
22 aa-long cationic peptide derived from magainin
(currently in Phase 3 clinical trials), has emerged as a
topical therapeutic option for mild infections associated
with diabetic foot ulcers. Pexiganan has MIC values
of 8–16 mg/mL against Escherichia coli, Klebsiella pneumo-
niae, Citrobacter koseri, Enterobacter cloacae, Acinetobacter
species and Pseudomonas aeruginosa (Table 1).
Designing AMPs with improved antimicrobial activ-
ity and selectivity
AMPs can theoretically be designed de novo or from
one or several preexisting sequences that can be used
as a template, with the aim of obtaining a sequence
with powerful activity and high selectivity [11,50–56].
Structure-activity relationship (SAR) studies form
an integral part of AMP research, where charge, second-
ary structure, and amphipathicity are the physicochemi-
cal properties most closely related to AMP activity
[25,54,57–60]. Some studies have focused on establish-
ing the MOA of natural and designed AMPs, highlight-
ing interactions with the phospholipids forming the
membrane over binding to receptors [44,45,61].
Membrane-AMP contact may induce changes, such
as conformational transition, self-association or oligo-
merization, in the peptide, thereby increasing specific
 4

Table 1. MIC Values in vitro for some AMPs which are currently being developed.
Administration MIC against Gram- MIC against Gram-
AMP Description Indication route Activity negative bacteria positive bacteria MIC against fungi Reference
Pexiganan A 22 aa-long Diabetic foot infection Topical Pathogens associated 8–16 mg/mLc 8–32 lg/mLc – [62–64]
magainin analog with diabetic foot (3.23–6.46 mmol/L) (3.23–12.9 mmol/L)
infection
(broad spectrum)
Novarifyn Synthetic antimicrobial MRSA, P. aeruginosa, C. Topical Mainly against 31.25 lg/mLb 62.5 lg/mLb – [65]
(NP432) difficile, A. Acinetobacter
baumannii and E. baumannii and
coli. infections Staphylococcus
aureus
Dalbavancin Lipoglycopeptide Acute bacterial Intravenous Gram-positive bacteria, – 0.03 mg/mLc – [62,66–68]
A. BARRETO-SANTAMARIA ET AL.

(approved by antibiotic infections of the mainly (0.016 mmol/L)

the FDA) skin and acute Staphylococcus
hematogenous aureus,
osteomyelitis including MRSA
Brilacidin Defensin mimetic Oral mucositis in Topical Multiple pathogens, 0.25–0.5 lg/mLc – [69]
patients undergoing Gram-negative and (0.27–0.53 mmol/L)
Chemo-radiation for Gram-positive
treating head and bacteria, including
neck cancer MRDS. Mainly
against
Staphylococcus
aureus
HB1345 Synthetic Acne, broad- Topical Mainly against 0.5–1 lg/mLa – [70]
lipohexapeptide spectrum antibiotic Propionibacterium
acnes
Surotomycin Cyclic lipopeptide Diarrhoea Oral Mainly against – <1 mg/mLc – [71,72]
Clostridium difficile (<0.6 mmol/L)
Lytixar (LTX-109) Synthetic antimicrobial Uncomplicated Gram- Topical Staphylococcus aureus 2–4 lg /mLa 4 lg/mLb – [73]
peptidomimetic positive skin (2.5–5.1 mmol/L)
infections, MRSA/
MSSA nasal colonies
AP138 Plectasin derivative MRSA Not specified Mainly against 2–4 lg/mLa [74]
implant infections Staphylococcus (0.45–0.9 mmpl/L)
aureus
Omiganan Indolicidin-derived 12 Papulopustular Topical Staphylococcus aureus – 16 lg/mLc – [75]
(CLS001) aa-long synthetic rosacea, acne (8.99 mmol/L)
cationic peptide vulgaris, vulvar
intraepithelial
neoplasia (VIN)
MU1140 Lantibiotic naturally Gram-positive bacteria Not specified Broad spectrum 0.5–32 lg/mLa – [76]
produced by the (MRSA, C. difficile) against Gram-
SMaRT strain parent positive bacteria
C16G2 35 aa-long designed Mouthwash having Topical Streptococcus mutans 4.2–5.2 mmol/La [77]
peptide derived selective elimination (17–21 lg/mL)
from the S. mutans of the oral
competence- cariogenic pathogen
stimulating peptide Streptococcus
(CSP) pheromone mutans
and novispirin bound
by a flexible tri-
glycine linker region
(continued)
Table 1. Continued.
Administration MIC against Gram- MIC against Gram-
AMP Description Indication route Activity negative bacteria positive bacteria MIC against fungi Reference
PAC-113 12 aa-long Oral candidiasis in HIV Topical Candida albicans – – 1.3–3.1 lg/mL [78]
antimicrobial seropositive patients (0.83–1.98 mmol/L)
peptide derived
from a naturally
occurring histatin
protein found
in saliva
Novamycin Poly-arginine based Yeast and Topical Mainly against 8–32 lg/mLb [79]
(NP339) cationic peptide mold infections Staphylococcus
aureus and
Aspergillus spp.
NovexatinR Cyclic, highly cationic Fungal nail infection Topical Saccharomyces – – 3 mmol/La (3.28 mg/mL) [80]
(NP213) peptide cerevisiae
(arginine/rich)
SGX942 Synthetic 5-aa peptide, Oral mucositis in head Intravenous Effective for tackling – – – [81]
innate defense and neck cancer. Gram-negative (i.e.
regulator (IDR), Complement for Burkholderia
lacking direct antibiotics. pseudomallei) and
antimicrobial Gram-positive (i.e.
activity Staphylococcus
aureus)
bacterial infections
p2TA (AB103) 10 aa-long designed Necrotizing soft Intravenous Attenuates T-cell CD28 – – – [82]
synthetic peptide tissue infections signaling in
Contains D-aa exacerbated
immune responses
during bacterial
infection. Effective
in mice challenged
with E. coli bacteria
The range of MIC values concerning several microorganisms is shown for broad-spectrum peptides.
a
MIC, bMIC100, cMIC90, dMIC50.
The values calculated from the reported MIC and the peptide sequence’s molecular weight are shown in brackets.
CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES
5
6 A. BARRETO-SANTAMARIA ET AL.
cell toxicity [83]. This is why circular dichroism has been
used for studying the structure of AMPs and their rela-
tionship with biological activity. These assays are car-
ried out by simulating the microorganisms’ membrane
conditions using media containing trifluoroethanol,
anionic surfactant sodium dodecyl sulfate, or more
complex components such as liposomes, lipopolysac-
charides (LPS) or whole bacteria [36,84,85]. These stud-
ies are the basis for developing useful strategies to
modify physicochemical properties of AMPs and
improve their antimicrobial potential, as shown below.
Sequence. Amino acid composition and primary
sequence are critical for the peptides’ antimicrobial activ-
ity since they determine the sequence’s structural and
physicochemical properties. Peptides having antimicro-
bial activity have been reported to contain mainly leu-
cine, glycine, and lysine in their sequences, and having
arginine or lysine with tryptophan favors the peptides’
insertion into the membrane of the pathogen [86–88]. In
addition, modifying aa side-chains can affect the anti-
microbial activity of AMPs, as occurs with arylation of
tryptophan residues, which has been shown to increase
the antimicrobial activity of a human lysozyme-derived
AMP [89]. N-methylation of peptide backbone amides
can lead to derivatives having similar antimicrobial activ-
ity but less hemolytic activity [90], whilst partially replac-
ing L-aa with D-aa can reduce hemolytic activity and
increase antimicrobial activity [36,91].
Charge. Although neutral [92] and anionic [93] AMPs
have been reported, positively-charged peptides (þ1 to
þ9) are usually more active because they are electro-
statically attracted by microbial membranes that
are negatively-charged [94]; this gives rise to a subcat-
egory of AMPs called cationic antimicrobial peptides
(CAMPs) [95,96].
AMP charge can be increased by replacing
uncharged aa with positively-charged aa or replacing
acid residues with positively-charged or amide-type res-
idues; the latter approach has even conferred anti-
microbial activity on originally inactive peptides [96].
Amidating the C-terminal extreme is one strategy
for increasing peptide charge without modifying its
sequence; it consists of leaving a –CONH2 group
(neutral at physiological pH) instead of –COOH
(negatively-charged at physiological pH), thereby
increasing a peptide’s net charge by þ1 [87]. Such an
amidation strategy has also stabilized the secondary
structure of peptides having a significant helical struc-
ture element content, thus favoring interaction with the
phospholipid bilayer, which may lead to increased
antimicrobial and hemolytic activity of the modified
peptides [97].
Increasing the positive charge of a molecule can
reduce its hemolytic activity [14]; however, when such
charge is increased (particularly in a peptide’s hydro-
philic region), this can result in increased antimicrobial
and hemolytic activity [98].
Structure. AMPs can have or assume differing second-
ary structures, including a-helix, b-sheet, extended or
mixed structures [99,100]. AMPs with an a-helical struc-
ture are usually more active on microbial membranes
but can have activity on red blood cell (RBC) mem-
branes as well [13,97]. It has been reported that altering
helix peptide structure by introducing D-aa, prolines or
glycines into their sequences can reduce hemolytic
activity [36,45,101,102]. However, such modifications do
not enhance selectivity for microorganisms in all cases,
since selectivity may be modified only moderately by
the structural changes, or antimicrobial activity may
decrease [101,103]. Disulfide bond formation for struc-
tural alteration has been more effective in some cases,
because it leads to stronger structural change, as seen
in modified LK peptide analogs where hemolytic activ-
ity has been reduced while antimicrobial activity has
not been greatly affected [103].
Hydrophobicity. A peptide’s hydrophobicity refers to
the percentage of hydrophobic aa in its sequence. This
characteristic enables water-soluble AMPs to bind (via
hydrophobic aa) to the membrane and divide the lipid
bilayer. Although a molecule’s optimum hydrophobicity
value varies according to its other characteristics, the
hydrophobicity of most natural AMPs is around 50%
[104]. Increasing peptide hydrophobicity (by aa substi-
tution or lipid conjugation) could induce greater
antimicrobial activity because it favors interaction with
phospholipid membranes [105–107] and it could
increase sequence selectivity and stability if non-natural
hydrophobic aa are involved [102]. However, increasing
hydrophobicity too much could lead to the formation
of peptide aggregates that would reduce its capability
for antimicrobial action [98]. Increasing cationic and
amphipathic peptide hydrophobicity could increase
affinity for zwitterionic phospholipids (neutral) on the
mammalian cell membrane, thereby increasing hemo-
lytic activity [35].
Amphipathicity. Amphipathicity is a feature whereby
peptides have a spatial aa distribution conferring both a
hydrophilic and a hydrophobic face. This property is
measured as a peptide’s hydrophobic moment (lH);

values range from 0 to 3.26, and it is calculated according
to a standard scale of hydrophobicity for every aa [108].
Amphipathicity has been shown to be very import-
ant for AMP activity and selectivity for peptides having
an a-helix structure but also for peptides having a
b-sheet structure [11,13,109,110]. The increased amphi-
pathicity of helical peptides (i.e. greater definition of
hydrophilic and hydrophobic faces) has been associated
with activity on membranes and reducing it could lead
to peptides having reduced hemolytic activity [14].
However, there are some discrepancies regarding the
effect of perfect or imperfect amphipathicity on AMP
function and selectivity; designed peptides having
imperfect amphipathicity could be more active and
selective than those having perfect amphipathicity [25].
Computational AMP design
AMP activity and selectivity result from many intercon-
nected properties, thereby hampering any prediction
regarding the effect on the physical-chemical properties
caused by modifying one aa and also on their anti-
microbial and hemolytic activity [106]. To overcome
such complexity, designing AMPs has been supported
by AMP sequences and their characteristics being
deposited in databases, as well as by using computa-
tional tools for predicting properties of peptide sequen-
ces and their possible activity on the membranes of
microorganisms [86,111,112].
Several databases have been created to date for
managing the available information on AMPs; they
sometimes include tools for analyzing and predicting
sequences having antimicrobial activity. For example,
the Yet Another Database of Antimicrobial Peptides
(YADAMP), the Database of Anuran Defense Peptides
(DADP) and the Linking Antimicrobial Peptides (LAMP)
database specialize mainly in peptides isolated from
specific sources [86].
The Collection of Anti-microbial Peptide 3 (CAMP3)
database (created in 2010), with 10,247 AMP sequences
[113], together with the Antimicrobial Peptide Database
3 (APD3) (created in 2003), with 2747 antimicrobial
sequences from the six kingdoms (mainly animal and
vegetal), have been the most used to date. APD3 has
rigorous parameters for introducing new sequences,
which can be only from natural sources, thereby confer-
ring great reliability on the data deposited there [114].
APD3 includes a secondary structure and antimicro-
bial activity predictor; it can also calculate some pepti-
des’ physical-chemical characteristics, such as their
composition, charge, and hydrophobicity. It has set
ranges in which such properties must be maintained
for increasing the probability of an AMP being
CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES 7
obtained, i.e. charge of 5 to þ10, hydrophobicity of
10–80%, and length of 5–174 aa (preferably less than
60 aa) [86].
In general, predictive methods developed from
these databases have been based on parameter
space strategy, database filtering technology, align-
ment, and machine learning (i.e. artificial neural net-
works, genetic algorithms, support vector machine, and
hidden Markov models), among others (Figure 2)
[113–120]. Methods based on machine learning and
combined methods have played important roles in
such approaches, thereby providing greater scope for
making predictions [121,122].
The predictors used for peptide computational
design are usually based on information regarding
hydrophobicity, charge distribution, and size [86]; how-
ever, some new approaches have brought greater pre-
cision to designing AMPs. For example, an innovative
membrane-pore-forming peptide prediction tool/
descriptor, based on the peptides’ topological hydro-
phobic distribution, gives more in-depth information
about aa spatial distribution features and patterns, as
well as about hydrophobicity, charge, and length [123].
A guideline for designing a computational model for
designing effective nontoxic antibacterial peptides has
been suggested recently; it includes molecular docking
and molecular dynamics simulations for improving AMP
activity predictions [124].
However, it should be noted that, in spite of their
great usefulness for obtaining AMPs, such methods for
rational peptide design in silico are strongly influenced
by the information encoded in the sequences depos-
ited in the databases, thereby limiting their capability
for identifying novel sequences beyond those described
for AMPs in the pertinent literature [119]. Even after
overcoming the stage of optimizing peptide activity
and selectivity, there is still a long way to go before
they can be used as therapeutic agents [43].
Tailor-made?
Although the physicochemical characteristics of candi-
date sequences are fundamental when designing new
AMPs, their activity and selectivity also depend on tar-
get cell composition, particularly the outer membrane
surface which forms the initial pathogen-AMP contact
point. Such targets must thus be considered when
designing and optimizing AMPs.
AMPs can act against bacteria, fungi, parasites or
viruses via different MOA, including anionic internal tar-
gets such as DNA [125], RNA [126] or regulatory
enzymes [127]. However, the main mechanism that
AMPs use is disruption of the membrane, not by
8 A. BARRETO-SANTAMARIA ET AL.
Figure 2. Databases and prediction methods regarding the computational design of peptides. The main AMP databases are shown
on the left-hand side of the Figure. The main methods for predicting AMP activity are shown on the right-hand side; they are based
on the information stored in the databases. ANN: Artificial Neural Networks; GA: Genetic Algorithms, SVM: Support Vector Machine;
HMM: Hidden Markov Models.
binding to specific receptors but by interacting with
the phospholipids forming it [44,45,128], and they act
in a similar way against RBC and other animal cells
[15,61,129]. AMPs have a clear preference for nega-
tively-charged lipids; they have the greatest affinity for
phosphatidylglycerol (PG), followed by phosphatidic
acid (PA), phosphatidylserine (PS) and cardiolipin (CL)
[130,131]. However, other non-phospholipid compo-
nents such as wall components or LPS should not be
ruled out as they may be crucial for AMP activity [8].
Mammalian cells. A good peptide candidate must
have minimal or no activity against mammalian cells so
that it can be used therapeutically without any restric-
tions [14,54]. However, because microorganisms share
certain membrane characteristics and components with
mammalian cells, many AMPs are poorly selective, i.e.
they are active against microorganisms and mammalian
cells (toxicity) [14,25,132].
Mammalian cell membranes are rich in zwitterionic
phospholipids phosphatidylcholine (PC) and phosphati-
dylethanolamine (PE), negatively-charged phospholi-
pids PS and phosphatidylinositol (PI), and sphingolipids
mainly containing choline (sphingomyelin) [133]. These
lipids are segregated on opposite faces of the bilayer,
producing asymmetry [134]; PE, PI, and PS are preferen-
tially located on the inner face of erythrocyte mem-
branes whilst the external face is enriched by PC and
sphingolipids [134,135]; this means that the cell surface
tends to have a net neutral charge [136].
Eukaryotic membranes also contain glycolipids and
sterol; the sterol in mammalian cells is cholesterol,
which is found symmetrically on both sides of the
bilayer, while the glycolipids are preferentially located
on its outer face [134,136]. The loss of fragmentation
activity caused by antibacterial peptides (including pex-
iganan) in assays involving phospholipid vesicles has
been related to an increase in the vesicles’ cholesterol
content, so sterol seems to be crucial for discrimination
between bacterial and animal cells [137]. The PS con-
tent of mammalian cells varies from 8–15% of the total
molar content of phospholipids; this becomes translo-
cated to the membrane’s outer face because of aging,
injury or disease, thereby increasing its distribution and
producing a negative charge on its surface. This PS
translocation has deep implications regarding cell func-
tion [134,138]. Such negative surface charges enable
cationic AMPs to affect the cells of microorganisms
and/or cells altered by injury or disease, while AMPs
have low toxicity for healthy mammalian cells.
Bacteria. The bacterial membrane is formed by nega-
tively-charged phospholipids such as PG, CL (lacking in
mammalian cells) and PS (found in mammalian cells),
thereby favoring initial electrostatic interaction with
positively-charged aa of cationic AMPs [136,139].
Hydrophobic interactions subsequently occur between
AMP apolar aa and the hydrophobic tails of the phos-
pholipids making up the membrane; a peptide thus
becomes inserted into the lipid bilayer and disrupts it,
 CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES 9

Figure 3. The components of target cells involved in AMP activity. AMPs are active against different microorganism and animal
cells, where various phospholipid and non-phospholipid components are crucial for defining their activity and selectivity. Anionic
cell components are important for the initial electrostatic interactions between an AMP and a target cell. The arrow next to the
phospholipid membrane composition indicates a reduction in phospholipid component abundance. Three membranes can be dis-
tinguished in parasitic cells: (1) the parasite’s membrane (P), (2) the parasitophorous vacuole (PV), and (3) the host cell (H). Viral
genetic information is covered by the nucleocapsid (nucleocap); this nucleocapsid, in turn, is surrounded by a phospholipid mem-
brane in covered viruses that is not present in naked ones.

with or without pore formation [44,129]. However,
AMPs must first cross certain barriers that protect the
bacteria to reach the bacterial cytoplasmic membrane
(which differs between Gram-negative and Gram-posi-
tive bacteria) (Figure 3). Gram-negative bacteria have a
permeable external membrane and a more robust
internal membrane that gives rise to a thin peptidogly-
can cell wall in the middle; this is a polysaccharide
formed by a peptide and N-acetyl muramic acid and N-
acetylglucosamine linked by b-1,4 bonds [140].
The outer membrane of Gram-negative bacteria has
an asymmetric composition; proteins such as porins
and LPS (a complex glycolipid acting as the first contact
site of AMPs) are on the external face whilst lipopro-
teins are present on the inner face. There is a high zwit-
terionic phospholipid PE content and somewhat lower
levels of negatively-charged phospholipid PG [141].
The LPS of the external face involve three compo-
nents: a hydrophobic lipid portion called lipid A, a central
polysaccharide, and a variable terminal polysaccharide,
which includes sugar acids that provide the molecule
with a negative charge. Initial interaction is thus facili-
tated through the positively-charged aa of cationic AMPs
[141,142], thereby enabling their contact with the
10 A. BARRETO-SANTAMARIA ET AL.
external membrane and subsequent passage through
permeabilization of the lipid bilayer [143,144]. Increasing
the positive charge of AMPs seems to favor interaction
with LPS; e.g. MBI-27 (26 aa) and MBI-28 (28 aa), peptides
active against Gram-negative bacteria (Pseudomonas aer-
uginosa), interact with LPS, but MBI-28 is more powerful
due to two additional lysine residues in the C-terminal
region [145].
The internal membrane also has high PE content
(but less than that of the outer membrane) and has a
relatively low amount of PG; Escherichia coli contains
60–80% PE and 15–20% PG, along with CL in some
cases [141,146]. The activity of some AMPs may be
related to the bacterial inner membrane PE content
that could, in turn, be related to these peptides’ low
selectivity, because they also act on human cells such
as RBC, which contain PE [132,134].
Gram-positive bacteria have a single membrane cov-
ered by a thick wall of peptidoglycan and an outermost
protein layer called the S layer [140,142] (Figure 3). Some
AMPs (particularly lantibiotics, a family of small polycyclic
antimicrobial peptides such as nisin, that are too short to
pass through the membrane) act as complexes with lipid
II (bacterial wall precursor) and thus inhibit synthesis of
the wall in Gram-positive bacteria [147].
The PE content is low in the plasmatic membrane of
Gram-positive bacteria and there is a large amount of
PG so they have a higher negative charge on their sur-
face; e.g. the PE content of Staphylococcus aureus is less
than 10%, compared to 80% PG and 10% CL [146]. The
higher negative charge on the membrane of Gram-posi-
tive bacteria could be related to the greater activity of
AMPs on this type of bacteria. In fact, one limitation of
most AMPs is their lack of activity or poor activity
against Gram-negative pathogens that cause many hos-
pital infections [9]. This is reflected in the activity of
AMPs found in clinical studies, which are mostly against
Gram-positive bacteria (Table 1).
The outer surface of Gram-positive bacteria contain
teichoic (TA) and lipoteichoic (LTA) acids; LTA, in par-
ticular, cross the wall and become exposed on the bac-
terial surface. They play an important role in bacterial
growth and physiology and contribute towards mem-
brane homeostasis (cell osmoprotection) and bacterial
virulence [148]. It has been suggested that LTA may be
important for the activity of AMPs because modifica-
tions like D-alanylation can confer resistance on the
bacteria by increasing the density of its wall [149]. It
has also been proposed that AMPs interact with the
wall’s negatively charged components, even with TA
and LTA; however, interaction with LTA may not be
favorable for AMP activity as this may reduce
antimicrobial efficacy since AMPs can become retained
in the wall. This would inhibit their accumulation on
the membrane that is needed for disruption in the lipid
bilayer [150].
A fundamental difference between bacterial and
mammalian cells is the transmembrane potential, which
refers to the electrochemical gradient produced by the
velocity of ion exchange through the membrane. The
transmembrane potential in normal mammalian cells is
90 to 110 mV compared to 130 to 150 mV in
pathogenic bacteria; this difference could be important
in the recognition by and selectivity of AMPs [104].
Bacteria are able to induce resistance to AMPs due to
changes in their membrane composition; such changes
could be due to flippase expression which can modify
cytoplasmic membrane composition, making it more sta-
ble against AMPs [151]. For example, when an AMP inter-
acts with PG-containing membranes, bacteria can increase
CL or aminoacylated lipid levels. CL is a negatively-
charged phospholipid that may protect lipid membranes
from permeabilization [152], and the aminoacylated lipid
can introduce positive charges (lysines) that reduce the
affinity of the AMPs for the membrane [151]. Changes in
membrane rigidity or a reduction of its negative charge
by translocation of positively-charged phospholipids from
the internal face have been found to be related to bacter-
ial resistance against AMPs [152]. The two-component
regulatory systems (TCS) are important in the develop-
ment of bacterial resistance. They perceive a stimulus in
the environment and activate signal transduction to
induce modifications in the membrane, making it more
resistant to the action of AMPs [153]. Strategies must thus
be sought, taking into account the TCS, to reduce the
appearance of resistance to AMPs.
Fungi. Fungi are eukaryotic microorganisms; their cells
are thus very similar to human cells, making it more dif-
ficult to develop drugs having selective toxicity against
them. The fungal lipid bilayer mainly consists of sphin-
golipids, PE, and PI; such components are also present
in mammalian cells. However, fungal sphingolipids
mainly contain inositol as monosaccharide and are dis-
tinct from those in mammalian cells [154]; the sphingo-
lipid glucosylceramide has particularly been proposed
as an important target for AMP selective activity against
fungi (Figure 3) [154].
Fungal phospholipid composition is also asymmetric;
e.g. the internal face of the plasma membrane of
Saccharomyces cerevisiae is enriched with PE, PI and PS,
as in erythrocytes [135]. However, ergosterol is these
microorganisms’ main neutral lipid; this is a vital lipid
that could be involved in antifungal AMP activity and

selectivity since it is not present in mammalian cells
[114,155,156]. Some AMPs having antifungal activity do
have toxicity against mammal cells and this has been
associated with their preference for cholesterol [157].
This suggests the high specificity of some AMPs that
may be selective for a particular sterol (cholesterol or
ergosterol) despite their great structural similarity [158].
Other characteristics unique to fungi could be
important for AMP activity. For example, one compo-
nent of yeast cell walls is chitin, a polysaccharide con-
sisting of N-acetylglucosamine units linked by b-1,4
bonds [159]. The interaction of antibiotics with chitin
has been related to a loss of membrane integrity in
fungi so that the interaction with chitin could enhance
AMP activity [160]. For example, the NP-1 peptide binds
to chitin and causes leaks through the membrane,
resulting in fungal death [161].
The yeast cell wall consists mainly of intertwined
b-1,3-glucan fibrils that form a network of glucans cov-
ering the cell surface; there is also a-galactomannan,
progressively filling the intrafibrillar spaces, and the
polysaccharide, laminarin, which is in the wall’s external
layer. b-1,3-glucan fibrils have b-1,6-glucan branching
which appears to be involved in the interconnection
with other wall components (including chitin) [162].
Glucan is more abundant than mannan; e.g. glucan
makes up 60–65% of total polysaccharides in Candida
albicans, while mannan constitutes just 20–25% [163]. It
has been reported that AMPs may have selective anti-
fungal activity that is attributed to their binding to glu-
can, particularly to laminarin, thereby inhibiting cell
wall synthesis and generating its thinning [164]. It has
been observed that AMP interaction with fungal wall
components favors subsequent electrostatic interaction
with negatively-charged phospholipids like PI, which
can lead to membrane disruption and/or cell entry
where AMPs can affect intracellular targets [155].
Proteins such as mannoprotein and enzymes charac-
teristic of these microorganisms could also serve as select-
ive AMP targets [154]. This has been reported for defensin
NaD1 from the tobacco plant, which seems to interact
with mannoprotein of the cell wall hyphae and requires
wall components to be able to exert its action on the
membrane and then enter the cell to accelerate cell death
[164]. Peptides such as lactoferrin can even reach the
mitochondria in fungal cells, leading to cell death by
apoptosis [165]. D-aa, particularly short peptides 2–10 aa-
long, may favor passage through the fungal wall [10].
Viruses. The presence or lack of an envelope enables a
virus to be classified as covered or naked. The envelope
is a membrane formed by glycoprotein-containing
CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES 11
phospholipids; an intermediate layer called the nucleocap-
sid is made up of proteins and contains a virus’ genetic
information. Naked viruses do not have this membranous
envelope, so the nucleocapsid forms its surface. It has
been proposed that AMPs having antiviral activity exert
such action through three main mechanisms: (1) inhibiting
viral binding and viral cell membrane fusion by viral aggre-
gation; (2) altering the viral envelope by lipid perturbation
in the membrane; and (3) inhibiting viral replication
[166,167]. Some AMPs act as important immunomodula-
tors for counteracting viral infections [168]. Substituting a
single aa in some peptides having antiviral and immuno-
modulatory activity, e.g. replacing an arginine with a lysine
in retrocyclin-1 [169] or leucine with a glycine in LL-37
derivative GI20, has led to increased antiviral activity [170].
Hydrophobicity and charge have been highlighted as
important properties, particularly in hapivirins and diprovir-
ins, where their increase has given rise to peptides having
greater antiviral activity [168].
AMPs are more effective against a covered than a
naked virus [167] because membrane disruption is a
viable neutralization mechanism [166].
Interestingly, the composition of mammalian mem-
brane cells infected by viral particles does not vary sig-
nificantly from that of uninfected cells [171]; although
viral membranes are derived from host cells, they can
differ significantly by lipid or protein translocation
towards the viral surface and by reduced membrane
fluidity. A virus can be selective for the host cell mem-
brane regions used in forming its cell envelope, prefer-
ring certain lipid rafts. For example, the cholesterol
content of the viral membrane can be increased com-
pared to that of host cell, and this has been shown to
be important for a virus’ infective capacity [171]. PC
content (the most abundant zwitterionic phospholipid
in mammalian cells) usually becomes reduced and PS
and sphingolipid content increased in the viral mem-
brane, which could be related to AMP activity [172].
Antiviral AMPs such as human neutrophil peptide 1
(HNP1) (a defensin that acts against several naked viral
species such as HPV and covered species such as HIV)
[173,174], can inactivate covered viruses by interacting
with O- or N-glucans in viral surface glycoproteins in a
lectin-dependent manner [167]. They can thus alter the
ability of glycoproteins to bind to their receptors on tar-
get cells. Although not a lot is known about naked
viruses, it has been suggested that such peptides could
inhibit virion escape from endocytic vesicles but not vir-
ion binding or internalization [167].
Parasites. AMPs can be active against parasites or
against cells invaded by such parasites [175]; their
12 A. BARRETO-SANTAMARIA ET AL.
activity occurs mainly through action against the mem-
brane of eukaryotic cells [176,177]. The peptides’ ability
to inhibit malarial parasite growth and bacteria has a
certain similarity since modifying active AMPs against
bacteria and parasites affects both activities [178].
Host cell protein and lipid permeability, fluidity and
composition become altered when parasites like
Plasmodium falciparum invade [179], thereby inducing
plasma membrane reorganization and altered cytoadher-
ence in invaded cells; this could be associated with PS
exposure on the membrane’s external face [180]. PS con-
fers a negative charge which leads to AMP activity against
their membrane; e.g. peptide NK-2 is active against the
membrane of P. falciparum-infected RBCs, subsequently
crosses the parasitophorous vacuole, and causes damage
to the merozoite membrane [176]. Such PS exposure is
not produced by all parasites; e.g. the membrane com-
position of macrophages infected with Leishmania amazo-
nensis amastigotes (oval and non-flagellated parasite in
intracellular stage of development) is very similar to that
of non-infected cells, but there is a significant increase in
lysophosphatidylcholine (LPC) content while PC and PE
content becomes reduced [181]. The parasitophorous
vacuole’s membrane has a similar composition to that of
the host cell membrane but with a higher concentration
of PC, which is the most abundant phospholipid, followed
by PE, PI, and PS and smaller amounts of sphingolipids
and LPC, as well as host cell membrane and parasite pro-
tein components (Figure 3) [181].
It seems that even though parasitic cells have similar
membrane composition to mammalian cells, AMPs
could have a greater affinity for the parasite membrane
than for infected cells; this occurs with a dermaseptin
S4 derivative against P. falciparum [182]. Although the
parasites’ plasma membrane is rich in zwitterionic PC
(40% of total phospholipid content in Leishmania mem-
brane) and PE (10%), AMPs interact weakly with these
phospholipids, so the interaction could depend on PS
(1%) and traces of PG, i.e. lipids with which such inter-
action is stronger [131]. It has been suggested that
increasing the positive charge density of AMPs
increases their selectivity against parasite membranes
and the membranes of parasite-infected cells, as seen
with the AMP NK-2 that are active against free trypano-
somes or within glioblastoma cells [175].
AMP antiparasitic activity could also involve inter-
action with ergosterol on the membrane of fungi and
parasites such as Leishmania and nematodes; ergosterol
has been shown to be the target of some non-peptide
antibiotics [183].
The diversity of composition of the target cell high-
lights the complexity involved in designing AMPs.
Although it has been suggested that the positive
charge of AMPs is responsible for the initial interactions
with anionic components on the target cells, the prefer-
ence of AMPs to interact with one component or
another goes beyond just this particular property. The
selectivity of these sequences seems to be the product
of multiple factors encompassing both the physico-
chemical characteristics of AMPs and the composition
of the cells with which they can interact. However, stud-
ies of AMPs and the components with which they inter-
act in bacteria, parasites, viruses, fungi, and mammal
cells have extended our knowledge of their MOAs,
which should help in the design of AMPs that are better
targeted and enable faster optimization of new candi-
dates [118,122,124].
For example, it has been suggested that peptides
that inhibit exotoxin production in S. aureus could pre-
vent colonization by pathogenic S. aureus while not
interfering with colonization by normal microbiota
(particularly lactobacilli). In addition, the increasing
inhibitory capability of these peptides is related to their
increasing charge [184].
On the other hand, optimizing the selectivity of
AMPs so that they bind exclusively to pathogens has
been done successfully without affecting commensal
organisms by attaching them to targeting peptide
regions. Peptides belonging to this class of molecule
are called specifically targeted antimicrobial peptides
(STAMPs); they enable pathogens to be killed and they
produce little or no effect on a host’s normal microbiota
[185,186]. A multiple-headed STAMP that has activity
against Pseudomonas aeruginosa and Streptococcus
mutans has been shown to eliminate both species from
a mixed planktonic culture and have little impact on
other bacteria because the targeting region enables
their nearly exclusive interaction with these two bacter-
ial species while the antimicrobial region causes bacter-
ial death [187]. Such strategies targeting exclusive
components of pathogenic microorganisms could over-
come the limitations of selectivity of AMPs, thus ena-
bling personalized treatment against infectious diseases
and mitigating the development of resistance and dam-
age to beneficial microbiota [188].
Multi-purpose AMPs
Studies of AMPs have shown that these peptides have
other types of biological activity apart from their anti-
microbial activity, thereby reaffirming their impressive
therapeutic potential. For example, cationic amphi-
pathic peptide [Leu]10-Dec-NH2 has a short sequence
(SLLSLIRKLLT-NH2) that has antimicrobial and anticarci-
nogenic activity, with high selectivity [189]; LL-37-derived

short AMPs KR-12-a5 and KR-12- a5 (5-DK) have antibio-
film and anti-inflammatory activity [36]; and peptide
AR23 and its analog A(A1R, A8R, I17K) have antibiofilm
and antimicrobial activity [14].
AMPs having antibiofilm activity. Antibiofilm activity
expands the antimicrobial capability of AMPs and pro-
vides an advantage over conventional antibiotics
against bacteria that form organized bacterial com-
plexes (biofilms), which can be resistant to antibiotics
and make conventional treatment against them ineffi-
cient [14,36,190]. This means that AMPs can be used
alone or in combination with conventional antibiotics
as effective therapies against bacterial biofilm-forming
strains. AMPs can degrade the biofilm and exert their
antimicrobial action, and they can cause planktonic
bacterial membrane disruption and facilitate the entry
of antibiotics into the cell [191–194].
AMPs having anticancer activity. Cationic antibacter-
ial peptides can also act against cancer cells; this may
be because the membranes of these two types of cells
(bacterial and cancer cells) have a negative net charge.
The membrane asymmetry of cancer cells is lost and PS
becomes exposed on the external face; this confers a
negative charge and facilitates AMP activity against them
[195]. Antifungal peptides that can bind to PI, i.e. phos-
phatidylinositol 4,5-bisphosphate (PIP2), on fungi have
also been reported; these peptides can also bind to PIP2
in tumor cells and trigger cell lysis [196]. Some AMPs
affect internal targets in cancer cells [197]; i.e. in mito-
chondria, an increase in negative charge from increased
amounts of the anionic lipid CL on their internal and
external membranes could mediate electrostatic inter-
action with positively-charged peptides and trigger apop-
tosis [198]. Anticancer AMPs are considered to be the
next generation of chemotherapeutic drugs due to such
properties as their short target cell interaction time that
reduces the probability of resistance being produced,
their potential low toxicity that reduces possible side
effects, and their good tumor penetration [199].
AMPs having anti-inflammatory activity. Sequences
such as peptide LL-37 or lactoferrin-derived peptides
can neutralize the inflammatory potential of pathogen
components [200,201]. An example of this would be
neutralizing LPS by directly binding to it or preventing
its binding to host cell receptors. In this way, the cell
signaling pathway triggered by Toll-like receptor
ligands in antigen-presenting cells is blocked, culminat-
ing in the blockage of expression of many proinflamma-
tory cytokines (IL-1, IL-6, TNF-a), chemokines (IL-8)
CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES 13
[202], and other immune mediators. In addition, anti-
inflammatory molecules such as IL-10 also become
blocked during the inflammatory resolution stage
[107,201–204]. The hydrophobicity and positive charge
of AMPs can increase their binding capability to LPS
[205]. The antimicrobial activity of AMPs in vivo thus
results from their ability to interact directly with patho-
gens and cause cell damage as well as their ability to
modulate innate components of host immunity by
decreasing the inflammatory response caused by
pathogenic organisms and reducing sepsis [201].
From the laboratory to the therapeutic industry
Although around 12,000 articles focused on the devel-
opment of new AMPs have been published annually
between 2012 and 2016 [206], few peptides have
advanced to the clinical stage or entered therapeutic
use [9,10]. It has been argued that, in some cases, the
FDA has played a crucial role in the precarious progress
of AMPs in the industry, whilst in others, it has been
due to pharmaceutical companies’ commercial interests
that has led them to discard promising AMPs and to
focus on more profitable chronic diseases [9]. In 2012,
the FDA launched its Antibacterial Drug Development
Task Force, which facilitates the development of antibi-
otics and supports the use of innovative assays and
alternative measures to assess clinical efficacy, yet still
maintains prior criteria on the need to demonstrate a
candidate’s superiority over currently available antimi-
crobials [207]. Two programs have emerged since
then. Combatting Antibiotic Resistance in Europe
(COMBACTE), launched in 2013, included development
of innovative assays for new antibacterial agents [208],
and the Global Action Plan on Antimicrobial Resistance,
approved by the 68th World Health Assembly in 2015,
included, among its strategic objectives, to increase
investment in new medicines, diagnostic tools and vac-
cines [4,209]. These types of initiatives have renewed
the possibilities for the development of AMPs within
the pharmaceutical industry and extended the pano-
rama regarding the development of new peptides for
treating infections. In 2016, it was anticipated that the
world market for antimicrobial peptides would continue
to grow at a 7.5% compound annual rate up to
2024 [210].
Limitations of AMPs and strategies for
overcoming them
A major difficulty in developing AMPs has been that
peptides isolated from natural sources were evaluated
14 A. BARRETO-SANTAMARIA ET AL.
Figure 4. Designing and optimizing peptide sequences for
obtaining AMPs. Once candidate peptides or sequences have
been selected, an early design step involves improving their in
vitro activity and selectivity based on changes in their physical-
chemical properties. This is followed by optimizing AMPs, focus-
ing on improving their stability in physiological conditions and
on reducing their production costs. The optimized sequences
are then submitted to preclinical and clinical studies where they
must be shown to be safe and effective in vivo before they can
be used as therapeutic agents against infectious diseases.
and then brought too quickly to preclinical and clinical
studies without the peptides having been fully opti-
mized [9]. A long and expensive process thus ended,
leaving them excluded from the therapeutic industry
and slowing down pharmaceutical development.
Improved strategies for identifying unsuitable candi-
dates early on and reducing the number of failures dur-
ing clinical trials have been sought for some time [211].
Design and modification strategies for overcoming AMP
limitations during early stages are supported by the
versatility of peptide-type molecules, making them
viable alternatives for developing new generations of
antibiotics (Figure 4), where the earliest limitation of
AMPs is their toxicity on mammalian cells. However,
even if such limitations are overcome, there are other
drawbacks, as with all peptide-type drugs, mainly due
to their low stability in physiological conditions and
their production costs.
Stability
Most AMPs cannot be administered orally because they
are rapidly degraded by stomach acids or intestinal pep-
tidases, and are not absorbed to fulfill their biological
function [212]. Although some cyclical and/or linear pep-
tides can be administered orally, most are administered
parenterally (by injection); their use also is limited by
their instability in host conditions [213]. Different strat-
egies have been adopted to increase the half-life of
AMPs in plasma [26], such as cutting sequences or clip-
ping them to limit a peptide’s enzymatic degradation;
this also significantly reduces production costs [10,214].
Another strategy involves identifying possible molecular
excision sites and replacing relevant aa or protecting
them by peptide folding or cyclization [90,215–217].
Stapling is carried out by forming covalent bridges
between side-chains using hydrocarbons, triazole, azo-
benzene, thiol, lactam, hydrazine or thioether [218–220].
Substituting natural aa with N-substituted glycines
(peptoids), lysines and arginines, or b-naphthylalanine
or trans-L-4-hydroxyproline has also been suggested as
a useful strategy for developing anti-infective agents
that are resistant to proteolytic degradation [221–224].
Modifications such as acylation, introduction of albu-
min binding elements, or conjugation with antibodies
have been used to increase peptide half-life by
binding to circulating albumin [225,226]. Isolating AMPs
directly from natural blood sources has been used as a
new approach for obtaining active and stable pepti-
des [227]. Developing alternate administration
routes, such as intranasal and transdermal [228], or
alternative formulation methods by using liposomes,
micelles, or nanoparticles [229] could increase

possibilities for using these and other peptide-type
therapeutic agents.
Production cost
Some studies have focused in obtaining AMPs having
shorter sequences to reduce their production costs and to
improve their stability in physiological conditions [10,230].
An example is lactoferricin, a 25 aa-long AMP with a 6 aa
activity minimal motif [231], or 37 aa-long human catheli-
cidin with a 12 aa activity minimal motif [232].
Biosynthesis approaches to produce recombinant
peptides are economical and easily scalable in principle
since AMPs can be produced as recombinants or by
chemical synthesis [32,33]. One such approach uses E.
coli as the expression system, but this approach has dif-
ficulties concerning yield and cost because AMPs could
have activity against the bacteria expressing it.
Furthermore, AMPs could have a strong interaction
with anionic components of these bacteria and hamper
the purification process [233]. Work is currently focus-
ing on developing more efficient strategies and expres-
sion systems for the large-scale production of AMPs,
such as using the yeast, Pichia pastoris, which reduces
processing costs by omitting cell lysis for obtaining the
aa sequence [234], or expression systems based on
plants such as barley [235].
Production by chemical synthesis offers a wide range
of possibilities for the design and modification of peptides
that are not available when using recombinant technolo-
gies, e.g. including non-natural aa and strategies for mak-
ing these molecules more stable in physiological
conditions [45]. Solid-phase peptide synthesis (SPPS), a
rapid approach for AMP production [236], enables pep-
tide production in small samples for laboratory assays but
has been considered unprofitable on a large scale, with
an estimated $100 to $1000 cost for 1 mg of AMP [234].
Despite this, large-scale SPPS has become a viable tech-
nology for producing small- and medium-sized thera-
peutic peptides ranging from around 5 to 50 aa, whilst
convergent synthesis is an option for the obtaining pepti-
des having more than 50 aa [212]. Optimizing SPPS meth-
ods has gained relevance to produce modified peptides
which cannot be obtained by recombinant technologies
and for obtaining cleaner products; however, work must
continue on improving methods for producing peptides
on a large scale [237,238].
Conclusions
AMPs represent promising agents for treating infections
caused by microorganisms due to their rapid action
against them; various modification strategies shown to
CRITICAL REVIEWS IN CLINICAL LABORATORY SCIENCES 15
be successful for optimizing peptide sequences have
been used for addressing the limitations regarding their
therapeutic use.
Although new antimicrobial development has been
delayed, usually for economic reasons, it is now recog-
nized that the development of new compounds must
be promoted due to the rapid increase in the resistance
of microorganisms to traditional antibiotics, and gov-
ernment programs have been developed for this pur-
pose. The essential characteristics of AMPs (their
activity, selectivity, stability in physiological conditions
and low production cost) must thus be considered from
the start so that advances can be assured during their
development stages. Useful strategies are also needed
to improve the design of the sequences to be used as
therapeutic agents, thereby avoiding arduous research
with candidates that must be discarded.
There are no universal standards for designing
selective AMPs; however, the design and experimental
evaluation of current peptide analog sequences has
enabled information to be obtained regarding possible
modifications that should lead to sequences with
improved activity, selectivity and diverse properties. It is
not enough to consider only a peptide’s properties
when designing AMPs since their activity and selectivity
result from interaction with multiple components of the
microorganism. Their activity on the membrane of the
microorganism must also be considered because AMPs
may interact with multiple targets that are necessary
for their antimicrobial activity to be effective.
Strategies for improving a peptide’s half-life under
physiological conditions and reducing production costs
are also necessary to produce AMPs that will be thera-
peutically useful.
Disclosure statement
No potential conflict of interest was reported by the authors.
ORCID
Adriana Barreto-Santamarıa http://orcid.org/0000-0003-
3485-6934
Manuel E. Patarroyo http://orcid.org/0000-0002-1119-6040
Hernando Curtidor http://orcid.org/0000-0002-6556-7812
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