Download Antimicrobial peptides: an overview of a promising class

DOI: 10.2478/s11535-007-0010-5
Review article
CEJB 2(1) 2007 1–33
Antimicrobial peptides: an overview
of a promising class of therapeutics
Andrea Giuliani∗, Giovanna Pirri, Silvia Fabiole Nicoletto
Research & Development Unit,
SpiderBiotech S.r.l.
10010 Colleretto Giacosa, Italy
Received 25 January 2007; accepted 8 February 2007
Abstract: Antibiotic resistance is increasing at a rate that far exceeds the pace of new development of
drugs. Antimicrobial peptides, both synthetic and from natural sources, have raised interest as pathogens
become resistant against conventional antibiotics. Indeed, one of the major strengths of this class of
molecules is their ability to kill multidrug-resistant bacteria. Antimicrobial peptides are relatively small
(6 to 100 aminoacids), amphipathic molecules of variable length, sequence and structure with activity
against a wide range of microorganisms including bacteria, protozoa, yeast, fungi, viruses and even
tumor cells. They usually act through relatively non-specific mechanisms resulting in membranolytic
activity but they can also stimulate the innate immune response. Several peptides have already entered
pre-clinical and clinical trials for the treatment of catheter site infections, cystic fibrosis, acne, wound
healing and patients undergoing stem cell transplantation. We review the advantages of these molecules
in clinical applications, their disadvantages including their low in vivo stability, high costs of production
and the strategies for their discovery and optimization.
c Versita Warsaw and Springer-Verlag Berlin Heidelberg. All rights reserved.
Keywords: Antimicrobial peptides (AMPs), mode of action, therapeutic use, proteases stability,
dendrimeric peptides
1
Introduction and biological activity
Antibiotic resistance has become a global public-health problem, thus it is imperative
that new antibiotics continue to be developed.
Antimicrobial peptides (AMPs) are an evolutionarily conserved component of the innate immune response, which is the principal defence system for the majority of living or∗
E-mail: [email protected]
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ganisms, and are found among all classes of life ranging from prokaryotes to humans [1, 2].
They represent a new family of antibiotics that have stimulated research and clinical interest [3] as new therapeutic options for infections caused by multidrug-resistant bacteria.
The diversity of AMPs discovered is so great that it is difficult to categorize them except
broadly on the basis of their secondary structure [4, 5]. Basically, AMPs can be classified
in four major classes: β-sheet, α-helical, loop and extended peptides [6], with the first two
classes being the most common in nature. In addition to the natural peptides, thousands
of synthetic variants have been produced which also fall into these structural classes. A
rich collection of all known AMPs can be found in the Antimicrobial Peptide Database
(http://aps.unmc.edu/AP/main.php). The Antimicrobial Peptide Database offers an interface to predict the antimicrobial activity of any submitted sequence, based on a simple
residue analysis and count method and some useful statistical information on peptides
sequence, function and structure.
AMPs are generally defined as peptides of less than 100 amino acid residues with
an overall positive charge (generally +2 to +9), imparted by the presence of multiple
lysine and arginine residues and a substantial portion (≥30% or more) of hydrophobic
residues. Currently, more than 800 AMPs with several different sequences have been
isolated from a wide range of organisms. A list of some well-studied AMPs is reported in
Table 1. A common feature shared among the cationic AMPs is their ability to fold into
amphipathic conformations, often induced by interaction with membranes or membrane
mimics. Alongside their direct antimicrobial activities against Gram-positive and Gramnegative bacteria, these peptides play additional roles as antibiotics against fungi [7]
and protozoa [8] with micromolar or submicromolar minimal inhibitory concentrations
(MIC) [6].
Magainins, magainin 2 and PGLa, are among the best studied AMPs. They are linear
peptides that were originally isolated from the skin of the African frog Xenopus laevis.
Magainins are α-helical ionophores that possess two important activities: a broad antimicrobial spectrum and an anti-endotoxin activity [9]. Moreover they have been shown
to lyse hematopoietic tumor and solid tumor cells by targeting cell membrane via a nonreceptor pathway with little toxic effect on normal blood lymphocytes [10]. AMPs may
also possess immunomodulatory activity when involved in the clearance of infection, including the promotion of wound healing [11], they can act as chemokines and/or induce
chemokine production, inhibiting lipopolysaccharide (LPS)-induced pro-inflammatory cytokine production and recruiting antigen-presenting cells, which can initiate responses by
the adaptive immune system [12]. A number of AMPs such as defensins have multiple
functions in host defense [13]. They are a highly complex group of 3-5 kDa open-ended
cysteine-rich peptides widely distributed in nature and found both in vertebrates and
invertebrates. On the basis of their size and pattern of disulfide bonding, mammalian
defensins are classified into three classes: α-, β- and θ-defensins, with the majority of
research focused on the first two classes. Depending on the species, α- and β-defensins
are found in neutrophils, epithelial cells, certain macrophage populations and Paneth
cells of the small intestine [14]. Human defensins can act as chemoattractants for mono-
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3
cytes [15, 16], stimulating their TNF and IL-1 expression [17], and they can enhance the
expression of IL8 in lung epithelial cells [18]. Moreover, defensins could exert chemotactic
activity not only towards nonspecific phagocytes but also towards cells engaged in adaptive immunity response [19]. Plant defensins are structurally related to defensins found
in other organisms including humans. These AMPs are small (45–54 amino acids), highly
basic cysteine-rich peptides that are apparently ubiquitous throughout the plant kingdom
and play a fundamental role in defense against bacteria and fungi. To date, sequences of
more than 80 different plant defensin genes from different plant species are available [20].
The second large family of AMPs in mammals is represented by cathelicidins. They
are AMPs bearing an amino-terminal cathepsin L inhibitor domain (cathelin). The
C-terminal 37 domain of the only human cathelicidin h-CAP 18, LL-37, is an amphipathic, helical peptide that exerts broad antimicrobial activity [21]. LL-37 has been
found to have additional defensive roles such as regulating the inflammatory response
and chemo-attracting cells of the adaptive immune system to wound or infection sites,
stimulation of angiogenesis and chemotaxis of neutrophils, monocytes and T-cells. LL-37
is found throughout the body: in epithelial cells of testis, gastrointestinal and respiratory
tract, in skin and in leukocytes [21].
AMPs such as mellitin and cecropin, isolated from bee venom and hemolymph of
moths respectively, are capable of inhibiting cell-associated production of HIV-1 by suppressing HIV-1 gene expression [22]. A synthetic tachyplesin (an antimicrobial peptide
present in leukocytes of the horseshoe crab Tachypleus tridentatus) conjugated to the
integrin homing domain RGD showed to inhibit the proliferation of both cultured tumor
and endothelial cells [23] alongside other AMPs, such as defensins [24], cecropin [25],
lactoferricin [26] and lactoferrin [27] that showed a similar anti-tumor activity. The selective disruption shown by AMPs for cancer cell plasma membranes is primarily ascribed
to the negative outer surface charge, imparted by the slightly higher levels of anionic
phosphatidylserine (PS) [28] as compared to normal eukaryotic cells. Recent data supported this hypothesis showing that a number of human cancer cells were preferentially
killed by NK-2, an AMP derived from an antibacterial effector protein from porcine NKand T-cells, showing a strong correlation with the amount of cell surface PS [29]. The
presence of O-glycosylated mucines (high molecular weight glycoproteins) that create
an additional negative charge on the cancer cell’s surface may have an additional role
in the selective cytotoxicity of the peptides [30]. If internalized inside eukaryotic cells,
AMPs could interact with the mitochondrial membranes inducing membrane potential
depolarization and tumoral cell death via the mitochondrial pathway of apoptosis [31].
Certain AMPs have been shown to inhibit the replication of enveloped viruses such
as influenza A virus [32], vesicular stomatitis virus (VSV) and human immunodeficiency
virus (HIV-1) [33, 34]. The generally accepted mechanism of antiviral action is a direct
interaction of these peptides with the envelope of the virus, leading to permeation of
the envelope and, eventually, lysis of the virus particle, analogous to the pore-formation
model suggested for antibacterial activity. Other mechanisms for antiviral action have
also been proposed: T22 ([(Tyr5,12,Lys7)-polyphemusin II]) a 18 amino acids peptide
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analogue of polyphemusin II, an AMP isolated from the hemocyte debris of American
horseshoe crabs (Limulus polyphemus), was found to specifically inhibit the ability of
T cell line-tropic HIV-1 to induce cell fusion [35]. It appears as though many peptides
initially defined as “AMPs” have been shown to have several alternative functions in vivo.
A large amount of experimental work aiming to understand AMPs structure, peptidemembrane interactions and their relationships with the peptides’ biological actions have
been carried out reflecting the increasing interest in this area. In particular, Ramamoorthy et al. used specific membrane model systems and nuclear magnetic resonance (NMR)
to characterize structure and dynamics of several AMPs such as pardaxins [36–38], MSI-78
(Pexiganan) and MSI-594 synthetic magainin analogues [39, 40], MSI-843 lipopeptide [41],
granulysin [42], tachyplesin [43] and a cyclic peptide subtilosin A [44]. In particular, the
studies on pardaxin [36, 38] have shown the role of cholesterol on inhibiting the membrane
disruption and the role of the peptide on the formation of cholesterol-rich domains. Other
studies on the carboxy-amide of pardaxin (P1a), have also observed the change in membrane disruption mechanism, from barrel-stave to carpet type, on membranes of varying
composition [38]. It is likely that other AMPs acting through composition-dependent
mechanisms could be isolated in the future since operating by more than one mechanism
could represent an evolutionary advantage. A study on tachyplesin has shown the role of
the Cys-Cys bond on structure, folding and function of the peptide in search of analogues
with improved or adequate antimicrobial activity and cell selectivity. Since the absence
of disulfide bonds could make an AMP easier and less expensive to produce, the study
provides useful findings in designing AMPs for pharmaceutical applications that retain a
significant antimicrobial activity and are free from Cys residues [43].
This review will be focused on the current knowledge of AMPs, with emphasis on the
mechanism of action, their applicability as therapeutic agents and several strategies for
their isolation and optimization.
2
Mode of action
The molecular mechanism of membrane permeation and disruption of AMPs depends on
a number of parameters such as the amino acid sequence, membrane lipids and peptide
concentration. Until now, the majority of experiments have focused primarily on the
interaction of cationic peptides with model membrane systems. Additional studies have
also been conducted on whole microbial cells predominantly utilizing membrane potential
sensitive dyes and fluorescently labeled peptides. These studies have indicated that all
AMPs interact with membranes and tend to divide peptides into two mechanistic classes:
1) membrane disruptive (barrel stave, toroidal, carpet and micellar aggregate models)
and 2) non-membrane disruptive (intracellular targets) [45]. A number of models for
antimicrobial peptide membrane permeabilization have been proposed even if it should
be stated that there is not universal consensus among investigators in this regard.
Bacterial membranes are negatively charged with lipids bearing phospholipid headgroups such as phosphatidylglycerol (PG), cardiolipin (CL), or phosphatidylserine (PS).
A. Giuliani et al. / Central European Journal of Biology 2(1) 2007 1–33
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Table 1 Description of some well-studied AMPs originating from a wide range of sources,
selected as representative examples of their structural class.
On the contrary, mammalian membranes are enriched in zwitterionic phospholipids (neutral in net charge) as phosphatidylethanolamine (PE), phosphatidylcholine (PC), or sphingomyelin (SM) [46]. Moreover, the presence of cholesterol, a major constituent of
mammalian cellular membranes, can reduce the activity of AMPs stabilizing the lipid
bilayer or by directly interacting and neutralizing them [47]. Composition likely provides an important determinant by which AMPs selectively target microbial versus host
membranes.
The mechanism of action of AMPs on Gram-negative bacteria will be discussed in
this review since it has been largely studied. An overview of the interaction of peptide
with a Gram-negative bacterial envelope is shown in Figure 1. The initial association of
peptides with the bacterial membrane occurs through electrostatic interactions between
the cationic peptide and anionic lipopolysaccharide, a major Gram-negative bacterial
component that is stabilized by interactions between divalent cations such as Mg2+ and
Ca2+ [48]. Removal of these ions or their displacement by cationic AMPs facilitates
the formation of destabilized areas through which the peptide translocates the outer
membrane in a process termed self-promoted uptake [49]. At this point, AMPs associate
with the outer monolayer of the cytoplasmic membrane inserting into the membrane
interface and then are proposed to create micelle-like aggregates with associated water,
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leading to a disruptive overall effect. In a mechanism non-disruptive for the membrane,
the AMP molecules can also pass through and dissociate from the membrane, binding to
cellular polyanions such as DNA and RNA or inhibiting other intracellular targets [48]. In
a mechanism non-disruptive for the membrane, the AMP molecules can also pass through
and dissociate from the membrane, binding to cellular polyanions such as DNA and RNA
or inhibiting other intracellular targets [48].
Fig. 1 The proposed mechanism of action for antimicrobial peptides in Gram-negative
bacteria. AMPs are proposed to associate with the negatively charged surface of the
outer membrane by either neutralizing the charge over a patch of the outer membrane
or by strongly interacting to the divalent cation binding sites on LPS. The listed models
explaining the mechanisms of membrane permebilization include: (A) carpet, (B) barrelstave, (C) wormhole or toroidal, and (D) aggregate channel. The net effect of A to D
is that some monomers will be translocated into the cytoplasm and can dissociate from
the membrane and bind to cellular polyanions such as DNA and RNA, inhibit enzymatic
activity such as protein synthesis or chaperone assisted protein folding. Lantibiotics such
as nisin can inhibit the transglycosylation of lipid II, which is necessary for the synthesis
of peptidoglican.
The several mechanistic models introduced in Figure 1 (the “barrel-stave”, “carpet”,
“wormhole or toroidal” and “aggregate channel” models) are described in detail below.
A. Giuliani et al. / Central European Journal of Biology 2(1) 2007 1–33
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2.1 Permeabilization mechanisms
Some peptides may act against microorganisms through a relatively diffuse manner,
termed the “carpet mechanism” that involves initial peptide binding to the membrane
surface to form high density clusters that cover the surface (Figure 1A). It is generally
accepted that peptide activity is not detergent-like [50] occurring by phospholipid displacement that changes in membrane fluidity and/or reductions in membrane barrier
properties that subsequently lead to membrane disruption. As in other models, peptides initially bind to the membrane mainly via electrostatic interactions, carpeting the
phospholipid bilayer [51]. When a threshold peptide density is reached, the membrane
is subjected to significant curvature strain leading to disruption. From this perspective,
membrane dissolution occurs in a dispersion-like manner that does not involve channel formation, and peptides do not necessarily insert into the hydrophobic membrane core. For
example, polarized attenuated total reflectance Fourier-transform infrared (ATR-FTIR)
spectroscopy studies on cecropin P1, a peptide derived from moth hemolymph indicates
that this peptide initially orients parallel to the membrane and does not enter the hydrophobic environment thereby destabilizing phospholipid packing. Yet, this peptide is
an efficient antimicrobial agent that causes membrane disruption due to a concentrated
layer of peptide monomers on the surface [46, 52].
The barrel stave mechanism is proposed for a selected group of peptides. This model
involves the perpendicular insertion and aggregation of a relatively small number of individual peptides (or peptide complexes) also referred as staves in a barrel-like ring inside
the membrane leading to a transmembrane pore or channel with a cylindrical structure
(Figure 1B). In this mechanism, the hydrophobic surfaces of α-helical or β-sheet peptides
face outward, toward the acyl chains of the membrane, whereas the hydrophilic surfaces
form the pore lining [53]. The initial step in a barrel-stave pore formation involves peptide
binding at the membrane surface, most likely as monomers and after a conformational
phase transition, the hydrophobic portion of the peptide is inserted into the membrane to
an extent corresponding to the hydrophobicity of the membrane outer leaflet while electrostatic interactions facilitates this process. When bound peptide reaches a threshold
concentration, peptide monomers self-aggregate and insert deeper into the hydrophobic
membrane core. Aggregation allows for a minimal exposure of the peptide hydrophilic
residues to the hydrophobic membrane interior, as the peptides adopt a transmembrane
configuration. The pore is a dynamic structure and the continued recruitment or lose
of peptide monomers results in a relatively polydisperse pore size. A minimal length of
∼22 amino acids is required to transverse the lipid bilayers with α-helical peptides, or
∼8 amino acids if the peptide adopts a β-sheeted structure [54]. Alamethicin (Alm) is
a well-studied example of a peptide that inserts and aggregates within cell membranes
to form conducting channels [55–57]. Alamethicin-induced membrane conductance has
been measured to proceed as a pattern of multistate conductance levels. This finding
suggests the existence of pores with openings of various diameters (2-3 Å), corresponding
to channels composed of 4-6 transmembrane-spanning peptides. However, there remain
relatively few peptides for which there is compelling evidence of a barrel-stave mechanism,
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despite this model having been proposed more than a decade ago.
More recent studies often support the toroidal pore model [56, 58]. This model is an
extension of the transmembrane helical bundle in which the pores are lined with peptides and lipids. A number of studies have been devoted to the elucidation of both the
structure of these peptide pores and the process of pore formation, mainly by the groups
of Matsuzaki [59] and Huang [56]. A pore structure that involves lipids and peptides
was first proposed by Matsuzaki et al. [60] based on the observation that magainin 2
induces rapid lipid flip-flop coupled with pore formation. The presence of negatively
charged phospholipids in the pore-lining reduces the repulsive interactions due to the
high positive density of charges of the peptides [61] and could explain the modulation in
ion selectivity due to alterations of membrane composition [62]. The model postulates
that peptide and lipid together form well-defined pores, with the membrane also curving inward to form a hole with the head groups facing towards the center of the pore
while the peptides line this hole. Although the formation of a formal toroidal channel
has not actually been demonstrated, the model predicts that the membrane will exhibit
positive curvature strain (Figure 1C). The formation of the toroidal pore and translocation of the peptide depends on a critical peptide-lipid ratio. An intolerable increase
in peptide concentration would reduce the stability of the pore as a result of enhanced
electrostatic repulsion (Coulomb energy) between the positive charged side chains [63].
A study representing a step forward in understanding self-association in determing magainin activity and selectivity has been performed by the group of Ramamoorthy using
NMR experiments on two synthetic variants of magainins (MSI-78 and MSI-594) solubilized in dodecylphosphocholine (DPC) micelles. The results supported the α-helical
conformation for both peptides but different tendencies to self associate: dimerization for
MSI-78 (antiparallel dimer formation) and a pronounced conformational heterogeneity
for MSI-594. For MSI-78, dimerization screens the hydrophobic residues reducing the
affinity for mammalian membranes which contain zwitterionic lipids in the outer leaflet,
while exposing positive charges. MSI-78 propensity to form stable dimers make it more
selective and active toward the negatively charged bacterial membranes if compared to
MSI-594 [39]. Examples of AMPs, other than magainins, that are proposed to form this
type of pore include mellitin and LL-37 [56, 64–66].
The barrel stave, the carpet and the toroidal models predict that the killing activity
of AMPs occurs due to perturbation of membrane integrity. However, several studies indicate that permeabilization is necessary but may not be enough to explain antimicrobial
activity [67]. Planar lipid bilayer model studies showed that transient conductance events
indicative of pore formation were only seen at high concentrations of peptides and high
transmembrane voltages [3], with little correlation between their magnitude and the bactericidal potency. In fact, some peptides, e.g. Gramicidin S, caused maximal membrane
depolarization at a concentration well below their minimal inhibitory concentrations indicating that membrane depolarization alone is not necessarily the crucial event in the
killing of microorganisms. To overcome this discrepancy, the aggregate channel model
was proposed [48]. After binding to the phospholipid head groups, the peptides insert
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9
into the membrane and then cluster into unstructured aggregates allowing the dynamic
formation of pores that span the membrane for short periods (Figure 1D). AMPs can
also enter the intracellular space through this mechanism. Once inside, the peptides
home in on their intracellular targets to exert their killing activities [3]. NMR studies
with indolicidin revealed an interesting fold, comprised of a hydrophobic core flanked by
two positively charged regions, suggesting the insertion into the hydrophobic core of the
membrane and formation of channel aggregates [68].
2.2 Alternative mechanism of action: intracellular targets
Permeabilization, per se, does not invariably result in bacterial death. Thus, although
AMPs interact with microbial cell membranes, creating pores that lead both to leakage of
ions and metabolites and depolarization as described above, it is likely that these effects
contribute to mechanisms by which AMPs exert their effects. However, a mounting
body of evidence supports additional or complementary mechanisms. Several studies
investigated the relationship between bacterial membrane permeabilization and cell death
revealing that cell killing may proceed with relatively little membrane disruption and
suggesting that AMPs may interact with putative key intracellular targets.
Several studies with different AMPs have shown that all-L and all-D enantiomers
are not of equal activity pointing to a stereo-specific interaction [69]. For example,
the all-D isomers of apidaecin [70] and drosocin [71] are no longer active. Indolicidin
was proposed to inhibit DNA synthesis leading to filamentation in Escherichia coli [72].
Some AMPs were found to interfere with the metabolic processes of microbes, an example is the glycine-rich attacins that showed to block the transcription of the omp
gene in E. coli [73], whereas magainins and cecropins induce selective transcription of
its stress-related genes micF and osmY at non-bactericidal concentrations [74]. PR-39,
a porcine proline/arginine-rich antimicrobial peptide, is able to bind the cell membrane
of E. coli without affecting its integrity [75] and kill bacteria by blocking both DNA
and protein synthesis [76]. Buforin II, a histone H2A-derived antimicrobial peptide originally isolated from the Asian toad, inhibits the cellular functions of E. coli by binding
to DNA and RNA after penetrating the cell membranes [77]. Two bovine bactenecins,
Bac5 and Bac7, inhibit protein and RNA synthesis of E. coli and Klebsiella pneumoniae and inhibit their respiration inducing a drop in ATP content [78]. The class of
short, proline-rich antimicrobial peptides secreted by insects, such as pyrrhocoricin, apidaecin, and drosocin, are thought to kill bacteria by entering cells and inhibiting the
molecular chaperone DnaK [79]. DnaK is a 70-kDa molecular chaperone that functions
co-translationally and post-translationally to promote protein folding and to inhibit the
formation of toxic protein aggregates. The killing of bacteria and DnaK binding are
related events, as an inactive pyrrhocoricin analogue made of all -D-amino acids failed
to bind to DnaK. These inhibitors could represent an important new class of antimicrobial agents since they demonstrated to selectively bind to the prokaryotic but not to the
eukaryotic chaperone [80].
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Alternative mechanisms of action may act independently or synergistically with membrane permeabilization [3]. Nevertheless, the interaction with the bacterial cell membrane
appears to be the killing mechanism of the vast majority of AMPs [81].
3
Potential as therapeutics
AMPs have many of the desirable features of a novel antibiotic class [82] and can complement conventional antibiotic therapy. Moreover, they show synergy with classical
antibiotics, neutralize endotoxin and are active in animal models. Minimal inhibitory
concentration (MIC) and minimal bactericidal concentrations (MBC) often coincide (less
than a two-fold difference), indicating that killing is generally the highly desirable bactericidal mode of action. Most pharmaceutical effort has been devoted to the development of
topically applied agents, such as the magainin 2 analogue Pexiganan [83], largely because
of the relative safety of topical therapy and the uncertainty surrounding the long-term
toxicology of any new class of drug administered systemically. The main hurdles that have
impeded the development of AMPs as systemic therapeutics include the experience that
many of the naturally occurring peptides (such as magainins), although active in vitro,
are effective in animal models of infection only at very high doses. The proximity of
these doses to the toxic doses of the peptide reflects an unacceptable margin of safety
that could justify their use. Moreover, since AMPs share features with eukaryotic nuclear
localization signal peptides and are able to translocate into cells, they are responsible for
more subtle toxicities, including apoptosis, mast-cell degranulation or extracellular DNA
transfer, and need further studies [82] relative to their use in systemic therapy. AMPs
now involved in pharmaceutical development are shown in Table 2. Pexiganan (Magainin
Pharmaceuticals, since renamed Gaenera, USA), a 22-aminoacid analogue of magainin 2,
was the first antimicrobial peptide to undergo commercial development as an antibiotic
cream for the topical treatment of diabetic foot ulcers named LocilexT M . In 1999 FDA
approval was denied because Pexiganan showed insufficient evidence of efficacy despite
unanimous agreement about the drug’s performance in phase II trials [84] at which stage
most drugs (60-70%) fail due to low efficacy. Iseganan (Intrabioitcs Pharmaceuticals,
USA), a pig protegrin-based peptide, in clinical trials failed to prevent or reduce oral
mucositis in patients receiving radiotherapy for head-and-neck malignancy [85] and to
prevent ventilator-associated pneumonia. The most advanced antimicrobial peptides are
currently two indolicidin-based peptide variants MX-226 (Omiganan pentahydrochloride
1% gel) and MX-594AN developed by Migenix (Canada). MX-226 is a topical antibiotic
for the prevention of catheter-related infections. In August 2005, Migenix, in collaboration with Cadence Pharmaceuticals, initiated a confirmatory Phase III clinical trial with
a primary endpoint being the reduction of local catheter site infections. MX-594AN is
a novel, topical antibiotic for the treatment of acne vulgaris (Phase IIb completed in
2003) licensed to Cutanea Life Sciences in late 2005. Xoma (USA) has two peptides
in its pipeline derived from bactericidal/permeability-increasing protein (BPI), a human
host-defense protein that is one of the body’s early lines of defense against invading mi-
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11
croorganisms, known for its ability to kill bacteria and to neutralize the action of LPS.
XOMA 629 showed activity against Propionibacterium acnes and other skin flora but
preliminary Phase II trial results were inconclusive as to clinical benefit of XOMA 629
compared to vehicle gel (placebo). XOMA 629 is currently under preclinical studies to
design and evaluate new formulations to optimize its skin penetration. XOMA expects
R
to enter the clinic with the reformulated drug in 2007. NEUPREX
(rBPI21 , Opebacan)
is an injectable formulation of rBPI21 , a modified recombinant fragment of BPI under
clinical development for several disease indications: pediatric patients requiring open
heart surgery with cardiopulmonary bypass, severe burn patients, meningococcal sepsis
and allogeneic hematopoeitic stem cell transplants. Inimex Pharmaceuticals (Canada)
is developing AMPs that do not directly kill bacteria but selectively trigger the body’s
innate defenses without causing inflammation at subtherapeutic doses when compared
to conventional antibiotics. Preliminary data demonstrated that combination therapy
of these immunostimulating peptides significantly lowered bacterial load in vivo in a
murine Staphylococcus aureus infection model [86]. Additional studies are ongoing to
explore this new approach to treat topical and systemic infections such as chemotherapyinduced neutropenia and hospital pneumonia. P113 (developed by Periodontix, USA,
then acquired by Demegen, USA) is a 12-amino-acid cationic peptide based on histatins,
naturally occurring AMPs in the saliva [86] that demonstrated excellent in vitro activity against Candida albicans and common Gram-positive and Gram-negative pathogens.
Demegen licensed P113 to Pacgen (Canada) for treatment as a mouth rinse for oral
candidiasis in HIV patients (approval for Phase I/II clinical study received on March
2006). P113 with D-amino acids gave rise to P113D, a compound that demonstrated to
be less amenable to enzymatic degradation and maintained the antimicrobial activity of
the parent compound is planned to enter clinical trials as an inhalation treatment for
Pseudomonas aeruginosa lung infections in cystic fibrosis patients. AM-Pharma (The
Netherlands) is focused on the development of lactoferricin-based peptides (11-mer peptide from the N-terminus of human lactoferricin, hLF-11) for the prevention of infections
in patients undergoing hematopoietic stem-cells transplantation (Phase I completed). In
September 2003, the FDA approved Daptomycin, a cyclic anionic lipopeptide antibiotic,
for the treatment of complicated skin and skin-structure infections. The compound was
originally discovered by researchers at Eli Lilly & Company in the 1980s, who designated the compound LY 146032. Due to the adverse effects on skeletal muscle Lilly
ceased development. The rights to LY 146032 were subsequently acquired by Cubist
Pharmaceuticals in 1997, which subsequently marketed the drug under the trade name
R
CUBICIN
. Daptomycin is active against Gram-positive bacteria only. It has proven in
vitro activity against enterococci (including glycopeptide-resistant enterococci (GRE)),
staphylococci (including methicillin-resistant S. aureus), streptococci and corynebacteria. Interestingly, its function is dependent on calcium (Ca2+ ). Indeed, the association of
Ca2+ with Daptomycin permits it to interact with bacterial membranes with effects that
are similar to those of the cationic AMPs. Polymyxins (polymyxin B and polymyxin E)
are old cyclic cationic lipopeptides (they were discovered in 1947 entered into clinical
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use in 1960) that are rapidly bactericidal against Gram-negative bacteria: Acinetobacter,
P. aeruginosa, Klebsiella, Enterobacter, E. coli, Citrobacter, Morganella, Haemophilus
influenzae and some strains of Stenotrophomonas maltophilia [87, 88]. Polymyxins have
been used for topical application with cultured skin on burns, for operative wound irrigation, for selective decontamination of the digestive tract, for superficial eye infections
and for the prevention of minor wound infections. Polymyxins are most often combined
with other agents to provide broad-spectrum antibiotics, as with the classical neomycin
sulfate and bacitracin combination [89]. Unfortunately, polymyxins are too toxic (mainly
nephrotoxicity, neuromuscular blockage and neurotoxicity) at clinically used doses to be
used systematically [90]. As almost no novel agents have been introduced to combat
multiple-antibiotic-resistant organisms, clinicians maybe forced to rediscover colistin and
colistimethate (colistin methanesulphonate sodium, colistin sulphomethate sodium), a
pro-drug derivative used systematically in intravenous infections of cystic fibrosis patients [90]. Given the concern for potential toxicity, the use of colistin and polymyxin B
should be viewed as a treatment of last resort for patients who have serious infections
caused by multidrug-resistant Gram-negative pathogens for which no other therapeutic
options exist. However, the significant recent increase in the data gathered on polymyxins, focusing on their chemistry, activity, mechanism of action, toxicity, pharmacokinetic
and optimum dosing strategies have led to the conclusion that these molecules have a
better therapeutic index than was reported in old studies.
Several companies are conducting preclinical studies on AMPs. HelixBiomedix (USA)
is developing an array of AMPs within several pharmaceutical programs ranging from
topical anti-infective, wound healing and cystic fibrosis. Lytix Biopharma (Norway) is
developing ultra-broad spectrum synthetic antimicrobial peptidomimetics (SAMPsTM ),
effective against bacteria (including MRSA, methicillin resistant staphylococcus aureus)
as well as fungi. SB006 and its derivatives are developed by SpiderBiotech (Italy). These
molecules are tetrabranched synthetic peptides isolated by phage-display selection that
show potent, protease-stable activity against Gram-negative, multidrug resistant clinical isolates [91]. Preclinical testing has shown that SB006 has a good safety profile
and activity in several murine models of infection (unpublished data). PepTx (USA),
an early stage pharmaceutical company, is developing preclinical compounds (PTXseries) based on β-sheet-forming peptide 33mers [92] originally designed by incorporating
β-conformation stabilizing residues from the β-sheet domains of α-chemokines and functionally important residues from the β-sheet domain of human neutrophil bactericidal
protein (BPI), PTX002 and its derivatives showed a broad spectrum of antimicrobial
(Gram-negative bacteria in the nanomolar range and Gram-positive bacteria in the submicromolar range) and anti-endotoxin activity. Lantibiotics are a unique class of peptide
antibiotics produced by Gram-positive bacteria. They are highly modified peptides rich
in lanthionines, a nonproteinogenic aminoacid composes of two alanine residues that are
crosslinked on their β-carbon atoms by a thioether linkage, combining the beneficial biological interactions and versatility of peptides with desirable pharmaceutical properties
such as stability in the systemic circulation. Novacta Biosystems (England) is exploiting
A. Giuliani et al. / Central European Journal of Biology 2(1) 2007 1–33
13
its innovative technological platform to develop optimized analogues of the lantibiotic
Mersacidin as antibacterial agents against Gram-positive infections using a biosynthetic
engineering approach, which will allow the primary amino acid sequence of the molecule
to be extensively modified. The first defensin ever found in fungi, Plectasin, was discovered by a team from Denmark-based biotech company Novozymes and researchers from
Georgetown University Medical Center and the David Geffen School of Medicine at UCLA
in 2002 from a fungus, Pseudoplectania nigrella, that grows on the shady floors of northEuropean pine forests. Plectasin showed to be active at rates comparable to penicillin
and vancomycin against S. pneumoniae, a bacterium responsible for a lot of diseases,
including community-acquired pneumonia, sepsis and otitis media. Plectasin may represent the first of a powerful new class of antibiotics and antivirals and is currently under
preclinical development [93].
The broad antimicrobial spectrum of AMPs positions them for several other applications such as ’chemical condoms’ to limit the spread of sexually transmitted diseases or
imaging probes for bacterial and fungal infections, exploiting their affinity for microbial
membranes [94]. Moreover, AMPs can enhance the potency of existing antibiotics in vivo,
probably by facilitating access of antibiotics into the bacterial cell resulting in a synergic
effect [95]. The successful demonstration that amphiphilic peptides covalently bonded to
a water-insoluble resin retain antimicrobial activity [96] led to the suggestion of their use
in the area of therapeutic medical devices such as intravenous catheters.
For infections with viruses or parasites that can hide in the host cells, dermaseptin
peptides were shown to be active toward human erythrocytes infected by the malaria parasite Plasmodium falciparum [97]. Novel formulations and delivery systems may also be
necessary in the future to modulate the action of AMPs with a low therapeutic index. In
the past, an encouraging result has been obtained in an animal model for fungal infection,
where the high cytotoxicity of indolicidin for host cells was overcome by administration
of liposomally encapsulated material [98]. A revolutionary alternative in exploiting the
potential of AMPs is described in a recent work by Raqib et al. [99] where they propose
the use of sodium butyrate in the treatment of shigellosis, an infection of the gastrointestinal tract by bacteria of the genus Shigella, in an experimental infection in rabbits.
This study led to the hypothesis that butyrate improves the outcome in shigellosis as a
direct consequence of the LL-37 induction after the interaction of butyrate with rectal
lumen.
Multi-drug resistance (MDR) is the major mechanism of drug resistance in malignant tumour cells, most commonly arising from the over-expression of P-glycoprotein,
a membrane-bound efflux pump that actively removes many conventional anticancer
drugs [100]. A few AMPs were found to be cytotoxic against MDR cancer cells [101]
and to have minimized side effects when compared to other chemotherapeutic agents
that are currently available [100]. Morevoer, AMPs have other important advantages:
they kill cancer cells rapidly, are unaffected by classical chemotherapy resistance mutations, do not easily select chemotherapy-resistant variants, show synergy with classical
chemotherapy (e.g. doxorubicin, etoposite, cisplatin), destroy primary tumors and pre-
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A. Giuliani et al. / Central European Journal of Biology 2(1) 2007 1–33
vent metastases [102]. In addition, due to their mechanism of action, AMPs are independent of cell proliferation and can be employed to destroy nondividing (dormant) tumor
cells. To be clinically used, these peptides need to combine high and specific anticancer
activity with stability in serum. Several studies addressed these issues by targeting them
to specific sites using homing domains [23], by vector-mediated delivery of genes encoding AMPs into tumor cells [103] or by designing and synthesizing derivatives of natural
AMPs through optimization of amphipathicity, deletions and/or substitutions of amino
acids [100].
Table 2 Commercial development of antimicrobial peptides.
A. Giuliani et al. / Central European Journal of Biology 2(1) 2007 1–33
4
15
Limitation as therapeutics
4.1 Cost aspects
The principal reason why the pharmaceutical industry has been reluctant to promote the
clinical use of this class of antibacteral therapeutics is the high production cost of peptides.
The cost price of synthetic peptides is five to twenty times as high as that of conventional
antibiotics. As a typical example, for the treatment of infections with the currently
known AMPs about 1 mg/kg of body weight per day per patient would be necessary,
with a cost of about $50-400 per gram [104] far too expensive for everyday usage in less
developed countries. Despite this drawback, the most popular example of a peptidebased drug produced in multi-tons is the T-20 peptide (Fuzeon, Roche), which is the first
HIV fusion-inhibitor peptide produced at such scale by a combination of solid phase and
solution phase methodologies. The decision of Roche to go fully synthetic, instead of
doing the 26-mer peptide recombinantly, and the following massive investment lowered
the cost of all synthesis reagents, making industrial peptide synthesis more feasible. In
general, the demand for higher quality peptides, the growing popularity of microwavebased peptide synthesis technology, and innovative strategies to enhance delivery and
bioavailability of peptide drugs, are driving a radical improvement in production efficiency.
The future is likely to be a landscape where pharma companies will increasingly move
peptide drugs through their pipelines into late-stage development and where it will be
possible to produce cost effective, high quality peptides.
One of the cheapest alternatives to synthetic peptides is production by recombinant
expression methods using microorganisms that are resistant to the produced peptide antibiotic. Plectasin, the first fungal defensin with an interesting therapeutic potential, can
be produced in a fungal expression system effectively and at high yields, the same system is currently used for industrial scale production of other proteins by Novozyme [93].
Although the costs may be lowered by a factor hundred, we must not underestimate
the technical difficulties in producing large amounts of peptides. At the current production efficiency, for each 100 kg of recombinant peptide one million liters of fermentation
mixture will be necessary [5]. This is acceptable for the production of proteins with a
high specific activity, for instance hormones or recombinant vaccines, while it could be
not economically feasible, not for the ton-scale production of AMPs. One of the most
successful and efficient method for peptide production in an E. coli expression system
has been already described [105]. A fusion protein containing an antimicrobial peptide,
designated P2, at its carboxy terminus and bovine prochymosin at its amino terminus
was genetically engineered. Bovine prochymosin was chosen as the fusion partner because
of its complete insolubility and host protective activity from trom the toxic effect of P2
peptide. The choice of the fusion partner is generally critical for stability, yield and subsequent purification steps. As with many protein production methods, the production of
AMPs by recombinant means for large-scale production could be an economically viable
and promising alternative.
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4.2 Antimicrobial peptide resistance
Unlike conventional antibiotics which microbes readily circumvent, it has been claimed
that acquisition of resistance by a sensitive microbial strain against AMPs is surprisingly
improbable due to the deep changes in membrane structure that would be needed to
confer effective resistance [1], their great diversity and the fact that AMPs have remained
effective against bacterial infections for at least 108 years. Anyway, certain microbial
pathogens are inherently more resistant to AMPs due to stable structural or functional
properties or pathogenesis strategies. For example, resistant species of genera such as
Morganella and Serratia express an outer membrane that lacks the appropriate density of acidic lipids to provide peptide-binding sites. Other resistant species, such as
Porphyromonas gingivalis, secrete digestive proteases that destroy peptides [1]. In many
species of Gram-negative bacteria, the charge on the outer membrane is modulated by the
PhoPQ regulon, a two-component system that uses a sensor (PhoQ) and an intracellular
effector (PhoP) [106]. Under potentially lethal stress conditions, the PhoP/PhoQ regulon affects antimicrobial peptide sensitivity through modulation of the PmrA regulon,
which controls a bank of genes that mediate decoration of the outer membrane with the
positively charged moieties of ethanolamine and 4-aminoarabinose [107] yielding global
protein, phospholipid, and lipopolysaccharide modifications. Other virulent bacteria rely
upon elaboration of a capsule as a means of adherence to tissue or avoidance of opsonization and phagocytosis such as the highly anionic capsular exopolysaccharide produced by
virulent strains of P. aeruginosa [108].
Attempts at inducing Pexiganan resistance in E. coli and S. aureus by chemical mutagenesis have been up to now unsuccessful. Anyway, population models of the evolutionary
genetics of resistance have cast doubt on this claim. A very recent study has documented
the experimental evolution of resistance to Pexiganan through continued selection in the
laboratory. In this selection experiment, 22/24 lineages of E. coli and P. fluorescens
independently evolved heritable mechanisms of resistance to Pexiganan when propagated
in medium supplemented with this antimicrobial peptide for 600-700 generations [109].
The study demonstrated for the first time that resistance can evolve rapidly whenever
bacterial populations are consistently exposed to elevated levels of AMPs. Even if the
study raise several questions about the safety in developing and introducing AMPs in
clinical practice, we must take into consideration that the conditions experienced in nature by bacteria are quite different and, at the moment, we cannot predict the time-scale
required for the appearance of resistant pathogens.
4.3 Susceptibility to proteolysis
Peptides are relatively sensitive toward proteolytic degradation. The rapid clearance of
peptides and unfavorable pharmacokinetics, due to proteolytic degradation, may severely
restrict their applicability. In this context, it is noteworthy that clinical trials of peptide therapeutics have been generally focused on topical applications [5]. It has been
A. Giuliani et al. / Central European Journal of Biology 2(1) 2007 1–33
17
suggested that complexation with negatively charged or lipophilic proteins may either facilitate clearance from the circulation [67] or protect AMPs against excessive proteolytic
breakdown in vivo [110] even if it remains unclear to which extent complexation is a beneficiary effect for activity in vivo. To increase peptide half-life, many strategies involving
several formulations, ways of administration and different levels of chemical modification
are possible [111, 112]. The introduction of D-amino acids (whenever their mode of action
doesn’t require specific interaction with chiral targets), amidation at the N-terminus, nonnatural amino acids, and peptide cyclization are the most common strategies to increase
peptide stability. The peptaibols for instance, a family of peptides characterized by short
chain lengths (≤20 residues), C-terminal alcohol residues and high levels of non-standard
amino acids, are quite resistant against proteolysis because they contain high levels of
non-standard amino acids, principally α-amino-isobutyric acid, isovaleric acid (Iva) and
the amino acid hydroxyproline (Hyp) [113]. Other uncommon amino acids can also be
inserted to prevent binding to the active sites of proteolytic enzymes [114] or the peptide
bonds may be modified in such a way that proteolytic enzymes can no longer cleave them,
e.g. by alkylation of the nitrogen atoms [115]. Since there are few studies about pharmacokinetics of AMPs and the effects of all the above-mentioned modifications, considerable
additional research on these issues is urgently needed.
5
Optimization strategies and high throughput screening
of AMPs
While it is generally easy to design AMPs endowed with bactericidal activity using a
mixture of positive charged and hydrophobic aminoacids, it has been proven to be difficult
to improve other features such as in vivo stability, salt sensitivity and toxicity in view of
their clinical applications.
In an attempt to improve microbicidal activity of a selected peptide, general optimization strategies may be followed including cyclization [116], introduction of fluorine
atoms or trifluoromethyl groups [117], increase of positive charge or hydrophobicity [118]
or improving segregation of polar and hydrophobic residue starting from an helical projection [119]. Recent studies have investigated the effect of different basic residues on
peptide antibacterial activity and cell selectivity [120]. The primary amine of Lys interacts less electrostatically with zwitterionic phospholipids than the guanidinium group of
Arg, suggesting that Arg-to-Lys substitution would increase selectivity against negatively
charged phospholipids of bacterial membranes and cause the peptide to be less lytic to
erythrocytes. All the findings suggest that the activity of AMPs is determined by a subtle
combination of factors such as sequence, hydrophobicity and position of cationic residue.
At the same time, a large number of studies have attempted to establish more precise quantitative structure-activity relationship (QSAR) between the primary sequence
of AMPs and their activity toward selected microorganisms. Unfortunately, a general
statement is almost impossible to obtain due to the complexity of the target (LPS and
cell membranes) and the mechanism of action lacking a defined target. However, since
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A. Giuliani et al. / Central European Journal of Biology 2(1) 2007 1–33
the peptides are amenable to extensive chemical modification, several design strategies
have been pursued by several groups with interesting results. One of the most classical
approaches is the synthesis of combinatorial libraries built around an existing 18-mer
lytic peptide composed of leucine and lysine residues and on a deconvolution strategy
directed toward activity specificity [121]. This approach allows measuring the effect of
different chemical composition and properties of bacterial, fungal and mammalian cell
membranes on the induced conformations of peptides using CD spectroscopy and get insights into the physical and chemical factors that are important for membrane targeting.
Many studies have shown that the amphipathic structure is a key factor for the biological
function of AMP rather than the secondary structure. In another interesting approach
[122] the effect of the covalent attachment of aliphatic acids with different lengths (10,
12, 14 and 16) to the N-terminus of a biologically inactive peptide have been evaluated.
The data revealed that these modifications generally endowed the resulting lipopeptides
with lytic activity while the selectivity against bacteria, fungi and human erythrocytes
was influenced by the length of the fatty acid chain. The results suggested that working
solely on the hydrophobic element of an AMP without mutating the primary sequence
can be an effective strategy in the development of new lipopeptides with increased activity and, maybe, a better pharmacokinetic profile and resistance to proteases. To improve
the antimicrobial profile of a peptide Otvos et al. [123] synthesized chimeric dimers in
which pyrrhocoricin’s potent DnaK-binding domain was connected to drosocin’s superior
cell penetrating module obtaining a new class of antimicrobials that kill bacteria with a
combination of DnaK inhibition and membrane disintegration. The first successful creation of a synthetic, target-specific antimicrobial peptide has been reported by Eckert
et al. [124]. A rationally designed Pseudomonas-specific targeting moiety (KH), functionally analogous to the targeting domain found in nisin, had been added to a general
killing AMP (novispirin G10). The targeting domain KH, selectively delivered the killing
domain (G10) to several Pseudomonas spp. resulting in an overall enhancement in killing
kinetics and selectivity.
The rational design of new AMPs could be performed using a linguistic model [125].
This approach consists of treating the peptide sequence as a formal language and building
a set of regular grammars to describe this language and create new, unnatural sequences
where each amino-acid is analogous to words in a sentence. To find a set of regular
grammars a Teiresias algorithm, a tool for the discovery of rigid patterns (motifs) in biological sequences, can be applied on a set of natural AMP sequences [126]. Overlapping
grammars describing common arrangement of aminoacids can be put together to create
new 20-mer sequences that correspond to the antimicrobial syntax. Designed sequences
are scored and filtered to reduce redundancy and similarity to natural AMPs and then
experimentally tested on various bacteria. Even if this approach is really original, it
has some limitations. First, amino acid residues that are poorly conserved may be neglected with this approach. Second, the approach seems to be less useful when applied
on large sequences. Hilpert et al. [127] set up a design strategy that seems to be a costand time-effective approach for the systemic investigation of pharmacologically relevant
A. Giuliani et al. / Central European Journal of Biology 2(1) 2007 1–33
19
AMPs. The authors described a new technical approach based on the spot-synthesis on
cellulose supports of 229 variants of Bac2A [128], a small size, linear bovine bactenecin.
Peptides can be cleaved from the support by ammonia gas, inserted and serially diluted
into a microtiter plate and finally incubated with an indicator P. aeruginosa strain with
a luciferase gene cassette incorporated as viability indicator. Sequence scrambling of
Bac2A led to activities, as confirmed by minimal inhibitory concentration analysis, ranging from superior or equivalent to Bac2A to inactive. The system is estimated to allow
the screening of 50,000 peptides per pipetting robot per year. Seldom has a method
been developed to tap the potential of huge amount of genomic and proteomic data. A
unique indexing method based on Fourier transformation (FT) to mine this rich source
of data for the presence of peptides with potential antimicrobial activity has been developed by Nagarjan and co-workers [129]. FT methods has been extensively used in many
bioinformatic applications, ranging from structural studies of proteins [130] to the analysis of genomic sequences to study periodicities in DNA sequences and detect probable
coding regions [131]. All the different groups of peptide sequences were retrieved from
the Antimicrobial Peptide Database and coded with numerical indexes based on their
physicochemical properties such as hydrophobicity, polarity, charge, the presence of cysteines and amino acid distribution based on APD statistics. A method was established
using a property-based sequence coding approach and tested mining randomly 10,000
different sequences of 16 residues in length and generating 3 hits that were submitted to
APD’s simple activity prediction tool to check and validate the prediction accuracy. This
perspective could be impressive whenever the method could be tuned to include all the
different classes of AMPs, virtually generating and data mining the whole peptide space
for the AMPs.
The development of peptide phage libraries represents a major advancement in the
discovery of new lead compounds for antibacterial therapy [132, 133]. An evaluation
of phage-display-derived peptides as ligands for a broad range of bacterial enzymes was
reported [134]. A series of well-characterized enzymes with diverse structures, functions
and established biochemical assays were selected and a series of peptides that bound
specifically to each target were isolated by phage display. These peptides were shown to
be effective as inhibitors of enzymatic function in vitro [135]. Thus, isolated peptides can
be used as inhibitors to evaluate the function of the enzyme or for antibiotic discovery
efforts (i.e., as a lead compound for peptidomimetic design). Since it is well known that
lipid A accounts for all the toxic effects of endotoxin [136], in a recent study, Thomas et al.
reported the results of obtaining lipid A binding peptides by biopanning of a repertoire
of a random pentadecapeptide library displayed on the filamentous M-13 phage [137].
Twelve selected peptides were isolated and their primary structure revealed no consensus
sequence between the peptides suggesting that the lipid A binding motif is not sequencespecific which is in accord with the high variability seen with the naturally occurring
endotoxin binding peptides.
The use of peptides identified by phage display to inhibit the function of target proteins inside a bacterial cell has also been reported [138]. Peptides that bind to essential
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A. Giuliani et al. / Central European Journal of Biology 2(1) 2007 1–33
targets of known function were selected using phage display were subsequently expressed
as fusions to glutathione-S-transferase (GST) under the control of a tightly regulated
promoter and resulted in the inhibition of bacterial growth by inactivation of the specific
target protein.
An interesting approach could be the investigation of a phage display system for the
selection of peptides by biopanning of whole bacteria [91]. This could allow the selection,
from a large peptide library, of bacterial membrane ligands with antimicrobial activity.
Although its use for the identification of novel antimicrobial agents has not been fully
exploited, the findings reported so far indicate that peptide phage libraries could play a
promising role in the infectious disease area.
5.1 Dendrimeric peptides as novel antimicrobials
Multiple antigen peptide (MAP), consisting of a number of peptides covalently attached
to a branching lysine core (Figure 2), was originally developed by Tam as a peptidic
immunogen for preparation of antibody [139]. The in vitro and in vivo efficiency of
dendrimeric peptides like MAPs is generally ascribed to their multimeric nature, which
enables polyvalent interactions. In medical sciences, dendrimers are useful tools in several
applications as an antibody mimics, drug transport vectors, anticancer agents, magnetic
resonance imaging (MRI) contrast reagents, and so on [140].
Fig. 2 Schematic representations of three types of dendrimeric cores with three generation
of lysines (A,B,C). The first Lys is always linked to a βAla as a spacer. (A) Two branched
Lys; (B) four branched (Lys)2 Lys; (C) eight branched [(Lys)2 Lys]2 Lys. Shown in D is
the structural formula of B, with α and ε branches bearing peptides.
In a recent study, Bracci et al. proposed the synthesis of bioactive peptides in dendrimeric form in order to increase their half-life, due to acquired resistance to protease
and peptidase activity. Several experiments showed that the higher in vivo efficiency
A. Giuliani et al. / Central European Journal of Biology 2(1) 2007 1–33
21
of dendrimeric with respect to monomeric peptides was additionally due to a pharmacokinetic advantage, such as different clearance, or resistance to peptidase and protease
activity [141, 142]. This hypothesis was successfully verified by testing the stability of
monomeric and dendrimeric peptides in human plasma and serum [143]. Recently, lysine
cores have been used as synthetic scaffolds for the attachment of two to eight copies of
a tetrapeptide R4 (RLYR) or an octapeptide R8 (RLYRKVYG) with the aim of producing antimicrobial dendrimers. Both R4 and R8 contained a combination of basic
and lypophilic amino acids that have been found in protegrins and tachyplesins, natural
peptides with antimicrobial activity. Dendrimeric effect was manifested by high potency
in antimicrobial assays against 10 organisms in high- and low-salt conditions [144]. In
other work, a series of low-generation dendrimeric peptides containing basic (lysine) and
aromatic amino acids, was synthesized in an attempt to evaluate their antimicrobial potency [145]. All the dendrimeric peptides in which lysine was a starting and branching
element expressed moderate activity against S. aureus NCTC 4163, and E. coli NCTC
8196.
Production of AMPs obtained by selecting a large 10-mer phage peptide library
against whole E. coli cells was described by Pini et al. [91]. One selected peptide was
isolated and synthesized both in monomeric and dendrimeric tetrabranched form and the
antibacterial activity of the dendrimeric peptide showed to be much higher than that of
the monomeric form. Modification of the original sequence, by residue substitution or
sequence shortening, produced three different MAPs with enhanced stability to natural
degradation and improved bactericidal activity against a large panel of Gram-negative
bacteria. The same dendrimeric peptides showed high stability to blood proteases, low
hemolytic activity and low cytotoxic effects on eukaryotic cells, making them promising
candidates for the development of new antibacterial drugs.
The multivalency of peptide dendrimers appears to be desirable in the design of AMPs
for their ability to amplify cationic charges and hydrophobic clusters as the number of
dendrimer branches increases while the large hydrophobic core may enhance fusogenic
activity. A plausible mechanism is that amplification by a dendrimeric design increases
the local concentration of peptidic units mimicking the mechanisms of action through
which high-ordered AMPs exert their membranolytic effects [144]. Synthesis of AMP in
dendrimeric form may represent an unusual design but maybe a generally useful strategy
for increasing their in vitro and in vivo activity.
6
Conclusions
During the past two decades it has become evident that increasing bacterial drug resistance has created an urgent need for new classes of antibiotics. Even if a superbug
epidemic has not yet hit and the panel of traditional antibiotics can still manage drug
resistant pathogens, at the moment, AMPs seem to represent one of the most promising future strategies for defeating this threat. This statement is well represented by the
fact that AMPs are subject to an increasing number of academic studies. At industrial
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A. Giuliani et al. / Central European Journal of Biology 2(1) 2007 1–33
level, several companies worldwide are focused on the development of AMPs with several molecules both at the preclinical and clinical stage. This demonstrates that despite
the first clinical trials failing (Pexiganan and Iseganan), there is still a lot of general
optimism for their use in future clinical practice. Some of the challenges facing the development of peptidic drugs have already been overcome, starting from the industrial
production of T-20 peptide (Fuzeon) at the multi-ton scale production, incredibly boosting the production of new peptides on a large scale with beneficial effects on the cost of
all starting materials. Other challenges are common to other class of molecules that may
be defined as innovative. Several strategies have been devised to optimize AMPs with
promising activity, ranging from inclusion of non-natural aminoacids and a new method
applicable to high-throughput screening to multimerization of linear sequences. Even if
we cannot exclude the fact that resistance may evolve whenever bacterial populations
are consistently exposed to elevated levels of AMPs, this concern should not discourage
their further study and development; instead, they may help us to rationalize their use in
future, for example preventing the big mistake made in the past of distribution of large
amounts of antibiotics, and thereby minimizing the emergence of resistant organisms.
Competing interests statement
The authors declare competing financial interests.
A. Giuliani is an executive board member and minor shareholder of SpiderBiotech which
is developing antimicrobial peptides.
G. Pirri is head of research and minor shareholder of SpiderBiotech.
References
[1]
[2]
[3]
[4]
[5]
[6]
M. Zasloff: “Antimicrobial peptides of multicellular organisms”, Nature, Vol. 415,
(2002), pp. 389–395.
H.G. Boman: “Peptide antibiotics and their role in innate immunity”, Annu. Rev.
Immunol., Vol. 13, (1995), pp. 61–92.
M. Wu, E. Maier, R. Benz and R.E.W. Hancock: “Mechanism of interaction of
different classes of cationic antimicrobial peptides with planar bilayers and with
the cytoplasmic membrane of Escherichia coli ”, Biochemistry, Vol. 38, (1999), pp.
7235–7242.
R.M. Epand and H.J. Vogel: “Diversity of antimicrobial peptides and their mechanisms of action”, Biochim. Biophys. Acta, Vol. 1462, (1999), pp. 11–28.
W. van’t Hof, E.C.I. Veerman, E.J. Helmerhorst and A.V.N. Amerongen: “Antimicrobial peptides: properties and applicability”, Biol. Chem., Vol. 382, (2001), pp.
597–619.
R.E.W. Hancock and R. Lehrer: “Cationic peptides: a new source of antibiotics”,
Trends Biotechnol., Vol. 16, (1998), pp. 82–88.
A. Giuliani et al. / Central European Journal of Biology 2(1) 2007 1–33
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
23
F.V. Mohammad, M. Noorwala, V.U. Ahmad and B. Sener: “Bidesmosidic triterpenoidal saponins from the roots of Symphytum officinale”, Planta Med., Vol 61,
(1995), p. 94.
S.B. Aley, M. Zimmerman, M. Hetsko, M.E. Selsted and F.D. Gillin: “Killing of
Giardia lamblia by cryptdins and cationic neutrophil peptides”, Infect. Immun, Vol.
62, (1994), pp. 5397–5403.
M.G. Scott, H. Yan and R.E.W. Hancock: “Biological properties of structurally
related α-helical cationic antimicrobial peptides”, Infect. Immun., Vol. 67, (1999),
pp. 2005–2009.
M.A. Baker, W. L. Maloy, M. Zasloff and L.S. Jacob: “Anticancer efficacy of magainin 2 and analogue peptides”, Cancer Res., Vol. 53, (1993), pp. 3052–3057.
R.L. Gallo, M. Ono, T. Povsic, C. Page, E. Eriksson, M. Klagsbrun and M. Bernfield: “Syndecans, cell surface heparan sulfate proteoglycans, are induced by a
proline-rich antimicrobial peptide from wounds”, Proc. Natl. Acad. Sci. U.S.A, Vol.
91, (1994), pp. 11035–11039.
T. Ganz: “Defensins and host defense”, Science, Vol. 286, (1999), pp. 420–421.
V. Dhoplea, A. Krukemeyera and A. Ramamoorthy: “The human beta-defensin3, an antibacterial peptide with multiple biological functions”, Biochim. Biophys.
Acta, (2006), Vol. 1758, pp. 1499–1512.
D. Yang, A. Biragyn, L.W. Kwak and J.J. Oppenheim: “Mammalian defensins
in immunity: more than just microbicidal”, Trends Immun., Vol. 23, (2002), pp.
291–296.
M.C. Territo, T. Ganz, M. E. Selsted and R. Lehrer: “Monocyte-chemotactic activity of defensins from human neutrophils”, J. Clin. Invest., Vol. 84, (1989), pp.
2017–2020.
H.J. Huang, C.R. Ross and F. Blecha: “Chemoattractant properties of PR-39, a
neutrophil antibacterial peptide”, J. Leukoc. Biol., Vol. 61, (1997), pp. 624–629.
Y.V. Chaly, E.M. Paleolog, T.S. Kolesnikova, I.I. Tikhonov, E.V. Petratchenko
and N.N. Voitenok: “Neutrophil alpha-defensin human neutrophil peptide modulates cytokine production in human monocytes and adhesion molecule expression
in endothelial cells”, Eur. Cytokine Netw., Vol. 11, (2000), pp. 257–266.
S. Van Wetering, S.P. Mannesse-Lazeroms, J.H. Dijkman and P.S. Hiemstra: “Effect
of neutrophil serine proteinases and defensins on lung epithelial cells: modulation
of cytotoxicity and IL-8 production”, J. Leuok. Biol., Vol. 62, (1997), pp. 217–226.
D. Yang, O. Chertov, S.N. Bykovskaia, Q. Chen, M.J. Buffo, J. Shogan, M. Anderson, J.M. Schröder, J.M. Wang, O.M.Z. Howard and J.J. Oppenheim: “β-Defensins:
Linking Innate and Adaptive Immunity Through Dendritic and T Cell CCR6”, Science, Vol. 286, (1999), pp. 525–528.
B.P.H.J. Thomma, B.P.A. Cammue and K. Thevissen: “Plant defensins”, Planta,
Vol. 216, (2002), pp. 193–202.
24
A. Giuliani et al. / Central European Journal of Biology 2(1) 2007 1–33
[21] U.H. Durr, U.S. Sudheendra and A. Ramamoorthy: “LL-37, the only human member of the cathelicidin family of antimicrobial peptides”, Biochim. Biophys. Acta Biomembranes, Vol. 1758, (2006), pp. 1408–1425.
[22] M. Wachinger, A. Kleinschmidt, D. Winder, N. Von Pechmann, A. Ludvigsen, M.
Neumann, R. Holle, B. Salmons, V. Erfle and R. Brack-Werner: “Antimicrobial
peptides melittin and cecropin inhibit replication of human immunodeficiency virus
1 by suppressing viral gene expression”, J. Gen. Virol., Vol. 79, (1998), pp. 731–740.
[23] Y. Chen, X. Xu, S. Hong, J. Chen, N. Liu, C. B. Underhill, K. Creswell and L.
Zhang: “RGD-tachyplesin inhibits tumor growth”, Cancer Res., Vol. 61, (2001), pp.
2434–2438.
[24] B.L. Kagan, M.E. Selsted, T. Ganz and R.I. Lehrer: “Antimicrobial defensin peptides form voltage-dependent ion-permeable channels in planar lipid bilayer membranes“, Proc. Natl. Acad. Sci. U.S.A, Vol. 87, (1990), pp. 210–214.
[25] A.J. Moore, D.A. Devine and M.C. Bibby: “Preliminary experimental anticancer
activity of cecropins”, Pept. Res, Vol. 7, (1994), pp. 265–269.
[26] H.J. Vogel, D.J. Schibli, W. Jing, E.M. Lohmeier-Vogel, R.F. Epand and R.M.
Epand: “Towards a structure-function analysis of bovine lactoferricin and related
tryptophan and arginine-containing peptides”, Biochem. Cell Biol., Vol. 80, (2002),
pp. 49–63.
[27] Y.C. Yoo, S. Watanabe, R. Watanabe, K. Hata, K. Shimazaki and I. Azuma:
“Bovine lactoferrin and Lactoferricin inhibit tumor metastasis in mice”, Adv. Exp.
Med. Biol., Vol. 443, (1998), pp. 285–291.
[28] S.R. Dennison, M. Whittaker, F. Harris and D.A. Phoenix: “Anticancer α-Helical
Peptides and Structure / Function Relationships Underpinning Their Interactions
with Tumour Cell Membranes”, Curr. Protein Pept. Sci., Vol. 7, (2006), pp. 487–
499.
[29] H. Schröder-Borm, R. Bakalova and J. Andrä: “The NK-lysin derived peptide NK-2
preferentially kills cancer cells with increased surface levels of negatively charged
phosphatidylserine”, FEBS Lett., Vol. 579, (2005), pp. 6128–6134.
[30] N. Papo and Y. Shai: “Host defense peptides as new weapons in cancer treatment”,
Cell. Mol. Life Sci., Vol. 62, (2005), pp. 784–790.
[31] J. Pardo, P. Perez-Galan, S. Gamen, I. Marzo, I. Monleon, A.A. Kaspar, S.A. Susin,
G. Kroemer, A.M. Krensky, J. Naval and A. Anel: “A role of the mitochondrial
apoptosis inducing factor in granulysin-induced apoptosis”, J. Immunol., Vol. 167,
(2001), pp. 1222–1229.
[32] T. Murakami, M. Niwa, F. Tokunaga, T. Miyata and S. Iwanaga: “Direct virus
inactivation of tachyplesin I and its isopeptides from horseshoe crab hemocytes”,
Chemotherapy, Vol. 37, (1991), pp. 327–334.
[33] M. Masuda, H. Nakashima, T. Ueda, H. Naba, R. Ikoma, A. Otaka Y. Terakawa,
H. Tamamura, T. Ibutaka, T. Murakami, Y. Koyanagi, M. Waki, A. Matsumoto,
N. Yamamoto, S. Funakoshi and N. Fuji.: “A novel anti-HIV synthetic peptide T22 ([Tyr5,12,Lys7]-polyphemusin II)”, Biochem. Biophys. Res. Commun., Vol. 189,
A. Giuliani et al. / Central European Journal of Biology 2(1) 2007 1–33
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
[45]
[46]
25
(1992), pp. 845–850.
M. Morimoto, H. Mori, T. Otake, N. Ueba, N. Kunita, M. Niwa, T. Murakami
and S. Iwanaga: “Inhibitory effect of tachyplesin I on the proliferation of human
immunodeficiency virus in vitro”, Chemotherapy, Vol. 37, (1991), pp. 206–211.
T. Murakami, T. Nakajima, Y. Koyanagi, K. Tachibana, N. Fujii, H. Tamamura,
N. Yoshida, M. Waki, A. Matsumoto, O. Yoshie, T. Kishimoto, N. Yamamoto and
T. Nagasawa: “A small molecule CXCR4 inhibitor that blocks T cell line-tropic
HIV-1 infection”, J. Exp. Med., Vol. 186, (1997), pp. 1389–1393.
R.F. Epand, A. Ramamoorthy and R.M. Epand: “Membrane Lipid Composition
and the Interaction of Pardaxin: The Role of Cholesterol”, Protein Pept. Lett., Vol.
13, (2006), pp. 1–5.
F. Porcelli, B. Bethany, D.K. Lee, K.J. Hallock, A. Ramamoorthy and G. Veglia:
“Structure and Orientation of Pardaxin Determined by NMR Experiments in Model
Membranes”, J. Biol. Chem., Vol. 279, (2004), pp. 45815–45823.
K.J. Hallock, D.K. Lee, J. Omnaas, H.I. Mosberg and A. Ramamoorthy: “Membrane Composition Determines Pardaxin’s Mechanism of Lipid Bilayer Disruption”,
Biophys. J., Vol. 83, (2002), pp. 1004–1013.
F. Porcelli, B.A. Buck-Koehntop, S. Thennarasu, A. Ramamoorthy and G. Veglia: “Structures of the Dimeric and Monomeric Variants of Magainin Antimicrobial
Peptides (MSI-78 and MSI-594) in Micelles and Bilayers, Determined by NMR
Spectroscopy”, Biochemistry, Vol. 45, (2006), pp. 5793–5799.
A. Mecke, D.K. Lee, A. Ramamoorthy, B.G. Orr and M.M.B. Holl: “Membrane
Thinning Due to Antimicrobial Peptide Binding: An Atomic Force Microscopy
Study of MSI-78 in Lipid Bilayers”, Biophys. J., Vol. 89, (2005), pp. 4043–4050.
S. Thennarasu, D.K. Lee, A.Tan, U.P. Kari and A. Ramamoorthy: “Antimicrobial
activity and membrane selective interactions of a synthetic lipopeptide MSI-843”,
Biochim. Biophys. Acta, Vol. 1711, (2005), pp. 49–58.
A. Ramamoorthy, S. Thennarasu, A. Tan, D. Lee, C. Clayberger and A.M. Krensky:
“Cell selectivity correlates with membrane-specific interactions: A case study on the
antimicrobial peptide G15 derived from granulysin”, Biochim. Biophys. Acta, Vol.
1758, (2006), pp. 154–163.
A. Ramamoorthy, S. Thennarasu, A. Tan, K. Gottipati, S. Sreekumar, D.L. Heyl,
F.Y.P. An and C.E. Shelburne: “Deletion of All Cysteines in Tachyplesin I Abolishes
Hemolytic Activity and Retains Antimicrobial Activity and Lipopolysaccharide Selective Binding”, Biochemistry, Vol. 45, (2006), pp. 6529–6540.
S. Thennarasu, D.K. Lee, A. Poon, K.E. Kawulka, J.C. Vederas and A. Ramamoorthy: “Membrane permeabilization, orientation, and antimicrobial mechanism of
subtilosin A”, Chem. Phys. Lipids. Vol. 137, (2005), pp. 38–51.
J.P. Powersand and R.E.W. Hancock: “The relationship between peptide structure
and antibacterial activity”, Peptides, Vol. 24, (2003), pp. 1681–1691.
R. Yeaman and N.Y. Yount: “Mechanisms of antimicrobial peptide action and
resistance”, Pharmacol. Rev., Vol. 55, (2003), pp. 27–55.
26
A. Giuliani et al. / Central European Journal of Biology 2(1) 2007 1–33
[47] K. Matsuzaki: “Why and how are peptide-lipid interactions utilized for self-defense?
Magainins and tachyplesins as archetypes”, Biochim. Biophys. Acta, Vol. 1462,
(1999), pp. 1–10.
[48] R.E.W. Hancock and D.S. Chapple: “Peptide antibiotics”, Antimicrob. Agents
Chemother., Vol. 43, (1999), pp. 1317–1323.
[49] R.E.W. Hancock: “Peptide antibiotics”, Lancet, Vol. 349, (1997), pp. 418–422.
[50] J.M. Sanderson: “Peptide-lipids interactions: insights and perspectives”, Org.
Biomol. Chem., Vol.3, (2005), pp. 201–212.
[51] Y. Shai and Z. Oren: “From ”carpet” mechanism to de-novo designed diastereomeric
cell-selective antimicrobial peptides”, Peptides, Vol. 22, (2001), pp. 1629–1641.
[52] N. Sitaram and R. Nagaraj: “Interaction of antimicrobial peptides with biological
and model membranes: structural and charge requirements for activity”, Biochim.
Biophys Acta, Vol. 1462, (1999), pp. 29–54.
[53] E. Breukink and B. de Kruijff: “The lantibiotic nisin, a special case or not?”,
Biochim. Biophys. Acta, Vol. 1462, (1999), pp. 223–234.
[54] R.I. Lehrer and T. Ganz: “Cathelicidins: a family of endogenous antimicrobial
peptides”, Curr. Opin. Hematol., Vol. 9, (2002), pp. 18–22.
[55] M.S.P. Sansom: “Alamethicin and related peptaibols-model ion channels”, Eur.
Biophys., Vol. 22, (1993), pp. 105–124.
[56] L. Yang, T.A. Harroun, T.M. Weiss, L. Ding and H.W. Huang: “Barrel-stave model
or toroidal model? A case study on melittin pores”, Biophys. J., (2001), Vol. 81,
pp. 1475–1485.
[57] L. Beven, O. Helluin, G. Molle, H. Duclohier and H. Wroblewski: “Correlation
between anti-bacterial activity and pore sizes of two classes of voltage-dependent
channel-forming peptides”, Biochim. Biophys. Acta, Vol. 1421, (1999), pp. 53–63.
[58] L. Yang, T.M. Weiss, R.I. Lehrer and H.W. Huang: “Crystallization of antimicrobial
pores in membranes: magainin and protegrin”, Biophys. J., Vol. 79, (2001), pp.
2002–2009.
[59] K. Matsuzaki: “Magainins as paradigm for the mode of action of pore forming
polypeptide”, Biochim. Biophys. Acta, Vol. 1376, (1998), pp. 391–400.
[60] K. Matsuzaki, O. Murase, N. Fujii and K. Miyajima: “An antimicrobial peptide,
magainin 2, induced rapid flip-flop of phospholipids coupled with pore formation
and peptide translocation”, Biochemistry, Vol. 35, (1996), pp. 11361–11368.
[61] B. Bechinger: “The structure, dynamics and orientation of antimicrobial peptides in
membranes by solid-state NMR spectroscopy”, Biochim. Biophys. Acta, Vol. 1462,
(1999), pp. 157–183.
[62] R.A. Cruciani, J.L. Barker, S.R. Durell, G. Raghunathan, H.R. Guy, M. Zasloff and
E.F Stanley: “Magainin 2: A natural antibiotic from frog skin, forms ion channels
in lipid bilayer membranes”, Eur. J. Pharmacol., Vol. 226, (1992), pp. 287–296.
[63] K.A. Brogden: “Antimicrobial peptides: pore formers or metabolic inhibitors in
bacteria?”, Nature Rev. Microb., Vol. 3, (2005), pp. 238–250.
A. Giuliani et al. / Central European Journal of Biology 2(1) 2007 1–33
27
[64] K.J. Hallock, D.K. Lee and A. Ramamoorthy: “MSI-78, an analogue of the magainin
antimicrobial peptides, disrupts lipid bilayer structure via positive curvature strain”,
Biophys. J., Vol. 84, (2003), pp. 3052–3060.
[65] K.A. Henzler Wildman, D.K. Lee and A. Ramamoorthy: “Mechanism of lipid bilayer disruption by the human antimicrobial peptide, LL-37”, Biochemistry, Vol.
42, (2003), pp. 6545–6558.
[66] K.A. Henzler-Wildman, G.V. Martinez, M.F. Brown and A. Ramamoorthy: “Perturbation of the Hydrophobic Core of Lipid Bilayers by the Human Antimicrobial
Peptide LL-37”, Biochemistry, Vol. 43, (2004), pp. 8459–8469.
[67] T. Ganz and R.I. Lehrer: “Defensins”, Pharmacol. Ther., Vol. 66, (1995), pp. 191–
205.
[68] A. Rozek, C.L. Friedrich and R.E.W. Hancock: “Structure of the bovine antimicrobial peptide indolicidin bound to dodecylphosphocholine and sodium dodecyl
sulfate micelles”, Biochemistry, Vol. 39, (2000), pp. 15765–15774.
[69] S. Vunnam, P. Juvvadi and R.B. Merrifield: “Synthesis and antibacterial action of
cecropin and proline-arginine-rich peptides from pig intestine”, J. Pept. Res., Vol.
49, (1997), pp. 59–66.
[70] P. Casteels and P. Tempst: “Apidaecin-type peptide antibiotics function through
a non-poreforming mechanism involving stereospecificity”, Biochem. Biophys. Res.
Commun., Vol. 199, (1994), pp. 339–345.
[71] P. Bulet, L. Urge, S. Ohresser, C. Hetru and L. Otvös: “Enlarged scale chemical
synthesis and range of activity of drosocin, an O-glycosylated antibacterial peptide
of Drosophila”, Eur. J. Biochem., Vol. 238, (1996), pp. 64–69.
[72] Subbalakshmi and N. Sitaram: “Mechanism of antimicrobial action of indolicidin”,
FEMS Microbiol. Lett., Vol. 160, (1998), pp. 91–96.
[73] A. Carlsson, P. Engström, E.T. Palva and H. Bennich: “Attacin, an antibacterial
protein from Hyalophora cecropia, inhibits synthesis of outer membrane proteins in
Escherichia coli by interfering with omp gene transcription”, Infect. Immun., Vol.
59, (1991), pp. 3040–3045.
[74] J. Oh, Y. Cajal, E.M. Skowronska, S. Belkin, J. Chen, T.K. Van Dyk, R.M. Sasse
and M.K. Jain: “Cationic peptide antimicrobials induce selective transcription of
micF and osmY in Escherichia coli ”, Biochim. Biophys. Acta, Vol. 1463, (2000),
pp. 43–54.
[75] V. Cabiaux, B. Agerberth, J. Johansson, F. Homblé, E. Goormaghtigh and J.
M. Ruysschaert: “Secondary structure and membrane interaction of PR-39, a
Pro+Arg-rich antibacterial peptide”, Eur. J. Biochem., Vol. 224, (1994), pp. 1019–
1027.
[76] H.G. Boman, B. Agerberth and A. Boman: “Mechanisms of action on Escherichia
coli of cecropin P1 and PR-39, two antibacterial peptides from pig intestine”, Infect.
Immun., Vol. 61, (1993), pp. 2978–2984.
[77] C.B. Park, H. S. Kim and S.C. Kim: “Mechanism of action of the antimicrobial
peptide buforin II: buforin II kills microorganisms by penetrating the cell mem-
28
[78]
[79]
[80]
[81]
[82]
[83]
[84]
[85]
[86]
[87]
[88]
[89]
A. Giuliani et al. / Central European Journal of Biology 2(1) 2007 1–33
brane and inhibiting cellular functions”, Biochem. Biophys. Res. Commun., Vol.
244, (1998), pp. 253–257.
B. Skerlavaj, D. Romeo and R. Gennaro: “Rapid membrane permeabilization and
inhibition of vital functions of gram-negative bacteria by bactenecins”, Infect. Immun., Vol. 58, (1990), pp. 3724–3730.
L. Otvos Jr., O. Insug, M.E. Rogers, P.J. Consolvo, B.A. Condie, S. Lovas, P.
Bulet and M. Blaszczyk-Thurin: “Interaction between Heat Shock Proteins and
Antimicrobial Peptides”, Biochemistry, Vol. 39, (2000), pp. 14150–14159.
L.S. Chesnokova, S.V. Slepenkov and S.N. Witt: “The insect antimicrobial peptide,
L-pyrrhocoricin, binds to and stimulates the ATPase activity of both wild-type and
lidless DnaK”, FEBS Lett., Vol. 565, (2004), pp. 65–69.
O. Toke: “Antimicrobial Peptides: New Candidates in the Fight Against Bacterial
Infections”, Pept. Sci., Vol. 80, (2005), pp. 717–735.
R.E.W. Hancock and H.G. Sahl: “Antimicrobial and host-defense peptides as new
anti-infective therapeutic strategies”, Nature Biotechnol, Vol. 24, (2006), pp. 1551–
1557.
M. Zasloff: “The Commercial Development of the Antimicrobial Peptide Pexiganan”, In: K. Lohner (Ed.): Development of Novel Antimicrobial Agents: Emerging Strategies, Horizon Scientific Press, Wymondham, UK, 2001, pp. 261–270.
H.M. Lamb and L.R. Wiseman: “Pexiganan Acetate”, Drugs, Vol. 56, (1998), pp.
1047–1052.
A. Trotti, A. Garden, P. Warde, P. Symonds, C. Langer, R. Redman, T.F. Pajak,
T.R. Fleming, M. Henke, J. Bourhis, D.I. Rosenthal, E. Junor, A. Cmelak, F. Sheehan, J. Pulliam, P. Devitt-Risse, H. Fuchs, M. Chambers, B. O’Sullivan and K.K.
Ang: “A multinational, randomized phase III trial of iseganan-HCl oral solution
for reducing the severity of oral mucositis in patients receiving radiotherapy for
head-and-neck malignancy”, Int. J. Radiat. Oncol. Biol. Phys., Vol. 58, (2004), pp.
674–681.
Y.J. Gordon, E.G. Romanowski and A.M. McDermott: “A review of antimicrobial
peptides and their therapeutic potential as anti-infective drugs”, Curr. Eye Res.,
Vol. 30, (2005), pp. 505–515.
N. Markou, H. Apostolakos, C. Koumoudiou, M. Athanasiou, A. Koutsoukou, I.
Alamanos and L. Gregorako: “Intravenous colistin in the treatment of sepsis from
multiresistant Gram-negative bacilli in critically ill patients”, Crit. Care, Vol. 7,
(2003), pp. 78–83.
M.E. Falagas and S.K. Kasiakou: “Colistin: the revival of polymyxins for the
management of multidrug-resistant gram-negative bacterial infections”, Clin. Infect. Dis., Vol. 40, (2005), pp. 1333–1341.
A. Kubo, C.S. Lunde and I. Kubo: “Indole and (E)-2-hexenal, phytochemical potentiators of polymyxins against Pseudomonas aeruginosa and Escherichia coli ”,
Antimicrob. Agents. Chemother., Vol. 40, (1996), pp. 1438–1441.
A. Giuliani et al. / Central European Journal of Biology 2(1) 2007 1–33
29
[90] S.P. Conway, M.N. Pond, A.Watson, C. Etherington, H.L. Robey and M.H. Goldman: “Intravenous colistin sulphometate in acute respiratory exacerbations in adult
patients with cystic fibrosis”, Thorax, Vol. 52, (1997), pp. 987–993.
[91] A. Pini, A. Giuliani, C. Falciani, Y. Runci, C. Ricci, B. Lelli, M. Malossi, P. Neri,
G.M. Rossolini and L. Bracci: “Antimicrobial Activity of Novel Dendrimeric Peptides Obtained by Phage Display Selection and Rational Modification”, Antimicrob.
Agents Chemother., Vol. 49, (2005), pp. 2665–2672.
[92] K.H. Mayo, J. Haseman, E. Ilyina and B. Gray: “Designed beta-sheet-forming
peptide 33mers with potent human bactericidal/permeability increasing proteinlike bactericidal and endotoxin neutralizing activities”, Biochim. Biophys. Acta,
Vol. 1425, (1998), pp. 81-92.
[93] P.H. Mygind, R.L. Fischer, K.M. Schnorr, M.T. Hansen, C.P. Sönksen, S. Ludvigsen, D. Raventós, S. Buskov, B. Christensen, L. De Maria, O. Taboureau, D.
Yaver, S.G. Elvig-Jørgensen, M.V. Sørensen, B.E. Christensen, S. Kjærulff, N.
Frimodt-Moller, R.I. Lehrer, M. Zasloff and H.-H. Kristensen: “Plectasin is a peptide antibiotic with therapeutic potential from a saprophytic fungus”, Nature, Vol.
437, (2005), pp. 975–980.
[94] M.M Welling, A. Paulusma-Annema, H.S. Balter, E.K. Pauwels and P.H. Nibbering:
“Technetium-99m labelled antimicrobial peptides discriminate between bacterial
infections and sterile inflammations”, Eur. J. Nucl. Med., Vol. 24, (2004), pp. 292–
301.
[95] A. Giacometti, O. Cirioni, F. Barchiesi and G. Scalise: “In-vitro activity and killing
effect of polycationic peptides on methicillin-resistant Staphylococcus aureus and
interactions with clinically used antibiotics”, Diagn. Microbiol. Infect. Dis., Vol. 38,
(2000), pp. 115–118.
[96] S.L. Haynie, G.A. Crum and B.A. Doele: “Antimicrobial activities of amphiphilic peptides covalently bonded to a water-insoluble resin”, Antimicrob. Agents
Chemother. Vol. 39, (1995), pp. 301–307.
[97] J.K. Ghosh, D. Shaool, P. Guillaud, L. Ciceron, D. Mazier, I. Kustanovich, Y. Shai
and A. Mor: “Selective cytotoxicity of dermaseptin S3 toward intraerythrocytic
Plasmodium falciparum and the underlying molecular basis”, J. Biol. Chem., Vol.
272, (1997), pp. 31609–31616.
[98] I. Ahmad, W.R. Perkins, D.M. Lupan, M.E. Selsted and A.S. Janoff: “Liposomal
entrapment of the neutrophil-derived peptide indolicidin endows it with in vivo
antifungal activity”, Biochim. Biophys. Acta, Vol. 1237, (1995), pp. 109–114.
[99] R. Raqib, P. Sarker, P. Bergman, G. Ara, M. Lindh, D.A. Sack, K.M. Nasirul
Islam, G.H. Gudmundsson, J. Andersson and B. Agerberth: “Improved outcome in
shigellosis associated with butyrate induction of an endogenous peptide antibiotic.”,
Proc. Natl. Acad. Sci., Vol. 103, (2006), pp. 9178–9183.
[100] S. Kim, S.S Kim, Y.J. Bang, S.J. Kim and B.J. Lee: “In vitro activities of native
and designed peptide antibiotics against drug sensitive and resistant tumor cell
lines”, Peptides, Vol. 24, (2003), pp. 945-953.
30
A. Giuliani et al. / Central European Journal of Biology 2(1) 2007 1–33
[101] S.A. Johnstone, K. Gelmon, L.D. Mayer, R.E. Hancock and M.B. Bally: “In vitro
characterization of the anticancer activity of membrane-active cationic peptides. I.
Peptide-mediated cytotoxicity and peptide-enhanced cytotoxic activity of doxorubicin against wild-type and p-glycoprotein over-expressing tumor cell lines”, Anticancer Drug. Res., Vol. 15, (2000), pp. 151–160.
[102] C. Leuschner and W. Hansel: “Membrane Disrupting Lytic Peptides for Cancer
Treatments”, Curr. Pharm. Design, Vol. 10, (2004), pp. 2299–2310.
[103] D. Winder, W.H. Gunzburg, V. Erfle and B. Salmons: “Expression of antimicrobial peptides has an antitumour effect in human cells”, Biochem. Biophys. Res.
Commun., Vol. 242, (1998) pp. 608–612.
[104] A.K. Marr, W.J. Gooderham and R.E.W. Hancock: “Antibacterial peptides for
therapeutic use: obstacles and realistic outlook”, Curr. Opin. Pharmacol., Vol. 6,
(2006), pp. 468–472.
[105] C. Haught, G.D. Davis, R. Subramanian, K.W. Jackson and R.G. Harrison: “Recombinant Production and Purification of Novel Antisense Antimicrobial Peptide
in Escherichia coli ”, Biotech. Bioeng., Vol. 57, (1998), pp. 55–61.
[106] E.A. Groisman: “The ins and outs of virulence gene expression: Mg2+ as a regulatory signal”, Bioessays, Vol. 20, (1998), pp. 96–101.
[107] J.S. Gunn, S.S. Ryan, J.C. Van Velkinburgh, R.K. Ernst and S.I. Miller: “Genetic and functional analysis of a PmrA-PmrB-regulated locus necessary for
lipopolysaccharide modification, antimicrobial peptide resistance, and oral virulence
of Salmonella enterica serovar typhimurium”, Infect. Immun., Vol. 68, (2000), pp.
6139–6146.
[108] C. Friedrich, M.G. Scott, N. Karunaratne, H. Yan and R.E.W. Hancock:
“Salt-resistant alpha-helical cationic antimicrobial peptides”, Antimicrob. Agents
Chemother., Vol. 43, (1999), pp. 1542–1548.
[109] G.G. Perron, M. Zasloff and G. Bell: “Experimental evolution of resistance to an
antimicrobial peptide”, Proc. Biol. Sci., Vol. 273, (2006), pp. 251–256.
[110] O. Sørensen, T. Bratt, A.H. Johnsen, M.T. Madsen and N. Borregaard: “The human
antibacterial cathelicidin, hCAP-18, is bound to lipoproteins in plasma”, J. Biol.
Chem., Vol. 274, (1999), pp. 22445–22451.
[111] C. Adessi and C. Soto: “Converting a peptide into a drug: strategies to improve
stability and bioavailability”, Curr. Med. Chem., Vol. 9, (2002), pp. 963–978.
[112] M. Goodman, C. Zapf and Y. Rew: “New reagents, reactions, and peptidomimetics
for drug design”, Biopolymers, Vol. 60, (2001), pp. 229–245.
[113] A. Wiest, D. Grzegorski, B.W. Xu, C. Goulard, S. Rebuffat, D.J. Ebbole, B. Bodo
and C. Kenerley: “Identification of peptaibols from Trichoderma virens and cloning
of a peptaibol synthetase”, J. Biol. Chem., Vol. 277, (2002), pp. 20862–20868.
[114] A. Banerjee, A. Pramanik, S. Bhattacharjya and P. Balaram: “Omega amino acids
in peptide design: incorporation into helices”, Biopolymers, Vol. 39, (1996), pp.
769–777.
A. Giuliani et al. / Central European Journal of Biology 2(1) 2007 1–33
31
[115] J.M. Ostresh, S.E. Blondelle, B. Dörner and R.A. Houghten: “Generation and use of
nonsupported-bound peptide and peptidomimetic combinatorial libraries”, Methods
Enzymol., Vol. 267, (1996), pp. 220–234.
[116] A. Wessolowski, M. Bienert and M. Dathe: “Antimicrobial activity of arginineand tryptophan-rich hexapeptides: the effects of aromatic clusters, D-amino acid
substitution and cyclization”, J. Pept. Res., Vol. 64, (2004), pp. 159–169.
[117] D. Gimenez, C. Andreu, M. del Olmo, T. Varea, D. Diaz and G. Asensio: “The
introduction of fluorine atoms or trifluoromethyl groups in short cationic peptides
enhances their antimicrobial activity”, Bioorg. Med. Chem., Vol. 14, (2006), pp.
6971–6978.
[118] M. Dathe, J. Meyer, M. Beyermann, B. Maul, C. Hoischen and M. Bienert: “General
aspects of peptide selectivity towards lipid bilayers and cell membranes studied
by variation of the structural parameters of amphipathic helical model peptides”,
Biochim. Biophys. Acta, Vol. 1558, (2002), pp. 171–186.
[119] Z. Oren, J. Ramesh, D. Avrahami, N. Suryaprakash, Y. Shai and R. Jelinek: “Structures and mode of membrane interaction of a short alpha helical lytic peptide and
its diastereomer determined by NMR, FTIR, and fluorescence spectroscopy”, Eur.
J. Biochem., Vol. 269, (2002), pp. 3869–3880.
[120] S.-T Yang, S.Y. Shin, C.W. Lee, Y.-C. Kim, K.-S Hahm and J.I. Kim: “Selective cytotoxicity following Arg-to-Lys substitution in tritrpticin adopting a unique
amphipathic turn structure”, FEBS Lett., Vol. 540, (2003), pp. 229–233.
[121] S.E. Blondelle and K. Lohner: “Combinatorial libraries: a tool to design antimicrobial and antifungal peptide analogues having lytic specificities for structure-activity
relationship studies”, Biopolymers, Vol. 55, (2000), pp. 74–87.
[122] A. Malina and Y.Shai: “Conjugation of fatty acids with different lengths modulates
the antibacterial and antifungal activity of a cationic biologically inactive peptide”,
Biochem. J., Vol. 390, (2005), pp. 695–702.
[123] L. Otvos Jr., C. Snyder, B. Condie, P. Bulet and J.D. Wade: “Chimeric Antimicrobial Peptides Exhibit Multiple Modes of Action”, Int. J. Pept. Res. Ther., Vol. 11,
(2005), pp. 29–42.
[124] R. Eckert, F. Qi, D.K. Yarbrough, J. He, M.H. Anderson and W. Shi: “Adding
Selectivity to Antimicrobial Peptides: Rational Design of a Multidomain Peptide
against Pseudomonas spp.”, Antimicrob. Agents Chemother., Vol. 50, (2006), pp.
1480–1488.
[125] C. Loose, K. Jensen, I. Rigoutsos and G. Stephanopoulos: “A linguistic model for
the rational design of antimicrobial peptides”, Nature, Vol. 443, (2006), pp. 867–869.
[126] I. Rigoutsos and A. Floratos: “Combinatorial pattern discovery in biological sequences: The TEIRESIAS algorithm”, Bioinformatics, Vol. 14, (1998), pp. 55–67.
[127] K. Hilpert, M.R. Elliott, R.Volkmer-Engert, P. Henklein, O. Donini, Q. Zhou, D.F.
Winkler and R.E.W. Hancock: “Sequence requirements and an optimization strategy for short antimicrobial peptides”, Chem. Biol., Vol. 13, (2006), pp. 1101–1107.
32
A. Giuliani et al. / Central European Journal of Biology 2(1) 2007 1–33
[128] K. Hilpert, R. Volkmer-Engert, T. Walterand and R.E.W. Hancock: “Highthroughput generation of small antibacterial peptides with improved activity”, Nature Biotechnol., Vol. 23, (2005), pp. 1008–1012.
[129] V. Nagarajan, N. Kaushik, B. Murali, C. Zhang, S. Lakhera, M.O. Elasri and Y.
Deng: “A Fourier Transformation based method to mine peptide space for antimicrobial activity”, BMC Bioinformatics, Vol. 7, Suppl. 2, (2006).
[130] A.D. McLachlan: “Analysis of periodic patterns in amino acid sequences: collagen”,
Biopolymers, Vol. 16, (1977), pp. 1271–1297.
[131] S. Tiwari, S. Ramachandran, A. Bhattacharya, S. Bhattacharya and R. Ramaswamy: “Prediction of probable genes by fourier analysis of genomic sequences”,
Comput. Appl. Biosci., Vol. 13, (1997), pp. 263–270.
[132] D.J. Christensen, E.B. Gottlin, R.E. Benson and P.T. Hamilton: “Phage display
for target-based antibacterial drug discovery”, Drug Discov. Today., Vol. 6, (2001),
pp. 721–727.
[133] F. Sanschagrin and R.C. Levesque: “A specific peptide inhibitor of the class B
metallo-ß-lactamase L-1 from Stenotrophomonas maltophilia identified using phage
display”, J. Antimicrob. Chemother., Vol. 55, (2005), pp. 252–255.
[134] R. Hyde-DeRuyscher, L.A. Paige, D.J. Christensen, N. Hyde-DeRuyscher, A. Lim,
Z.L. Fredericks, J. Kranz, P. Gallant, J. Zhang, S.M. Rocklage, D.M. Fowlkes,
P.A. Wendler and P.T. Hamilton: “Detection of small-molecule enzyme inhibitors
with peptides isolated from phage-displayed combinatorial peptide libraries”, Chem.
Biol., Vol. 7, (2000), pp. 17–25.
[135] H. Grøn and R. Hyde-DeRuyscher: “Peptides as tools in drug discovery”, Curr.
Opin. Drug Disc., Vol. 3, (2000), pp. 636–645.
[136] C. Galanos, O. Luderitz, E.T. Rietschel and O. Westphal: “Newer aspects of the
chemistry and biology of bacterial lipopolysaccharides with special reference to their
lipid A component“, Int. Rev. Biochem. Vol. 14, (1977), pp. 239–334.
[137] C.J. Thomas, S. Sharma, G. Kumar, S.S. Visweswariah and A. Surolia: “Biopanning
of endotoxin-specific phage displayed peptides”, Biochem. Biophys. Res. Commun.,
Vol. 307, (2003), pp. 133–138.
[138] J. Tao, P. Wendler, G. Connelly, A. Lim, J. Zhang, M. King, T. Li, J.A. Silverman,
P.R. Schimmel and F.P. Tally: “Drug target validation: lethal infection blocked by
inducible peptide”, Proc. Natl. Acad. Sci. U.S.A., Vol. 97, (2000), pp. 783–786.
[139] J.P. Tam: “Synthetic peptide vaccine design: synthesis and properties of a highdensity multiple antigenic peptide system”, Proc. Natl. Acad. Sci. U.S.A., Vol. 85,
(1988), pp. 5409–5413.
[140] C.C. Lee, J.A. MacKay, J.M.J. Fréchet and F.C. Szoka: “Designing dendrimers for
biological applications”, Nature Biotechnol., Vol. 23, (2005), pp. 1517–1526.
[141] L. Bracci, L. Lozzi, A. Pini, B. Lelli, C. Falciani, N. Niccolai, A. Bernini, A.
Spreafico, P. Soldani and P. Neri: “A branched peptide mimotope of the nicotinic
receptor binding site is a potent synthetic antidote against the snake neurotoxin
alpha-bungarotoxin”, Biochemistry, Vol. 41, (2002), pp. 10194–10199.
A. Giuliani et al. / Central European Journal of Biology 2(1) 2007 1–33
33
[142] L. Lozzi, B. Lelli,Y. Runci, S. Scali, A. Bernini, C. Falciani, A. Pini, N. Niccolai, P.
Neri and L. Bracci: “Rational design and molecular diversity for the construction of
anti-alpha-bungarotoxin antidotes with high affinity and in vivo efficiency”, Chem.
Biol., Vol. 10, (2003), pp. 411–417.
[143] L. Bracci, C. Falciani, B. Lelli, L. Lozzi, Y. Runci, A. Pini, M. G. De Montis, A.
Tagliamonte, and P. Neri: “Synthetic peptides in the form of dendrimers become
resistant to protease activity”, J. Biol. Chem., Vol. 278, (2003), 46590–46595.
[144] J.P. Tam, Y.A. Lu and J.L. Yang: “Antimicrobial dendrimeric peptides”, Eur. J.
Biochem., Vol. 269, (2002), pp. 923–932.
[145] J. Janiszewska, J. Swieton , A.W. Lipkowski and Z. Urbanczyk-Lipkowska: “Low
molecular mass peptide dendrimers that express antimicrobial properties”, Bioorg.
Med. Chem. Lett., Vol. 13, (2003), pp. 3711–3713.