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A novel tool against multiresistant bacterial pathogens – lipopeptidemodification of the natural antimicrobial peptide ranalexin forenhanced antimicrobial activity and improved pharmacokinetics
Cornelius Domhan , Philipp Uhl , Anna Meinhardt ,Stefan Zimmermann , Christian Kleist , Thomas Lindner ,Karin Leotta , Walter Mier , Michael Wink
PII: S0924-8579(18)30101-8DOI: 10.1016/j.ijantimicag.2018.03.023Reference: ANTAGE 5414
To appear in: International Journal of Antimicrobial Agents
Received date: 16 November 2017Revised date: 29 March 2018Accepted date: 31 March 2018
Please cite this article as: Cornelius Domhan , Philipp Uhl , Anna Meinhardt , Stefan Zimmermann ,Christian Kleist , Thomas Lindner , Karin Leotta , Walter Mier , Michael Wink , A novel tool againstmultiresistant bacterial pathogens – lipopeptide modification of the natural antimicrobial peptideranalexin for enhanced antimicrobial activity and improved pharmacokinetics, International Journalof Antimicrobial Agents (2018), doi: 10.1016/j.ijantimicag.2018.03.023
This is a PDF file of an unedited manuscript that has been accepted for publication. As a serviceto our customers we are providing this early version of the manuscript. The manuscript will undergocopyediting, typesetting, and review of the resulting proof before it is published in its final form. Pleasenote that during the production process errors may be discovered which could affect the content, andall legal disclaimers that apply to the journal pertain.
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Highlights
The antimicrobial peptide ranalexin showed strong antimicrobial activity against Gram-
positive bacteria and Acinetobacter species.
Ranalexin is excreted via the kidneys within one hour in WISTAR rats.
Lipopeptide derivatives of ranalexin displayed enhanced antimicrobial activity, especially
against Gram-negative bacteria.
The investigated lipopeptide derivative accumulated in the liver of WISTAR rats.
Time-kill studies showed a fast concentration-dependent killing within one hour.
Cytotoxicity of the lipopeptides depends on the chain length of the fatty acid.
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A novel tool against multiresistant bacterial pathogens – lipopeptide modification of the natural
antimicrobial peptide ranalexin for enhanced antimicrobial activity and improved
pharmacokinetics
Cornelius Domhan1, Philipp Uhl2, Anna Meinhardt², Stefan Zimmermann3, Christian Kleist2, Thomas
Lindner2, Karin Leotta2, Walter Mier2, Michael Wink1*
1 Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, 69120 Heidelberg,
Germany 2 Department of Nuclear Medicine, Heidelberg University Hospital, 69120 Heidelberg, Germany 3 Department of Infectious Diseases, Medical Microbiology and Hygiene, Heidelberg University
Hospital, 69120 Heidelberg, Germany
* Corresponding author
Key words
Ranalexin, Antimicrobial peptides, Lipopeptides, Positron emission tomography, Multiresistant
bacteria, Drug development
Abstract
As evolutionary optimized defense compounds, antimicrobial peptides (AMPs) represent a powerful
tool against bacterial infections. Ranalexin, an AMP found in the skin of the American bullfrog (Rana
catesbeiana), is primarily active against Gram-positive bacteria with a minimum inhibitory
concentration (MIC) of 8 – 16 mg/L but shows weaker activity against Gram-negative bacteria (> 64
mg/L). By substitution of 6 N-terminal amino acids by decanoic (C10:0) to myristic (C14:0) acid, we
were able to develop lipopeptide derivatives with enhanced antimicrobial activity. The antimicrobial
capacity of the peptides was tested against different bacterial strains, including multiresistant clinical
isolates. C13C3lexin, the most potent derivative showed MICs of 2 – 8 mg/L against Gram-positive
bacteria and 2 – 16 mg/L against Gram-negative bacteria. In time-kill studies, it could be clearly
shown that ranalexin and the lipopeptide C13C3lexin function as concentration dependent, fast
acting substances against different bacteria. Cell viability assays revealed that cytotoxicity towards
human cells increases with the chain length of the attached fatty acid (IC50 13,65 – 108,10 µg/mL).
Furthermore, with positron emission tomography (PET)-imaging pharmacokinetics studies of 68Ga-
labeled ranalexin and its derivatives were performed for the first time. Here we could demonstrate
that ranalexin is rapidly cleared via the kidneys within 1 h post injection. In contrast, the lipopeptide
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showed strongly extended circulation within the bloodstream and a shift from renal to hepatic
accumulation characteristics. Therefore, the more favorable pharmacokinetics and enhanced
antimicrobial activity clearly demonstrate the potential of the lipopeptidic AMPs as novel bullets
against emerging multiresistant bacterial pathogens.
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1. Introduction
Prior to the introduction of antibiotics into medicine about 70 years ago, bacterial infections
represented a major cause of death. Unfortunately, antibiotic resistance developed and is now
spreading faster than the launch of new effective antimicrobial drugs into the market, potentially
leading to a health crisis in the near future [1]. Nowadays, increasing numbers of infections occur
caused by bacteria which are resistant to most of the antibacterial treatments currently available [2].
It is estimated that more than 2,000,000 illnesses and 23,000 deaths are caused by antibiotic
resistance in the United States per year [3]. The majority of severe infections are caused by bacteria,
generally referred to as “ESKAPE”, comprising Enterococcus faecium, Staphylococcus aureus,
Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species
[4]. Infections caused by the multidrug resistant A. baumannii belong to infections most difficult to
treat [5].
The development of new effective antibiotics is a costly endeavor, but to abandon this goal could
lead to severe health problems [6]. Thus, new approaches are urgently needed. Antimicrobial
peptides (AMPs) represent natural products, occurring from microorganisms to the human. AMPs
consist of 15 to nearly 50 amino acids and are generally positively charged [7]. Importantly, many
AMPs are amphiphilic and interact with biomembranes by disrupting the cell membrane. In contrast,
most of the established antibiotics act against targets like ribosomes or enzymes involved in the cell
wall synthesis [8]. Due to their broad-spectrum and bactericidal activities, AMPs are promising
therapeutic alternatives in the fight against multiresistant bacteria [9]. Naturally occurring peptides
are mostly not directly suitable for medical use as therapeutics because they show intrinsic
weaknesses, including poor chemical and physical stability and also a short circulating plasma half-
life [10]. Another drawback of many AMPs is their low efficacy against Gram-negative bacterial
pathogens [7]. Amphibians produce evolutionary optimized AMPS which serve as potent tools
against bacterial infections [11]. Ranalexin, an AMP from the skin of the American bullfrog, Rana
catesbeiana, consists of 20 amino acids (Figure 1) [12]. It is an amphipathic molecule with a cationic
heptapeptide ring at its carboxyl terminus and a hydrophobic region at its amino terminal end [12].
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These two regions are linked via a “hinge” molecule made of the amino acid proline. The cyclic
structure of ranalexin is based on a disulfide bond between the two cysteine residues. Ranalexin
combined with lysostaphin displayed synergistic inhibitory effects against clinical isolates of S. aureus
[13]. Combination of ranalexin with linezolid or polymyxin B displayed significant synergism against
multidrug-resistant bacteria [14]. Ghiselli et al. further tested a single dose treatment of ranalexin in
rats with septic shock, leading to prevention of bacterial growth, endotoxemia and mortality in the
animals [15].
In order to improve the antibacterial activity of ranalexin, modifications of the peptide are needed.
Acylation can significantly improve the antimicrobial potency of peptides [16]. In contrast, the
removal of the acyl chain almost completely destroys the antimicrobial activity of polymyxin B [16].
Due to the fact, that solid-phase peptide synthesis is quite expensive and complex, Aleinein et al.
established a method of expressing recombinant ranalexin in Escherichia coli and Pichia pastoris
which represents an alternative route for peptide production [14, 17].
In this present article, we report on the development of novel lipopeptides based on the naturally
occurring AMP ranalexin. We demonstrate their improved antimicrobial activity against a variety of
Gram-positive and Gram-negative bacteria, including multidrug-resistant clinical isolates. We also
evaluate the substances for their cytotoxicity on various vertebrate cell types. Furthermore, we
delineate their formidable pharmacokinetic profile in a Wistar rat model using positron emission
tomography (PET).
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Please insert in Table 1 here
2. Material and methods
2.1 Peptide synthesis
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Ranalexin and its lipopeptide derivatives (C14C3lexin, C13C3lexin, C12C3lexin C11C3lexin and
C10C3lexin), were synthesized by fluoren-9-methoxycarbonyl (Fmoc) chemistry with an Applied
Biosystems ABI 433A synthesizer as described in detail by Brings et al. [18]. For further information
see supplementary data.
2.2 Antimicrobial activity
Acinetobacter bohemicus DSM 100419, Escherichia coli K12 DSM 498, Pseudomonas fluorescens DSM
50090, Salmonella subterranea DSM 16208, Yersinia mollaretii DSM 18520, Bacillus licheniformis
DSM 13, Bacillus megaterium DSM 32, Bacillus subtilis DSM 10 and Staphylococcus auricularis DSM
20609 were purchased from the Leibniz Institute DSMZ German Collection of Microorganisms and
Cell Culture (Braunschweig, Germany). All other bacteria were obtained from the Department of
Medical Microbiology and Hygiene, Heidelberg University Hospital. The MIC values were determined
by broth microdilution method of the Clinical and Laboratory Standards Institute (CLSI, Wayne, PA,
USA) [19]. The protocol is described in detail in the supplementary information.
2.3 Time-kill studies
Time-kill studies were performed according to the guidelines of CLSI [20]. Briefly, Escherichia coli
ATCC 25922 and Staphylococcus aureus ATCC 25923 were adjusted to 1x 108 cfu/ml, diluted to 1x 106
cfu/ml and adjusted to 4x MIC, 2x MIC and 1x MIC of ranalexin, C13C3lexin and cefuroxime (potency
88.6%) (Sigma-Aldrich, Steinheim, Germany) as control. Also, an aliquot of the 1*106 cfu/mL dilution
was mixed to an equal volume of medium (cation-adjusted Mueller Hinton broth) for growth control.
After 0, 0.5, 1, 2, 4, 8 and 12 h aliquots were serially diluted (1:10) in sterile saline. 10 μl of each
dilution were spread onto agar plates. After 24 h of incubation at 37 °C, cfu were counted and the
viable units within the original inoculum were calculated.
2.4 Cell viability assay
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In order to assess cytotoxic effects of the AMPs against eukaryotic tissue, the human tumor cell lines
MCF-7 (breast adenocarcinoma, ACC-115), HeLa (cervical adenocarcinoma, ACC-57), C4-2 (prostate
carcinoma, ATCC CRL-3314), HepG2 (liver hepatocellular carcinoma, ACC-180), the immortalized
Chinese hamster (Cricetulus griseus) ovary cell line CHO-K1 (ACC-110) as well as human peripheral
blood mononuclear cells (PBMCs) were exposed to the peptides. AMP cytotoxicity was investigated
in vitro by an MTT viability assay [21]. The protocol is described in detail in the supplement.
2.5 Radiolabeling with 68Ga
For the PET scans, the DOTA-conjugated peptides were labelled with 68Ga which was eluted with 0.6
M hydrochloric acid (HCl) from an iThemba LABS 68Ge/68Ga generator (DSD Pharma, Pukersdorf,
Austria) as described previously [22] (further information see supplement).
2.6 Micro-PET Imaging in Wistar rats
The procedures in this study were approved by the Animal Care and Use committees at the
Regierungspräsidium Karlsruhe, Germany. Adult, male, Wistar rats (250 – 300 g) were purchased
from Janvier Labs (Le Genest-Saint-Isle, France). The animals were anaesthetized by isoflurane
inhalation and 80 – 100 MBq of the 68Gallium labelled peptide, dissolved in 100 µl of 0.9% NaCl were
injected into the tail vein. Imaging was performed with an Inveon small-animal PET scanner (Siemens,
Erlangen, Germany). Cumulative images from 0 – 20 min, 20 – 40 min and 40 – 60 min after injection
were taken. For the DOTA coupled lipopeptide one further image from 120 – 140 min after injection
was made. The data were normalized for animal weight and injected dose. Pictures and data are
given as standardized uptake value (SUV) [18].
3. Results
3.1 Peptide synthesis
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Pure batches of ranalexin and derived lipopeptides C10C3lexin, C11C3lexin, C12C3leixn, C13C3lexin
and C14C3lexin were obtained. Their sequences, molecular masses and the weight analyzed by
HPLC/MS are shown in Table 1. The chemical structures of ranalexin and C13C3lexin are illustrated in
Figure 1, delineating the design of novel lipopeptide based on the conjugation of saturated fatty acid
to a peptide moiety. Detailed LC-MS spectra can be found in supplementary Figures 1 - 4.
3.2 Antimicrobial susceptibility of clinical isolates
The results of the susceptibility testing of the bacterial strain E. faecium UL602570, P. aeruginosa
BL303790 and K. pneumoniae KL800877 with a variety of antibiotics are shown in Table 2. Through
molecular biological analyses diverse resistance genes were detected. E. faecium UL602570 contains
a vanA resistance gene. The metallo-β-lactamase gene blaVIM-2 was found in P. aeruginosa
BL303790. K. pneumoniae KL800877 contains a KPC-2 Klebsiella pneumoniae carbapenemase. The
results of the susceptibility testing of the different clinical Acinetobacter isolates, shown in
supplementary Table 1, were quite alarming. Only A. baumannii BL600773 and A. junii KL511137
showed a wide spectrum of susceptibility to established antibiotics. The other A. baumannii isolates
belong to the group of multiresistant Gram-negative bacteria (MRGN). A. baumannii BL600773;
KL210652; KL314822; SC300007; SR201346; SR300573 and UL515338 are 4-MRGN strains, which are
resistant to antibiotics of the groups of carbapenems, quinolones, amino glycosides and fourth
generation cephalosporins. A. baumannii KL309623; SC322333 and SC404997 belong to the 3-MRGN
strains, being resistant to 3 of the groups mentioned. The antimicrobial susceptibility testing here
confirmed previous findings, that the polymyxin antibiotic colistin is often one of the only few
remaining antibiotics effective against multiresistant A. baumannii strains.
Please insert Table 2 here
3.3 Antimicrobial activity of the AMPs
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The results of the MIC testing of AMPs against a representative selection of bacteria are shown in
Figure 2 and 3. Ranalexin shows a potent activity against all 12 tested Acinetobacter strains (4 – 8
mg/L), and also against Gram-positive bacteria (8 – 16 mg/L). Against the tested Gram-negative non-
Acinetobacter bacteria, ranalexin shows only low antimicrobial activity (16 - >64 mg/L). In contrast,
the lipopeptides derivatives are more potent than ranalexin. Especially, MIC values against the non-
Acinetobacter Gram-negative bacteria are significantly lower for C13C3lexin and C12C3lexin (2 – 16
mg/L) than the MIC values for ranalexin. Tridecanoic acid and dodecanoic acid seem to be the
preferred chain length for the antimicrobial activity. Gram-positive bacteria are less sensitive against
variations in the molecular structure of the antimicrobial peptides (Figure 3). The non-Acinetobacter
Gram-negative bacteria are very sensitive for variations of the chain length of the fatty acid. The
changes of the MIC values demonstrate this fact, especially by comparison up the values for
C11C3lexin (4 – 16 mg/L) and C10C3lexin (16 – 64 mg/L). Another important characteristic of
ranalexin and the lipopeptide derivatives is that they are not affected by resistance mechanisms
against established antibiotics. Even multiresistant strains like K. pneumoniae KL800877 or A.
baumannii SC300007 are sensitive to the lipopeptides. The MICs for the bacteria of the genus
Pseudomonas are of special interest since they are higher than the MICs of the other bacteria tested.
The MBC is mostly one to two potencies higher than the MIC (Table 3). This allows the assumption
that the mechanism of action of the peptides is in fact bactericidal.
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3.4 Time-kill studies
For estimation of time-kill kinetics, we performed time-kill studies against Gram-negative E. coli ATCC
25922 and Gram-positive S. aureus ATCC 25923 at different concentrations (4x MIC, 2x MIC and 1x
MIC). The results, illustrated in Figure 4, indicate that ranalexin and C13C3lexin are concentration
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dependent and fast acting, bactericidal substances. At a concentration of 4x MIC, no cfu could be
detected after 30 min of incubation. At a concentration of 2x MIC the lipopeptide C13C3lexin shows
an advantage compared to ranalexin. After 30 min no cfu could be detected for the lipopeptide,
whereas for ranalexin, no cfu could be detected only after 2 h for E. coli and after 4 h for S. aureus,
respectively. Both substances, ranalexin and C13C3lexin exhibited superior killing kinetics compared
to the established antibiotic cefuroxime (Figure 4).
Please insert Figure 4 here
3.5 Cell viability assay
Essential for the assessment of any potential clinical application, cytotoxicity of ranalexin and its
derived lipopeptides was investigated in a viability assay against different cancer as well as non-
cancerous cell types. The IC50 values, representing the specific concentrations of the respective
substances tested upon which 50% viability is observed within the chosen cell culture, are displayed
in Figure 5. Our study remarkably shows that the cytotoxicity of the lipopeptides is dependent on the
chain length of the fatty acid linked to the peptide. The AMPs carrying fatty acid moieties from C14
to C12 (IC50 12.74 – 32.07 µg/mL) exhibit higher cytotoxicity than ranalexin (IC50 21.61 – 32.29
µg/mL). In contrast, for C10C3lexin lower toxicity was observed (IC50 38.49 – 108.10 µg/mL). Even
though, the IC50 values differ between the different cell lines, they show a similar trend for ranalexin
as well as for its derivatives. Importantly, also non-tumorous cell lines were tested, such as the
hamster ovary cells CHO and human peripheral blood cells (PBMCs). Even though these cells show
similar sensitivity to the compounds tested as the tumor cells lines, native PBMCs are significantly
less vulnerable to the AMP C10C3lexin (IC50 108.10 µg/mL) carrying the shortest fatty acid moiety.
Please insert Figure 5 here
3.6 Micro PET Imaging
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For estimation of the pharmacokinetics, PET imaging of DOTA-labelled ranalexin and C13C3lexin was
performed in Wistar rats (Figure 6). The corresponding calculated SUV values after injection are
shown in the supplementary Figures 5 and 6. The PET images and the SUVs clearly show that
ranalexin is eliminated through the kidneys and excreted via the urine. Interestingly, the first picture
from 0 – 20 min reveal that a greater amount of radioactivity is localized in the renal pelvis and in the
bladder, followed by renal accumulation. 1 h post administration, most of the radioactivity is found
in the bladder, which indicates a short half-life of ranalexin within the body. In contrast, the
pharmacokinetics of C13C3lexin look very promising. The images demonstrate that the lipopeptide
accumulates within the liver and stays there for several hours. Appreciable amounts of radioactivity
in the bladder are first visible after 40 – 60 min. Most importantly, the modified peptide shows no
accumulation within the kidneys. The images strongly indicate a prolonged retention time of
C13C3lexin within the animal body. This is exemplarily shown by determination of the SUV of the
heart for both peptides over a period of 60 min post injection in Figure 7. The SUV, the area under
the curve (AUC) and the plasma half-life are significantly higher for the lipopeptide than for the
natural peptide. These results clearly indicate the remarkably improved pharmacokinetics of DOTA-
C13C3lexin compared to DOTA-ranalexin.
Please insert Figure 6 here
Please insert Figure 7 here
4. Discussion
The number of new antibiotic drugs currently in clinical phase 2 or 3 testings still remains sobering.
The lack of new antibiotics against multidrug resistant Gram-negative bacteria endanger patients [2].
Especially the antibiograms of clinical isolates (Table 2) illustrate this problem. The emergence of
MRGN bacteria and the lack of new antibiotics with an innovative mode of action caused a revival of
polymyxins [23]. Polymyxins are considered one of the few remaining last-line antibiotics against
infections with drug-resistant Gram-negative pathogens [24]. The antimicrobial susceptibility testing
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here confirmed previous findings, that the polymyxin antibiotic colistin is often one of the only few
remaining antibiotics effective against multiresistant A. baumannii strains. Most of multidrug
resistant infections, from intensive care units, are caused by A. baumannii and K. pneumoniae [25].
Thus, we included multidrug-resistant isolates of these species in our study. Besides these well-
known problems, multiresistant Gram-positive bacteria also occur more frequently. Although most of
the multiresistant Gram-positive infections can still be treated with vancomycin, it is necessary to
develop new, innovative antibiotics to counterattack currently increasing antimicrobial resistances in
Enterococci and Staphylococci [26].
In this study, we investigated the antimicrobial activity of ranalexin and its lipopeptide derivatives
C10C3lexin, C11C3lexin, C12C3lexin, C13C3lexin and C14C3lexin. The peptides were synthesized with
solid-phase peptide synthesis by fmoc-chemistry. Solid-phase peptide synthesis represents an
effective and fast method of for peptide production [27]. For ranalexin we confirmed a high activity
against Gram-positive bacteria but weaker activity against Gram-negative bacteria. The MIC values
were comparable with those found in previous publications [12]. For E. faecalis ATCC 29212, P.
aeruginosa ATCC 27853 and E. coli ATCC 25922 quality control ranges of MICs for vancomycin and
colistin, respectively are provided by the CLSI [19]. The MIC values of the positive controls in our
study are in accordance with those quality control MICs, which indicates the correct performance of
the broth dilution method. The approved lipopeptide antibiotics colistin and daptomycin possess N-
terminal fatty acids which are essential for their antimicrobial activity [28]. N-acylation of AMPs could
lead to enhanced antimicrobial activity [29]. In this study, we successfully demonstrated, that
shortened, acylated derivatives of ranalexin are significantly more potent against Gram-positive and
Gram-negative bacteria, compared with the naturally occurring peptide. The remarkably broad
spectrum of the antimicrobial activity of the lipopeptides indicates their potential as novel highly
effective antibiotics. A broad spectrum antibiotic could be especially used for the treatment of
bacterial infections with any unknown pathogenic strain [30].
We also demonstrated that acylation is a powerful tool in order to improve the activity against Gram-
negative bacteria but this class of bacteria is also very sensitive to changes in the length of the fatty
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acid. Even small variations had significant influence on antimicrobial activity. As a fact, Gram-
negative bacteria contain a thinner cell wall peptidoglycan layer but possess an additional outer
membrane which functions as a permeability barrier [31]. For this reason, it is more difficult for
antibiotics to reach the inner cell membrane. Acylation could facilitate the penetration through the
outer membrane [8]. Indeed, the synthesized lipopeptides carry the same amino acid sequence. The
only difference is the length of the fatty acid attached. In the cut-out of the HPLC profile of ranalexin
and the lipopeptides (Supplementary Figure 7) it is clearly visible that an increase in length of the
fatty acids causes a prolongation of the retention time. It is well known, that the retention time is
proportional to the hydrophobicity of the respective peptides [32]. Thus, C12C3lexin and C13C3lexin
probably possess the ideal hydrophobicity for penetrating the outer membrane of Gram-negative
bacteria. Furthermore, the peptides show a bactericidal mode of action and most importantly, AMPs
are not affected by classical mechanisms of resistance which has evolved against antibiotics attacking
differing targets, such as ribosomes, the cell wall or DNA [33]. The antimicrobial activity of ranalexin
and the lipopeptides are moderate against P. fluorescens DSM 50090, compared to other Gram-
negative bacteria. As a possible explanation, it can be assumed that P. fluorescens belongs to the
resident skin flora of R. catesbeiana and therefore also produces the antibiotic mupirocin [34, 35].
Time-kill studies confirmed the speed of bactericidal activity as another powerful advantage of the
peptides [36]. Established bactericidal antibiotics like cefuroxime need several hours for killing
infectious bacteria [37], whereas ranalexin and C13C3lexin showed improved bactericidal kinetics.
The superior bactericidal kinetics of C13C3lexin clearly highlights the potential of lipopeptides as fast
acting bactericidal substances. Because of their membrane-targeted mode of action, AMPs obviously
bear a potential for toxicity [38]. With respect to future clinical application, a fond estimation of
cytotoxicity of ranalexin and the lipopeptide derivatives against vertebrate cells is essential. Using a
cell viability assay, we were able to assess their cytotoxicity against several cell lines, and most
importantly, also against non-tumorous hamster and native human blood cells. While ranalexin
showed moderate toxicity (IC50 21.62 – 32.29 µg/mL) compared to other AMPs [39], the harmful toxic
activity of the lipopeptides obviously depends on the chain length of the fatty acid attached to the
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peptidic moiety. Fatty acids comprising chain lengths from C14 to C12 showed unfavorable toxicity
(IC50 12.74 – 32.07 µg/mL), whereas a chain length of C10, represented by decanoic acid, significantly
reduced the toxicity of the lipopeptide (IC50 38.49 – 108.10 µg/mL). Interestingly, human PBMCs
were shown to exhibit a dramatically lower sensitivity towards the C10C3lexin compound compared
to all other cell types tested. Although, in general, the influence of the fatty acid on toxicity is still
moderate compared to the influence on antimicrobial activity, our observations necessitate further
investigations to clearly reveal the impact of specific modifications of AMPs on both improved
antimicrobial activity and the reduction of potential adverse effects.
It was possible for the first time to estimate the pharmacokinetics of ranalexin in rats. The
biodistribution studies, performed with PET-imaging of DOTA-ranalexin and DOTA-C13C3lexin in
Wistar rats showed promising improvements with respect to pharmacokinetics. While the natural
peptide ranalexin is rapidly cleared via the kidneys and excreted through the urine, the synthetic
lipopeptide accumulates in the liver for a minimum of two hours and shows prolonged circulation
within the blood. Acylation has been proven to be a reliable tool for improving the half-life of a drug.
Telavancin, a lipoderivative of vancomycin, which was approved in 2009, has a half-life in humans of
9 h compared to 6 h of its origin vancomycin [30, 40]. The lately approved dalbavancin, another
lipoderivative of vancomycin, shows a half-life of over 300 h in humans [30]. Short half-lives of
antimicrobial peptides is a serious and well-known problem [38]. Therefore, we could show that the
modification of the antimicrobial peptide ranalexin by the replacement of particular amino acids with
tridecanoic acid leads to improved antimicrobial activity, prolonged half-life as well as specific
targeting into the liver. The SUV of the heart of a radiolabeled compound is a good indicator for the
residence time of a substance within the blood stream. The comparison of the SUVs of the heart of
DOTA-ranalexin and DOTA-C13C3lexin clearly evidences that the lipopeptide shows a longer plasma
half-life. Furthermore, the higher SUVs of the lipopeptide clearly demonstrate that the lipopeptide
shows superior pharmacokinetic characteristics compared with the natural peptide.
The lipopeptide derivatives studied contain only 14 amino acids instead of 20 amino acids of the
origin ranalexin. The costs for synthesizing the peptides are 5 to 20 times higher than for classical
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antibiotics produced by fermentation [33]. Therefore, modification of ranalexin by lipopeptides
shows another big advantage, because the production costs could be reduced. In conclusion, our
investigation shows that the examined lipopeptides represent one promising example that new
substances can be generated from evolutionary optimized natural products using the knowledge of
modern drug design.
The enhanced antimicrobial activity and the improved pharmacokinetics of the lipopeptide
derivatives of ranalexin could lead to promising new drug candidates for the management of
occurring microbial challenges. Nevertheless, the described novel class of lipopeptides is by no
means the end of the story. Further improvements are needed for the design of a clinically applicable
lipopeptidic AMP in order to find the optimal balance between effective antimicrobial activity on the
one hand and minimal detrimental impact on the treated patient on the other. Moreover, since the
main part of the lipopeptides comprises an amino acid backbone which is naturally prone to
degradation by proteases in the gut, the issue of oral bioavailability will also be an important task for
future developments of AMP therapeutics.
Declarations
Funding: No funding
Competing Interests: No conflict of interest
Ethical Approval: Not required
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References
[1] Ling LL, Schneider T, Peoples AJ, Spoering AL, Engels I, Conlon BP, et al. A new antibiotic kills pathogens without detectable resistance. Nature. 2015;517:455-9. [2] Boucher HW, Talbot GH, Bradley JS, Edwards JE, Gilbert D, Rice LB, et al. Bad bugs, no drugs: no ESKAPE! An update from the Infectious Diseases Society of America. Clin Infect Dis. 2009;48:1-12. [3] Centers for Disease Control and Prevention, Antibiotic Resistance Threats in the United States, https://www.cdc.gov/drugresistance/threat-report-2013/index.html; 2013 [accessed 17.10.23]. [4] Rice LB. Federal funding for the study of antimicrobial resistance in nosocomial pathogens: no ESKAPE. J Infect Dis. 2008;197:1079-81. [5] Maragakis LL, Perl TM. Acinetobacter baumannii: epidemiology, antimicrobial resistance, and treatment options. Clin Infect Dis. 2008;46:1254-63. [6] Hancock REW. The end of an era? Nature Reviews Drug Discovery. 2007;6:28-. [7] Fox JL. Antimicrobial peptides stage a comeback. Nat Biotechnol. 2013;31:379-82. [8] Makovitzki A, Avrahami D, Shai Y. Ultrashort antibacterial and antifungal lipopeptides. Proc Natl Acad Sci U S A. 2006;103:15997-6002. [9] Kanthawong S, Nazmi K, Wongratanacheewin S, Bolscher JG, Wuthiekanun V, Taweechaisupapong S. In vitro susceptibility of Burkholderia pseudomallei to antimicrobial peptides. Int J Antimicrob Agents. 2009;34:309-14. [10] Fosgerau K, Hoffmann T. Peptide therapeutics: current status and future directions. Drug Discov Today. 2015;20:122-8. [11] Simmaco M, Mignogna G, Barra D. Antimicrobial peptides from amphibian skin: what do they tell us? Biopolymers. 1998;47:435-50. [12] Clark DP, Durell S, Maloy WL, Zasloff M. Ranalexin. A novel antimicrobial peptide from bullfrog (Rana catesbeiana) skin, structurally related to the bacterial antibiotic, polymyxin. Journal of Biological Chemistry. 1994;269:10849-55. [13] Graham S, Coote PJ. Potent, synergistic inhibition of Staphylococcus aureus upon exposure to a combination of the endopeptidase lysostaphin and the cationic peptide ranalexin. J Antimicrob Chemother. 2007;59:759-62. [14] Aleinein RA, Schafer H, Wink M. Secretory ranalexin produced in recombinant Pichia pastoris exhibits additive or synergistic bactericidal activity when used in combination with polymyxin B or linezolid against multi-drug resistant bacteria. Biotechnol J. 2014;9:110-9. [15] Ghiselli R, Giacometti A, Cirioni O, Orlando F, Mocchegiani F, Pacci AM, et al. Therapeutic efficacy of the polymyxin-like peptide ranalexin in an experimental model of endotoxemia. J Surg Res. 2001;100:183-8. [16] Jerala R. Synthetic lipopeptides: a novel class of anti-infectives. Expert Opin Investig Drugs. 2007;16:1159-69. [17] Aleinein RA, Hamoud R, Schafer H, Wink M. Molecular cloning and expression of ranalexin, a bioactive antimicrobial peptide from Rana catesbeiana in Escherichia coli and assessments of its biological activities. Appl Microbiol Biotechnol. 2013;97:3535-43. [18] Brings S, Fleming T, De Buhr S, Beijer B, Lindner T, Wischnjow A, et al. A scavenger peptide prevents methylglyoxal induced pain in mice. Biochim Biophys Acta. 2016;1863:654-62. [19] CLSI. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard—Ninth edition. CLSI document M07-A9. Wayne, PA: Clinical and Laboratory Standards Institute; 2012. [20] NCCLS. Methods for determining bactericidal activity of antimicrobial agents; Approved guideline. NCCLS document M26-A [ISBN 1-56238-384-1]. NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087 USA, 1999. [21] Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65:55-63. [22] Wischnjow A, Sarko D, Janzer M, Kaufman C, Beijer B, Brings S, et al. Renal Targeting: Peptide-Based Drug Delivery to Proximal Tubule Cells. Bioconjug Chem. 2016;27:1050-7.
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[23] Falagas ME, Kasiakou SK. Colistin: the revival of polymyxins for the management of multidrug-resistant gram-negative bacterial infections. Clin Infect Dis. 2005;40:1333-41. [24] Ortiz de la Tabla V, Ortega A, Bunuel F, Perez-Vazquez M, Marcos B, Oteo J. Detection of the high-risk clone ST131 of Escherichia coli carrying the colistin resistance gene mcr-1 and causing acute peritonitis. Int J Antimicrob Agents. 2017;49:115-6. [25] Mezzatesta ML, Caio C, Gona F, Cormaci R, Salerno I, Zingali T, et al. Carbapenem and multidrug resistance in Gram-negative bacteria in a single centre in Italy: considerations on in vitro assay of active drugs. Int J Antimicrob Agents. 2014;44:112-6. [26] Biedenbach DJ, Arhin FF, Moeck G, Lynch TF, Sahm DF. In vitro activity of oritavancin and comparator agents against staphylococci, streptococci and enterococci from clinical infections in Europe and North America, 2011-2014. Int J Antimicrob Agents. 2015;46:674-81. [27] Made V, Els-Heindl S, Beck-Sickinger AG. Automated solid-phase peptide synthesis to obtain therapeutic peptides. Beilstein J Org Chem. 2014;10:1197-212. [28] Hu Y, Amin MN, Padhee S, Wang RE, Qiao Q, Bai G, et al. Lipidated peptidomimetics with improved antimicrobial activity. ACS Med Chem Lett. 2012;3:683-6. [29] Mak P, Pohl J, Dubin A, Reed MS, Bowers SE, Fallon MT, et al. The increased bactericidal activity of a fatty acid-modified synthetic antimicrobial peptide of human cathepsin G correlates with its enhanced capacity to interact with model membranes. Int J Antimicrob Agents. 2003;21:13-9. [30] Singh SB, Young K, Silver LL. What is an "ideal" antibiotic? Discovery challenges and path forward. Biochem Pharmacol. 2017. [31] Hancock RE, Rozek A. Role of membranes in the activities of antimicrobial cationic peptides. FEMS Microbiol Lett. 2002;206:143-9. [32] Yamagami C, Kawase K, Iwaki K. Hydrophobicity parameters determined by reversed-phase liquid chromatography. XV: optimal conditions for prediction of log P(oct) by using RP-HPLC procedures. Chem Pharm Bull (Tokyo). 2002;50:1578-83. [33] Andrea Giuliani GP, Silvia Fabiole Nicoletto. Antimicrobial peptides: an overview of a promising class of therapeutics. CENT EUR J BIOL. 2007;2:1 - 33. [34] Lauer A, Hernandez T. Cutaneous bacterial species from Lithobates catesbeianus can inhibit pathogenic dermatophytes. Mycopathologia. 2015;179:259-68. [35] Fuller AT, Mellows G, Woolford M, Banks GT, Barrow KD, Chain EB. Pseudomonic acid: an antibiotic produced by Pseudomonas fluorescens. Nature. 1971;234:416-7. [36] Mohamed MF, Hammac GK, Guptill L, Seleem MN. Antibacterial activity of novel cationic peptides against clinical isolates of multi-drug resistant Staphylococcus pseudintermedius from infected dogs. PLoS One. 2014;9:e116259. [37] Nielsen EI, Viberg A, Lowdin E, Cars O, Karlsson MO, Sandstrom M. Semimechanistic pharmacokinetic/pharmacodynamic model for assessment of activity of antibacterial agents from time-kill curve experiments. Antimicrob Agents Chemother. 2007;51:128-36. [38] Hancock RE, Sahl HG. Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies. Nat Biotechnol. 2006;24:1551-7. [39] Vaucher RA, da Motta Ade S, Brandelli A. Evaluation of the in vitro cytotoxicity of the antimicrobial peptide P34. Cell Biol Int. 2010;34:317-23. [40] Rotschafer JC, Crossley K, Zaske DE, Mead K, Sawchuk RJ, Solem LD. Pharmacokinetics of vancomycin: observations in 28 patients and dosage recommendations. Antimicrob Agents Chemother. 1982;22:391-4.
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Figure 1: Structure of ranalexin and C13C3lexin. Six N-terminal amino acids are substituted by tridecanoic acid
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Figure 2: (A) MICs of ranalexin and lipopeptides against a representative selection of Gram negative bacteria, including multiresistant clinical isolates. Lipopeptides, especially the most potent lipopeptide C13C3lexin, show greatly enhanced antimicrobial activity. A special case are the MICs against Acinetobacter. Ranalexin has potent antimicrobial activity against Acinetobacter, the lipopeptides show comparable MICs, independently from the chain length of the fatty acid. (B) MICs of ranalexin and lipopeptides against a selection of Pseudomonas strains. While the peptides show strong activity against the reference strain P. aeruginosa ATCC 27853, they exhibit reduced activity against P. aeruginosa BL303790* VIM-2 and P. fluorescens DSM 50090. * = multiresistant clinical isolates.
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Figure 3: Minimum inhibitory concentration (MIC) of ranalexin and lipopeptides against a
representative selection of Gram positive bacteria. The most potent lipopeptides C13C3lexin
and C12C3lexin show enhanced antimicrobial activity compared to ranalexin. *
multiresistant clinical isolate.
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Figure 4: Time-kill curves of ranalexin, C13C3lexin and cefuroxime at 4x MIC, 2x MIC and 1x MIC against S. aureus ATCC 25923 and E. coli ATCC 25922. The peptides are fast acting bactericidal substances. At a concentration of 4x MIC no living bacteria could be detected after 30 min of incubation. Apparently, the lipopeptide C13C3lexin has an enhanced bactericidal activity compared to ranalexin.
E. coli ATCC 25922 S. aureus ATCC 25923
Time-kill studies
Figure 4: Time-kill curves of ranalexin, C13C3lexin and cefuroxime at 4x MIC, 2x MIC and 1x MIC against S. aureus ATCC 25923 and E. coli ATCC 25922. The peptides are fast acting bactericidal substances. At a concentration of 4x MIC no living bacteria could be detected after 30 min of incubation. Apparently, the lipopeptide C13C3lexin has an enhanced bactericidal activity compared to ranalexin.
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Figure 5: Cytotoxic capacity of the AMP ranalexin and its derivatives against human cells. Several tumor cells lines of different origin (breast – MCF-7, liver – HepG2, cervix – HeLa, prostate – C4-2), the immortalized Chinese hamster ovary cell line CHO and freshly isolated human peripheral blood mononuclear cells (PBMCs) were cultured upon exposure to increasing concentrations (0.25 - 128 µg/mL) of ranalexin and its derived lipopeptides C10C3lexin, C11C3lexin, C12C3lexin, C13C3lexin and C14C3lexin, carrying fatty acids of various chain lengths (from C10 to C14). The concentrations of the respective AMPs at which 50% cell viability (IC
50) was still observed for the studied cell types, were
estimated by a colorimetric MTT-assay. Cytotoxicity of the lipopeptides depends on the length of the conjugated fatty acid. Lipopeptides containing fatty acids of short chain length, such as C10 and C11, show lower toxicity in comparison to the natural peptide.
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Figure 6: PET-images of DOTA-ranalexin (A) and DOTA-C13C3lexin (B) after intravenous injection into the tail vein. After one hour the major part of DOTA-ranalexin is found in the bladder and the renal pelvis. In contrast, the biodistribution of DOTA-C13C3lexin. After two hours of injection, a larger amount of the substance accumulates in the liver.
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Figure 7: Standard uptake volume (SUV) of DOTA-C13C3lexin and DOTA-ranalexin in the heart. The systemic availability of the lipopeptide is distinctly improved.
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Table 1: Amino acid sequences. theoretical mass [Da] and molecular weight [m/z] after LC-MS
analysis of the investigated peptides. 1
position of DOTA-coupling. 2
LC-MS spectra shown in supplementary data.
peptide sequence theoretical mass
[Da] m/z = 1 m/z = 2 m/z = 3 ranalexin
2
FLGGLIKIVPAMICAVTKKC 2013.1890 2014.1183 1052.5655 702.0463
C14C3lexin myristic acid -
KIVPAMICAVTKKC 1713.0239 1713.9708 856.9888 571.6620
C13C3lexin tridecanoic acid -
KIVPAMICAVTKKC 1699.0082 1698.9459 849.9778
C12C3lexin dodecanoic acid -
KIVPAMICAVTKKC 1684.9926 1684.9316 842.9707 -
C11C3lexin2
undecanoic acid -
KIVPAMICAVTKKC 1670.9769 1670.9769 835.9921 557.6638
C10C3lexin decanoic acid -
KIVPAMICAVTKKC 1656.9613 1656.9080 828.9588 DOTA-
ranalexin2 FLGGLIKIVPAMICAVTKK
1C
2489.3692 - 1245.6499 830.7695 DOTA-
C13C3lexin2
tridecanoic acid -
KIVPAMICAVTKK1C 2085.1884 - 1043.5614 696.0438
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Table 2: Susceptibility of the multiresistant isolates to common antibiotics. The isolates are resistant (R) to a multitude of common antibiotics. Only a few antibiotics remain susceptible (S) against these isolates. E. faecium is vancomycin-resistant (VRE). The isolate of P. aeruginosa is a producer of the metallo-β-lactamase VIM-2. K. pneumoniae is a producer of an K. pneumoniae carbapenemase (KPC-2). A. baumannii isolates are resistant to four major groups of antibiotics (4-MRGN). i = intermediate, nt = not tested
Enterococcus faecium UL602570
Pseudomonas aeruginosa BL303790
Klebsiella pneumoniae KL800877
Acinetobacter baumannii KL314822
Acinetobacter baumannii SC300007
VRE VIM-2 KPC-2 4-MRGN 4-MRGN Ampicillin R nt R nt nt Amoxycillin/ Clavulanate
R R R R nt Piperacillin nt R R nt R Piperacillin/ Tazobactam
nt R R R R Cefazoline R nt R nt nt Cefotaxime nt R R R R Ceftriaxone nt R nt R R Ceftazidime nt R R R R Imipenem nt R R R R Meropenem R R R R R Ciprofloxacin R R R R R Gentamicin R S I R R Tobramycin nt R R R R Tigecycline nt R I nt nt Trimethoprim/ Sulfamethoxazole
R R R S R Tetracycline S nt I R nt Vancomycin R nt nt nt nt Teicoplanin R nt nt nt nt Erythromycin R nt nt nt nt Linezolid S nt nt nt nt Colistin nt nt S nt S
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Table 3: Comparison of minimum inhibitory concentration (MIC) and MBC (minimum bactericidal concentration) against a representative variety of Gram-negative and Gram-positive bacteria including multidrug-resistant strains. MIC and MBC in [mg/L]. Obviously, MBC is mostly equal to MIC or only one potency higher, which indicates a bactericidal mode of action of the peptides. An
exception are MBC values of Pseudomonas, that are significantly higher than MIC values. 1
clinical
isolate, 2multiple drug resistance,
3 multiresistant Gram-negative bacteria, resistant to four different
groups of antibiotics, 4
enterohemorrhagic E. coli, 5
K. pneumoniae carbapenemase, 6
Verona
integron-encoded metallo-β-lactamase, 7
vancomycin-resistant Enterococcus, n.i. = no inhibitory activity at 64 mg/L, n.b. = no bactericidal activity at 64 mg/L.
ranalexin
C14C3lexin
C13C3lexin
C12C3lexin
C11C3lexin
C10C3lexin
MIC MBC MIC MBC MIC
MBC MIC MBC MIC MBC MIC MBC Gram-negative bacteria A. baumannii KL314822
1
MDR2
4-MRGN3 4 4 4 4 4 4 2 4 4 4 4 4
A. baumannii SC3000071
MDR2
4-MRGN3 4 8 4 8 2 2 4 4 4 4 4 4
E. coli ATCC 25922 16 32 8 8 4 8 4 4 4 8 8 8 E. coli O157:H7 ATCC 35150
EHEC4 32 32 8 16 4 8 4 8 4 16 8 32
K. pneumoniae KL8008771
MDR2 KPC
5 n.i. n.i. 16 32 8 16 8 8 8 16 32 64 P. aeruginosa BL303790
1
MDR2 VIM-2
6 n.i. n.i. 16 64 8 64 16 64 16 64 32 64 S. enterica DSM 554 64 n.b. 16 32 8 32 16 16 8 8 16 16 Y. mollaretii DSM 18520 n.i. n.i. 4 8 4 8 8 32 8 32 64 64 Gram-positive bacteria B. megaterium DSM 32 8 8 4 4 2 2 4 4 4 4 8 8 B. subtilis DSM 10 8 8 8 8 4 4 4 4 4 4 4 4 E. faecalis DSM 29212 16 16 8 8 4 4 4 4 8 8 8 16 E. faecium UL602570
1
MDR2
VRE7 16 16 4 8 4 4 4 4 4 8 16 16
S. aureus MRSA NCTC
10442 8 16 4 8 4 8 4 4 8 8 16 16 S. epidermidis ATCC 14990 16 16 16 16 8 8 4 4 4 8 8 8
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GRAPHICAL ABSTRACT
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