PEGylation of the peptide Bac7(1-35) reduces renal clearance while retaining antibacterial activity and bacterial cell penetration capacity

Article abstract of DOI:10.1016/j.ejmech.2015.03.028

The proline-rich antibacterial peptide Bac7(1-35) protects mice against Salmonella typhimurium infection, despite its rapid clearance. To overcome this problem the peptide was linked to a polyethylene glycol (PEG) molecule either via a cleavable ester bond or via a non-hydrolysable amide bond. Both the PEGylated conjugates retained most of the in vitro activity against S. typhimurium. In addition, the ester bond was cleaved in human serum or plasma, releasing a carboxymethyl derivative of Bac7(1-35) which accounts for a higher activity of this peptide with relative to the other, non-hydrolysable form. Both PEGylated peptides maintained the capacity of the unconjugated form to kill bacteria without permeabilizing the bacterial membranes, by penetrating into cells. They exploited the same transporter as unmodified Bac7(1-35), suggesting it has the capacity to internalize quite sizeable cargo if this is linked to Bac7 fragment. PEGylation allows the peptide to have a wide distribution in mice, and a slow renal clearance, indicating that this strategy would improve the bioavailability of Bac7, and in principle of other antimicrobial peptides. This can be an equally important issue to reducing cytotoxicity for therapeutic use of these antibacterials.

Full text of DOI:10.1016/j.ejmech.2015.03.028

European Journal of Medicinal Chemistry 95 (2015) 210e219
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
PEGylation of the peptide Bac7(1e35) reduces renal clearance while
retaining antibacterial activity and bacterial cell penetration capacity
Monica Benincasa a, Sotir Zahariev b, Chiara Pelillo a, Annalisa Milan a, Renato Gennaro a,
,
*
Marco Scocchi a
a Department of Life Sciences, University of Trieste, Via Giorgieri 5, 34127 Trieste, Italy
b International Centre for Genetic Engineering and Biotechnology, Padriciano 99, 34149 Trieste, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The proline-rich antibacterial peptide Bac7(1e35) protects mice against Salmonella typhimurium infec-
tion, despite its rapid clearance. To overcome this problem the peptide was linked to a polyethylene
glycol (PEG) molecule either via a cleavable ester bond or via a non-hydrolysable amide bond. Both the
PEGylated conjugates retained most of the in vitro activity against S. typhimurium. In addition, the ester
bond was cleaved in human serum or plasma, releasing a carboxymethyl derivative of Bac7(1e35) which
accounts for a higher activity of this peptide with relative to the other, non-hydrolysable form. Both
PEGylated peptides maintained the capacity of the unconjugated form to kill bacteria without per-
meabilizing the bacterial membranes, by penetrating into cells. They exploited the same transporter as
unmodified Bac7(1e35), suggesting it has the capacity to internalize quite sizeable cargo if this is linked
to Bac7 fragment. PEGylation allows the peptide to have a wide distribution in mice, and a slow renal
clearance, indicating that this strategy would improve the bioavailability of Bac7, and in principle of other
antimicrobial peptides. This can be an equally important issue to reducing cytotoxicity for therapeutic
use of these antibacterials.
Received 13 January 2015
Received in revised form
11 March 2015
Accepted 13 March 2015
Available online 19 March 2015
Keywords:
Antimicrobial peptide
Proline-rich
PEGylation
Antibacterial activity
Cell penetrating
Optical imaging
© 2015 Elsevier Masson SAS. All rights reserved.
1. Introduction
among the most interesting candidates for this role [1,2].
Proline-Rich Antimicrobial peptides (PR-AMPs) are a widespread
Antibiotic resistance has become a major public health problem
within the lifetime of most people living today. The number of
bacterial species and strains no longer susceptible to antibiotics is
growing up day by day. To counteract this situation new antimi-
crobials with novel mechanism of action are greatly needed. Anti-
microbial peptides (AMPs) from innate immunity of animals are
group of antimicrobial peptides (AMPs) present in mammalian
neutrophils, as peptides belonging to the cathelicidin family, and in
the hemolymph of several invertebrate species [3,4]. Despite their
different origin and evolution, they have common hallmarks, such as
i) a high content of arginine and proline residues, ii) activity directed
mainly against Gram-negative bacteria, iii) a remarkably low cyto-
toxicity towards eukaryotic cells, iv) the lack of extensive membrane
damaging effects, v) and reduced activity of the all-D enantiomers,
consistent with a mode of action based on recognition of stereo-
specific molecular targets and/or transporters [4,5].
Studies on the bovine PR-AMPs Bac7 have revealed important
aspects of the mode of action and the antimicrobial activity of this
peptide [6,7]. The 1e35 fragment has in vitro antimicrobial activity
against Gram-negative Escherichia coli, Salmonella typhimurium,
Klebsiella pneumonia and Enterobacter cloacae at concentrations
Abbreviations: Boc2O, Di-t-butyl dicarbonate; Et2O, ethyl ether; CFU, colony
forming unit; Cmc, S-carboxymethyl-cysteine; DIEA, N,N-Diisopropylethylamine;
HCTU, 2-(6-Chloro-1-H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexa-
fluorophosphate; OH-PEG-OMe, methoxy-polyethylene glycol; HRP, horseradish
peroxidase; IE, ion exchange; kDa, kilodalton; MeOH, Methanol; N2H4$H2O, hy-
drazine hydrate; NCL, native chemical ligation; NMP, N-methylpyrolidone; PA, pe-
troleum ether (fraction 40e60 ꢀC); Ph3PBr2, triphenyl phosphite dibromide; Phth,
phthaloyl; PI, propidium iodide; PrOH(2), 2-propanol; Py, pyridine; PyBOP, (Ben-
zotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate; RP-HPLC,
reversed phase high-performance liquid chromatography; SPB, sodium phosphate
buffer; SPPS, solid phase peptide synthesis; TA, thioanisole; TCEP.HCl, Tris(2-car-
boxyethyl)phosphine hydrochloride.
ranging from 1 to 10 mM, comparable to that of the natural peptide.
Furthermore, it remains active against antibiotic-multi-resistant
clinical isolates due to a mechanism of action that is different to
those of currently used antibiotics [6].
* Corresponding author.
E-mail address: mscocchi@units.it (M. Scocchi).
http://dx.doi.org/10.1016/j.ejmech.2015.03.028
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

M. Benincasa et al. / European Journal of Medicinal Chemistry 95 (2015) 210e219
211
Bac7(1e35) has the capacity to translocate into both bacteria [5,7]
and eukaryotic cells without cell damage [8,9]. Uptake into E. coli and
other Gramenegative bacteria is mediated, at least in part, by the
SbmA transporter [5,10], a dimeric inner membrane protein involved
in the transport of different types of peptides, in cooperation with
the cognate outer membrane protein YaiW [11,12], and has been
shown to target proteins involved with protein production (the
chaperone DnaK and ribosomal subunits) [3,4,13].
The therapeutic potential of Bac7(1e35) has been assessed in
mice infected with S. typhimurium, resembling a model of typhoid
fever infection [14]. No toxic effects were observed when the
peptide was administered to the mice i.p. up to 75 mg/kg and it
significantly increased the survival rates and reduced the bacterial
load in liver and spleen of infected animals [14]. However, its
circulating lifetime was low as it was easily removed by murine
kidneys due to its small size and/or was degraded. The peptide
reached kidneys and bladder by 1 and 3 h, respectively, after in-
jection and was totally excreted within 24 h [14].
SigmaeAldrich (USA), Biosolve Ltd (Netherlands), Alexis (USA) and
Advanced ChemTech (USA). Fmoc-protected amino acids were
obtained from Novabiochem (Switzerland), Inbios (Italy), Iris
Biotech GmbH (Germany) or Bachem AG (Switzerland).
2.2. Purification methods
Analytical RP-HPLC was carried out on a Gilson HPLC System.
Samples were eluted with a linear gradient from A ¼ 0.1% TFA in
water to B ¼ 0.1% TFA in MeCN.
Preparative RP-HPLC was performed on a Waters RCM with
PrepPak Cartridge Delta-Pak 300 15RP18 (100 ꢁ 25 mm I.D.) at a
flow rate of 7 ml/min or on a Waters Prep LC universal base module
with a PrepPak Cartridge Delta-Pak 300 15RP18 (100 ꢁ 40 mm I.D)
column at a flow rate of 18 ml/min. Samples were injected manu-
ally and eluted from the column with a gradient slope from 0.6% to
0.8% B/min. Pure fractions, according to analytical RP-HPLC or ESI-
MS analysis, were pooled and freeze-dried. TFA was removed after
lyophilizing three times with 10 mM HCl solution.
Among the several antimicrobial peptides that are currently
undergoing clinical trials, most are being tested for topical use [15].
Very few are being considered for systemic therapy because of the
many hurdles that must be overcome [16]. First, peptide drugs have
short circulating half-lives, due to proteolytic digestion and rapid
kidney clearance, and are often antigenic [17,18]. They also tend to
show low therapeutic indices in vivo [16,19] in part due to a reduced
activity in the presence of serum and plasma components [20,21].
The polyethyleneglycol (PEG) moiety is frequently attached to
peptide and protein drugs (PEGylation) in order to improve the
in vivo efficacies of these drugs, by reducing their cytotoxicity and
immunogenicity or by prolonging the in vivo half-life [17,22,23]. It
has also been successfully used to modify AMPs [24e27]. For
example, a 5 kDa PEG linked to the N-terminus of tachyplesin
resulted in a compound with reduced cytotoxicity and sensitivity to
serum inhibition [26]. The cytotoxicity of the peptide magainin 2
was also significantly reduced after PEGylation [25]. N-terminally
PEGylated AMPs consisting of fragments belonging to human LL-37
and insect cecropin A also showed reduced toxicity towards lung
epithelial primary cell cultures [28]. However, to the best of our
knowledge, there are no studies relating to its use simply to
improve the pharmacokinetics of an internally acting AMP without
altering its activity.
IEX-HPLC was carried out on AKTA Basic 10 (Amersham Phar-
macia, Sweden) using one or two column in series, HiTrap™ SP HP
(5 mL, Pharmacia) equilibrated in 20 mM Na phosphate buffer pH
6.5. Elution was carried out with a NaCl gradient.
The eluent from analytical RP-HPLC or preparative RP-HPLC was
collected as fractions and aliquots analysed on Applied Biosystems
Sciex API 150EX or ion trap mass spectrometer (Amazon SL, Bruker)
for the correct peptide conjugate.
2.3. Peptide synthesis
All the peptides used in this study are shown in Scheme 1,
Scheme 2 and Table 1. The synthesis of Bac7(1e35) [Bac, in Table 1]
and its further conjugation to BODIPY® FL N-(2-aminoethyl) mal-
eimide fluorophore [Bac-BY in Table 1] are also described in the
Supplementary data. Small portions of the crude peptide resins
were cleaved, deprotected with a modification of the procedure as
described in the Supplementary data and purified by RP-HPLC to
furnish Bac7(1e35) (1) (Scheme 1 and Table 1). The C-terminally
extended Bac-Cys(H)eOH (2a) could be obtained in two ways,
directly synthesizing it in the solid state from Cys-substituted
chlorotrityl resin, or in solution from 1a via intermediate 3
(Scheme 1). In the latter case, N-terminally and side-chain pro-
tected crude peptide Boc-Bac(1e35)-OH (1a) was cleaved from the
resin with HFIP, and Bac-arylthioester (3) was prepared according
to the general method of Beyermann [29,30]. The free thiol group of
2a was than reacted with excess bromoacetic acid in the presence
of base to form the S-carboxymethylated Bac-derivative (2)
(Scheme 1 and Table 1).
Here, we describe the modification of Bac7(1e35) by linking it to
a long 20-kDa PEG chain to reduce the clearance rate of the peptide.
The different derivatives, one hydrolysable and the other stable,
were tested for their in vitro activity against S. typhimurium cells,
and the effects of human serum and plasma on the kinetics of
peptide release and its stability were investigated. Our linkage
strategy also allowed preparation of a fluorescent variant of the
PEGylated peptide, for use in optical imaging analysis to monitor its
biodistribution and permanence in the body of mice.
2.4. PEGylation of Bac7(1e35)
2. Materials and methods
Two sets of C-terminally PEGylated Bac7(1e35) were synthe-
sized: C-terminal esters e left column on Scheme 2, and C-terminal
amides e right column on Scheme 2. mPEG-OCOCH2Br (4) was
prepared by bromoacetylation of commercial HO-PEG-OMe
(mPEG-OH) with BrCH2COBr/DIEA, according to published pro-
cedures [31,32]. NH2-PEG-OMe (mPEG-NH2) (5) was prepared in 3
steps from mPEG-OH. Bromoacetylation of 5 with the activated
ester of bromoacetic acid BrCH2COOSu (Scheme 2) produced amide
6. Coupling of 5 with N, S-protected cysteine led to H-Cys(H)eNH-
PEG-OMe (7). Thioether ligation of H-Bac7(1e35)-Cys(H)eOH (2a e
Scheme 1) with excess of bromoacetyl-PEG-ester (4) or
bromoacetyl-PEG-amide (6) produce the expected C-terminal
PEGylated Bac7(1e35) ester [8, BacE-PEG] or amide [9, BacA-PEG,
see Table 1], with good yield.
2.1. Materials
20 kDa mPEG-OH was purchased from Nektar therapeutics
(Huntsville, AL, USA, Lot. 307360) or from Sunbio Chemicals Co.,
Ltd. (South Korea, Lot. C1OH-020-09135) and was dried before use
by azeotrope distillation from toluene. As indicated in the technical
specifications the polydispersivity is 452 36 residues.
Activating reagents, HCTU and PyBOP, were from Calbio-
chemeNovabiochem AG (Switzerland). Anhydrous DMF, NMP and
PIP were from Biosolve Ltd (The Netherlands); DIPEA, TFA, TA, TIPS
from Fluka Chemie AG (Switzerland). All other reagents and sol-
vents were reagent grade and were purchased from Fluka,

212
M. Benincasa et al. / European Journal of Medicinal Chemistry 95 (2015) 210e219
Scheme 1. Preparation of peptide precursors.
H-Cys(HeOC(O)eOPEGeOMe (10) was prepared with excellent
dithiothreitol, 40% glycerol, 0.05% bromophenol blue, 150 mM
TriseHCl, pH 7), incubated for 15 min at 60 ꢀC and analysed on a
16% Tricine/SDS gel. Proteins were then blotted onto nitrocellulose
membrane and incubated overnight with shaking in 40 mM
TriseHCl, pH 7.5, 5% non-fat milk, 0.1% Tween 20, 200 mM NaCl
(Blocking Solution, BS) at 4 ꢀC. Samples were incubated at room
temperature for 2 h with rabbit anti-Bac7(1e35) IgG diluted 1:1000
in BS, followed by an HRP-conjugated anti-rabbit IgG (1 h incuba-
tion). The detection of the chemiluminescence was performed us-
ing the commercially available kit ECL Plus Western Blotting
Detection Reagent (Pierce).
purity and yield by activation of an excess of Boc-Cys(Trt)-OH with
Boc2O/pyridine [33], followed by coupling to mPEG-OH, depro-
tection and purification of the ester. Similarly, mPEG-NH2 was
coupled to Boc-Cys(Trt)-OH, activated with HCTU/DIEA to obtain H-
Cys(H)eNH-PEG-OMe (7). An excess of H-Cys-OPEG-OMe (10) or H-
CyseNHePEG-OMe (7) were then reacted with Bac7(1e35)-
SPhNHAc (3) under conditions of native chemical ligation [34] and
respectively formed the C-terminal PEGylated ester Bac7(1e35)-Cys-
OPEG-OMe (11) and PEGylated amide Bac7(1e35)-Cys-NHPEG-OMe
(12). Michael addition of Alexa Fluor 680 maleimide or BODIPY® FL
N-(2-aminoethyl) maleimide to the free thiol group in 11 or 12
formed the corresponding succinimide adducts (13, 14) and (15),
according to modifications of a published procedure [14] or further
explained in the Supplementary data. The free thiol in Bac7(1e35)-
Cys(H)-OPEG-OMe (11) was reacted with an excess of bromoacetic
acid in the presence of base (as already described for 2 / 2a) and
formed S-carboxymethylated Bac7(1e35)-Cmc-OPEG-OMe (17).
A detailed description of all the reactions reported in Scheme 2
is available in the Supplementary data. The concentration of pep-
tides was determined using three methods: i) by accurate weighing
after lyophilisation; ii) using the method of Waddell [35]; iii) by
analytical RP-HPLC using an internal standard. PEGylated peptides
were also checked by acetic acid-urea PAGE for any eventual release
of free peptide (see Supplementary data).
2.8. In vivo biodistribution by time-domain optical imaging
In vivo biodistribution studies have been performed as previ-
ously described [14]. Two mice were injected intraperitoneally with
200
ml of BacE-PEG-Alexa680 in PBS corresponding to 6.9 nmol
AlexaFluor® 680, one monitored in the abdominal region and the
other in the renal region for 24 h. A blank image was acquired
before treatment of each animal and this was subtracted from the
images of the treated animal. The experiment was repeated twice.
Two-dimensional scanning regions of interest (ROI) were
selected and the laser power, integration time and scan step were
optimized according to the signal emitted. The data were recorded
as temporal point-spread functions, and the images were recon-
structed as fluorescence intensity and lifetime. All the experimental
procedures were performed according to the guidelines of the
European (86/609/EEC) and the Italian (D.L.116/92 and subsequent
addenda) laws and approved by the Italian Ministry of University
and Research as well as by the Animal Experimentation Committee
of the University Animal House.
2.5. Serum and plasma preparation
Murine serum and plasma were drawn from a pool of mice.
Human serum and plasma were taken from a pool of healthy do-
nors. Human and murine plasma was obtained with 2% (v/v) Na-
citrate as anticoagulant. Fluid samples were centrifuged at
13,000 g for 5 min and stored at ꢂ20 ꢀC until use.
2.9. Minimum inhibitory concentration
2.6. Bacterial strains and growth conditions
Broth microdilution susceptibility assay was performed as pre-
viously described [6]. Briefly, two-fold serial dilutions of peptides
were prepared in 96-well microplates in MHB with the addition of
human or murine serum or plasma to reach the final concentration
of 25% after bacteria addition, and 100% MHB as control. To study
the effect of the preincubation on BacE-PEG activity, bacteria were
added to each well containing peptide in 25% serum or plasma after
24 h at 37 ꢀC. The MIC (Minimum Inhibitory Concentration) was
taken as the lowest concentration of antimicrobial peptide result-
ing in the complete inhibition of visible growth after 24 h of in-
cubation at 37 ꢀC.
The strains used in this study were Salmonella enterica serovar
Typhimurium ATCC 14028 (S. typhimurium), E. coli BW25113 and
E. coli BW25113DsbmA, a deletion mutant for the sbmA gene. The
inoculum was incubated overnight at 37 ꢀC with shaking. For the
assays the overnight bacterial cultures were diluted 1:30 in fresh
MuellereHinton Broth (MHB) and incubated at 37 ꢀC with shaking
for approximately 2 h.
2.7. Bac7 stability and its release from PEGylated forms in serum
and plasma
2.10. Growth kinetics assay
Bac and BacE-PEG (120 mg) were incubated in 200 ml of 25% (v/v)
murine or human serum/plasma in PBS at 37 ꢀC. At different times,
aliquots were withdrawn, diluted 1:5 in sample buffer (12% SDS, 6%
Bacterial growth inhibition tests were performed using mid-
log phase bacterial cultures diluted in MHB to 1 ꢁ 106 CFU/ml,

M. Benincasa et al. / European Journal of Medicinal Chemistry 95 (2015) 210e219
213
H-Bac-Cys(BP)-NH-PEG-OMe 15
H-Bac-Cys(Y)-O-PEG-OMe
Y = AF680
13
14
Y = BP
BP/TCEP/
HEPES
pH 7.0
AF680/
TCEP/SPB
pH 7.0
BP/TCEP/
HEPES
pH 7.0
H-Bac-Cys(H)-O-PEG-OMe
11
H-Bac-Cys(H)-NH-PEG-OMe
12
SPB, TCEP,
EDTA, pH 7.3
SPB, TCEP,
3
3
EDTA, pH 7.1
H-Cys(H)-O-PEG-OMe 10
7
H-Cys(H)-NH-PEG-OMe
1) Boc-Cys(Trt)-OH/
HCTU/DIEA
2) TFA
1) Boc-Cys(Trt)-OH/
Boc2O/Py
2) TFA
1) Ph3PBr2/DCM
2) PhthNK/DMF
3) N2H4.H2O/MeOH
HO-PEG-OMe
NH -PEG-OMe
5
2
BrCH2COOSu
MeCN
BrCH2COBr/DIEA
in DCM
4
BrCH -COO-PEG-OMe
6
BrCH -CONH-PEG-OMe
2
2
SPB/TCEP/
EDTA/pH 7.0
SPB/TCEP/
EDTA/pH 7.0
2a
2a
H-Bac-Cys-OH
H-Bac-Cys-OH
CH -CONH-PEG-OMe
9
8
CH -COO-PEG-OMe
2
2
AF680: Alexa Fluor 680 Maleimide (Cys-succinimide adduct); BP: BODIPY® FL
N-(2aminoethyl)maleimide (Cys-succinimide adduct); HO-PEG-OMe
CH2CH2-(OCH2CH2)nav-OCH2CH2OMe, where nav = 452± 36
= HO-
Scheme 2. Amide or ester bond C-terminal PEGylation of Bac7(1e35).
in presence or absence of different concentrations of peptides.
loaded.
Bacterial growth was followed at 37 ꢀC with intermittent shaking
for 4 h by measuring every 10 min the absorbance at 620 nm
with a microplate reader (Tecan Trading AG, Switzerland). To
evaluate if the culture medium causes the release of free peptide,
BacE-PEG was incubated in MHB at 37 ꢀC for different times up to
The gel was stained by Coomassie blue and iodine staining for
protein bands and PEG species, respectively. For iodine staining, the
gel was stained with a 5% Barium chloride solution in milliQ water
(5% w/v BaCl2 in 100 ml milliQ water, ~240 mM HCl) for 15 min,
washed in distilled water for 30 min, floated in 0.1 M iodine solu-
tion (2% w/v KI, 1.27% w/v I2 in milliQ water) for 15 min and washed
in distilled water again [36].
4 h. At the end of incubation, 10 ml of each sample were loaded
on a 16% Tricine/SDS gel. As control, Bac in water or MHB was
Table 1
List of peptides used in this study.
Compound
Name used
Numbera
Bac7(1e35)
Bac7(1e35)-Cys(BODIPY-maleimide)
Bac
Bac-BY
1
e
Bac7(1e35)-Cys(H)eNH-PEG-OMe
BacA-PEG
BacA-PEG-BY
BacE-PEG
BacCMC
9
15
8
Bac7(1e35)-Cys(BODIPY-maleimide)eNHePEG-OMe
Bac7(1e35)-Cys(eCH2COeOPEG-OMe)-OMe
Bac7(1e35)-Cys(-CH2COOH)
2
Bac7(1e35)-Cys(BODIPY-maleimide-)-OeCH2COeOPEG-OMe
Bac7(1e35)-Cys(ALEXAFluorC2-maleimide-)-OeCH2COeOPEG-OMe
BacE-PEG-BY
BacE-PEG-Alexa
13
14
a
Numbers indicate the corresponding compounds described in Scheme 1 and 2.

214
M. Benincasa et al. / European Journal of Medicinal Chemistry 95 (2015) 210e219
2.11. Flow cytometric analysis
8e16
unconjugated peptide. The anti-salmonellae activity was evaluated
also by growth inhibition assays. At 2 M, both BacE-PEG and BacA-
PEG reduced bacterial growth by 75% after 4 h (Fig. 1A and B) while
unmodified Bac showed a similar growth inhibition at 0.5
(Fig.1C), confirming a difference in activity in line with the MIC. The
rapid antibacterial effect of the modified peptides, which initiated
without delay, suggested that the PEGylated forms per se were at
least in part responsible for the antibacterial activity in spite of the
steric hindrance due to the presence of the PEG group. To further
support this hypothesis, BacE-PEG was incubated in MHB for 4 h,
and the products analysed by SDS-PAGE. No free peptide was
detected (data not shown), confirming that short-term
mM, respectively 4- and 8-fold less active than that of the
Integrity of the cell membrane in Salmonella cells was assessed
by measuring the PI uptake by flow cytometry, as previously
described [7]. For the uptake evaluation, we used a cytofluorimetric
method described in Benincasa et al. [37]. Data analysis was per-
formed with the FCS Express3 software (De Novo Software, Los
Angeles, CA).
m
m
M
2.12. Confocal scanning laser microscopy (CSLM)
CSLM analysis were performed by using a Nikon C1eSI confocal
microscope. An oil immersion objective lens was used. The opti-
mum photomultiplier setting was determined in preliminary ex-
periments performed ad hoc, and then the same setting was used
for all samples. Salmonella typhimurium ATCC 14028 cells treated
0,4
A
for 2 h with 0.25
BY, or for 10 min with 0.25
following the same protocol used for the flow cytometric uptake
assay without any fixation. Ten l of each bacterial suspension were
mM Bac-BY, 1
m
M BacE-PEG-BYand 4
mM BacA-PEG-
no pepƟde
mM Polymyxin B, were prepared
0,3
1 μM
m
placed between two cover-glasses to obtain an unmovable mono-
layer of cells. The image stacks collected by CSLM were analysed
with the EZ-C1 Free Viewer (Nikon Corporation) and the Image J
0,2
0,1
1.40
g (Wayne Resband, National Institutes of Health, USA)
2 μM
software.
4 μM
0
3. Results
0
30
60
90
120
150
180
180
180
210
240
3.1. Preparation of PEGylated forms of Bac7(1e35)
time (min)
0,4
0,3
0,2
0,1
0
Bac7(1e35) (hereafter named Bac) was conjugated to 20 kDa
poly(ethylene glycol) (PEG) using two different strategies (Scheme
2). In one case the peptide was linked to the polymer through an
amide bond (BacA-PEG) (compound 9 in the Scheme), and in the
other via an ester bond (BacE-PEG) (compound 8) that in principle
allows the peptide to be released by blood esterases producing a
carboxymethyl derivative of Bac7(1e35) (BacCMC) (compound 2).
Corresponding fluorescent derivatives of BacA-PEG and BacE-PEG
have also been prepared to allow detection of the PEGylated pep-
tides in mice or when internalized into bacterial cells (Scheme 2).
All the compounds and their names are listed in Table 1.
B
no pepƟde
2 μM
4 μM
8 μM
0
30
60
90
120
150
210
240
3.2. Antimicrobial activity of PEGylated Bac
time (min)
Both amide- and ester-linked forms of the PEGylated peptide
0,4
0,3
0,2
0,1
0
showed
a good antibacterial activity against S. typhimurium
C
(Table 2). In particular, when assayed in MuellereHinton broth
(MHB), BacE-PEG showed MIC values of 4e8 mM and BacA-PEG of
no pepƟde
Table 2
Microdilution susceptibility assay of Bac7(1e35) and its PEGylated forms against
Salmonella typhimurium.
Peptide
MIC (
MuellereHinton Human
Serum Plasma
0.5e1
mM)
0.5 μM
Murine
Serum Plasma
1 μM
0
30
60
90
120
150
210
240
Bac
1e2
8e16
4e8
2
0.06e0.12 32
ꢃ32
>32
16
nd
nd
BacA-PEG
BacE-PEG
BacE-PEG (24 h)a
BacCMC
8e16
2
>32
64
nd
time (min)
2e4
1
1e2
0.25
Fig. 1. Growth kinetics of Salmonella typhimurium in the presence of BacE-PEG, BacA-
PEG and Bac. Bacterial growth in MHB was monitored at 620 nm. The dotted line in-
dicates the growth rate of the untreated bacterial cells, the continuous lines indicate
the growth rate of bacterial culture in the presence of the indicated concentrations of
BacE-PEG (A), BacA-PEG (B) and Bac (C). Growth kinetics are representative of three
independent experiments with identical results.
0.5e1
0.5
0.03e0.06 nd
MIC values are representative of three independent experiments with comparable
results.
a
Peptide was incubated for 24 h in the indicated media before the addition of
bacteria (see Materials and methods Section).

M. Benincasa et al. / European Journal of Medicinal Chemistry 95 (2015) 210e219
215
antimicrobial activity was due to the conjugated forms.
PEG than BacA-PEG.
Interestingly, in the presence of plasma (25%) the activity of all
the peptides against S. typhimurium was increased 4e8 fold
(Table 2). It is significant that the increase in activity occurred to the
same extent for both PEGylated and non PEGylated peptides and
we are currently unable to propose a reason for this effect, so that
further investigations are required to clarify it.
As in vivo experiments were carried out in a mouse model, the
effects of murine serum and plasma were also tested. In contrast to
what was observed in the presence of human body fluids, the
antibacterial activity of unconjugated Bac and of both the pegylated
forms was scarce in the presence of murine serum or plasma
(Table 2).
3.3. Release of Bac in human body fluids and stability of the native
peptide
The kinetics of release of Bac from BacE-PEG in the presence of
human serum and plasma is shown in Fig. 2. A peptide with the
expected size was detected using an anti-Bac7 antibody after 8 h of
incubation with either plasma or serum and release was nearly
complete within 72 h (Fig. 2A and B). On the other hand BacA-PEG
did not release free Bac within the same time period (data not
shown). To verify if Bac is stable and not degraded in the body fluids
after being released from PEG, a Western blot analysis of unmodi-
fied Bac incubated with human serum or plasma was carried out
(Fig. 2C). Results indicated that the Bac was not degraded in both
body fluids up to at least 72 h incubation.
3.5. Internalization into S. typhimurium cells
In order to evaluate whether the PEGylated derivatives retained
the same mechanism of action as the native peptide, we monitored
the cellular uptake using the fluorescent derivatives BacA-PEG-BY
and BacE-PEG-BY. First, however, we performed a permeabilization
assay to exclude that the PEGylated forms could damage the bac-
terial membranes. The percentage of propidium iodide (PI)-positive
bacterial cells treated with the PEGylated peptides at their MIC, or
slightly above MIC values, did not increase when compared to the
free peptide (<2% of PI positive cells, data not shown), suggesting
membrane permeabilization does not occur.
3.4. Effects of serum and plasma on the antibacterial activity of
PEGylated Bac
Bacterial susceptibility testing was performed in the presence of
human serum or plasma to evaluate their effects on the antibac-
terial activity of all the peptides (Table 2). Different results were
obtained depending on the type of body fluid. The addition of 25%
human serum to the medium did not modify the anti-salmonellae
activity of BacA-PEG (MIC ¼ 8e16
(MIC ¼ 0.5e1 M). Conversely, it resulted in an appreciable increase
M) likely due to the partial
mM) or of unconjugated Bac
m
When BacA-PEG-BY and BacE-PEG-BY were incubated with
S. typhimurium at 1 mM concentrations, the detected fluorescent
in the activity of BacE-PEG (MIC ¼ 2e4
m
release of the peptide (Table 2). In addition, when the assay was
carried out using an aliquot of BacE-PEG preincubated for 24 h with
25% human serum, the activity showed a further 2 fold increase,
signal indicated that they had been internalized into the cells, albeit
at a lower level than unPEGylated Bac-BY (Fig. 3A). Furthermore,
BacE-PEG-BY exhibited a higher level of internalization than BacA-
PEG-BY. Uptake assays were also carried out using Bac-BY, BacE-
PEG-BY and BacA-PEG-BY at a concentration corresponding to ¼ the
MIC values. Under this condition a similar amount of each peptide
was found inside the bacterial cells and with an analogous time-
dependence (Fig. 3B).
reaching a MIC of 1
BacCMC, the carboxymethyl peptide that would be obtained by ester
bond hydrolysis of BacE-PEG (0.5e1 M) (Table 2). Overall these
mM. This value is similar to that measured for
m
results show that in human serum an active peptide is released
from the polymer explaining the somewhat higher activity of BacE-
Fig. 2. Evaluation of Bac release from PEG conjugates in the presence of human biological fluids and Bac7 stability in human blood fractions. Western blot analyses of BacE-PEG in
human plasma (A) and human serum (B). BacE-PEG was incubated in 25% human plasma or serum up to 72 h at 37 ꢀC. Samples were then loaded on the gel to allow peptide
detection with a specific anti-peptide antibody. Western blot analysis of Bac incubated for different times at 37 ꢀC in 25% human serum or plasma (C). Lane 1: 0.5
mg Bac; lanes 2e6:
Bac after incubation with human or murine serum and plasma for the indicated times; lane 7: serum or plasma alone.

216
M. Benincasa et al. / European Journal of Medicinal Chemistry 95 (2015) 210e219
The localization of the peptides in S. typhimurium cells was
give BacE-PEG-Alexa. Biodistribution analyses were performed on
healthy mice by monitoring the availability of the labelled, PEGy-
lated peptide up to 24 h after its intraperitoneal administration and
performing ex vivo analysis at 24 h. Results showed that the 24 kDa
PEGylated form of Bac had a much wider distribution than the
unPEGylated peptide and was detected in different organs (kidney,
liver and peritoneal cavity) (Fig. 5A and B). Furthermore, it was still
present at 24 h post-injection, suggesting a slower renal clearance
compared to the unmodified peptide [14] and clearly due to the
increased molecular mass. Ex vivo analysis showed a significant
concentration of the PEGylated Bac in liver even at 24 h after in-
jection, as well as in kidney (Fig. 5C), indicating a prolonged in vivo
half-time for the peptide after its conjugation to PEG.
examined by confocal scanning laser microscopy (CSLM) (Fig. 3C). A
homogeneous distribution of Bac and of both the PEGylated de-
rivatives in the cell cytoplasm was observed. In contrast, the
membrane-acting antibiotic polymyxin B was visible mainly on the
cellular surfaces, as expected.
To evaluate whether the internalization of the PEGylated com-
pounds involves the same inner membrane protein transporter
SbmA responsible for Bac translocation, we repeated the uptake
assays using the E. coli BW25113DsbmA mutant strain with deleted
sbmA gene. The susceptibility of this mutant strain was significantly
decreased to both the PEGylated forms with respect to the wild-
type strain (Fig. 4A and B), and corresponded to a parallel reduc-
tion in the level of uptake of both BacA-PEG-BY and BacE-PEG-BY by
the mutated strain (Fig. 4C). This confirms the involvement of the
SbmA protein also in the internalisation of the 20 kDa-PEGylated
form of the peptide.
4. Discussion
AMPs are a promising class of antimicrobial biomolecules that
originated millions of years ago as part of innate immunity [38]. As
pharmaceuticals, however, AMPs have several disadvantages
including toxic side effects observed at higher doses (especially for
lytic AMPs targeting the membranes), and short circulation time in
blood, due to their degradation by blood-borne proteases and/or a
rapid clearance through the urinary tract. We have previously
shown that Bac is not toxic for eukaryotic cells [8] and well toler-
ated in a mouse toxicity model [14] but rapidly cleared by the
3.6. In vivo biodistribution
In vivo biodistribution analyses and evaluation of the perma-
nence of the peptide in mice were performed using another fluo-
rescent derivative of BacE-PEG. For this purpose, the maleimide
group of Alexa Fluor680© was linked to the free thiol group of the
cysteine residue located between the peptide and the PEG tail to
Fig. 3. Uptake studies of PEGylated forms of Bac. (A) Fluorescence of S. typhimurium cells exposed for 10 min to 1
mM Bac-BY, BacA-PEG-BY and BacE-PEG-BY, and analysed by flow
cytometry with (grey histograms) or without (empty histograms) incubation with Trypan Blue (TB) quencher for 10 min at 37 ꢀC. (B) Fluorescence of S. typhimurium cells exposed
for the indicated times to 0.25 mM Bac-BY, 4 mM BacA-PEG-BY and 1 mM BacE-PEG-BY (¼ the respective MIC values), and analysed by flow cytometry after incubation with Trypan
Blue (TB) quencher for 10 min at 37 ꢀC. For both plots, the Mean Fluorescence Intensity (MFI) value, with SD as error bars, is reported in the y axis. (C) Confocal microscopy images of
S. typhimurium treated with Bac-BY, Polymyxin B, BacE-PEG-BY and BacA-PEG-BY. All images are representative sections from the middle of the bacterial cell. Many fields were
examined and, for each experiment, over 95% of the cells displayed the pattern of the respective representative cell shown here.

M. Benincasa et al. / European Journal of Medicinal Chemistry 95 (2015) 210e219
217
kidney when injected intraperitoneally into mice.
In this work we have demonstrated that PEGylated derivatives
of Bac [i.e. Bac7(1e35)] retain an appreciable antimicrobial activity
and have substantially the same mode of action as the unmodified
peptide, while exhibiting a lower clearance rate. This chemical
modification could therefore considerably enhance its in vivo effi-
cacy. It is a further example of the usefulness of covalent attach-
ment to poly(ethyleneglycol) (PEG) chains of potential therapeutic
agents as a leading approach for overcoming limits, such as physical
and chemical instability, immunogenicity and rapid body clearance
[39].
In selecting the best strategy for linking PEG to Bac we consid-
ered the particular mode of action of this peptide. Bac has to
penetrate into bacterial cells to kill them [7] and the modification of
amino acids at its N-terminus markedly reduces its activity [40]. For
this reason we preferred to link the PEG tail to the C-terminus.
Furthermore, we decided to use an ester bond in order to obtain a
conjugate which was susceptible to blood esterases. As expected,
while stable in MH medium, the unPEGylated peptide was pro-
gressively released by BacE-PEG on a time-scale of several hours in
the presence of human serum or plasma. The released Bac peptide,
being very rich in proline, then itself remains stable in serum for at
least 72 h, and showed MIC values comparable to those of the
native peptide (Table 2).
The mechanism of action of unmodified Bac involves transport
by the SbmA membrane protein, so it was quite encouraging that
the PEGylated conjugates should continue to have an appreciable
anti-salmonella activity, and somewhat surprising for the BacA-PEG
conjugate which is linked via a stable amide bond. This suggested
that even in the ~24 kDa PEGylated form they could exert some
antimicrobial activity. In the case of BacE-PEG it is likely that the
this activity was due to both the PEGylated form and free released
peptide. The difference in anti-salmonella activity between BacE-
PEG and BacA-PEG is effectively only observed in presence of serum
and plasma, but not medium, and thus likely due to the partial
release of the peptide. The contribution to the antibacterial activity
of the intact form is suggested by that of BacA-PEG, showing an 8-
fold reduction of activity against S. typhimurium relative to Bac
(Table 2).
Both PEGylated peptides exhibited loss of activity when assayed
in murine plasma and serum with respect to human fluids. This
result is in agreement with previous observations showing signif-
icantly reduced in vitro and in vivo antibacterial activity in presence
of serum and plasma of mice, also due to peptide degradation [14].
The different influence of human and murine biological fluids on
the antibacterial activity of Bac could find an explanation in the
different composition of blood enzymes and/or proteins in the two
species. A number of proteases and carboxy esterases have in fact
been identified in murine blood that seem to be absent in human
blood [41].
Despite an at least five-fold increased in size, both BacE-PEG and
BacA-PEG conserve a mechanism of action mediated by their up-
take into target cells. The detection of cytoplasmic fluorescence in
salmonellae cells, particularly after incubation with the esterase-
stable BacA-PEG-BY, indicates that the PEGylated forms can still
translocate across the bacterial membranes. Internalization of the
PEGylated peptides was understandably lower than that of un-
conjugated Bac, in agreement with a reduced, but not abrogated,
activity. The fluorophore in the labelled derivatives was linked to
the peptide and not to the PEG molecule (Scheme 2), so for BacE-
PEG-BY it could not be excluded that the fluorescence inside the
bacterial cells was due only to peptide released from PEG on the
surface of the target cells and subsequently internalized, but this is
quite unlikely for a stable derivative such as BacA-PEG-BY.
We have conclusively shown that the inner membrane protein
Fig. 4. The involvement of the membrane protein SbmA in the activity/uptake of the
PEGylated peptides in E. coli. Growth kinetics of E. coli BW25113 and E. coli
BW25113
value. In the figure the continuous line indicates the wild-type E. coli BW25113 strain,
and the dotted line indicates the mutant E. coli BW25113 sbmA. Growth kinetics are
representative of three independent experiments with identical results. (C) Uptake of
M BacA-PEG-BY and 2 M BacE-PEG-BY into E. coli BW25113 and BW25113 sbmA
DsbmA in the presence of BacE-PEG-BY (A) and BacA-PEG-BY (B) at their MIC
D
4
m
m
D
strains. The Mean Fluorescence Intensity (MFI) value, with SD as error bars, is reported
in the y axis.

218
M. Benincasa et al. / European Journal of Medicinal Chemistry 95 (2015) 210e219
Fig. 5. Biodistribution of BacE-PEG-Alexa in healthy mice after i.p. injection. The animals were placed in a prone position (A) and supine position (B); fluorescence emission in the
regions of interest encompassing respectively the kidneys, the thorax and abdomen, were acquired at the indicated times. Panel B on the right shows the fluorescence emission of
the mouse in supine position at 24 h post injection and normalized with respect to the prescan acquired before peptide injection. (C) Ex vivo images: post-mortem images of mice at
24 h after i.p. injection of PEGylated BacE-PEG-Alexa. Imaging of the mice was performed immediately after sacrifice, after removal of the abdominal wall, and normalized. A specific
signal was detected in kidney, liver and the peritoneal cavity (panel on the left); ex vivo images of organs at 24 h after i.p. injection of PEGylated BacE-PEG-Alexa. Imaging of the
organs was performed immediately after sacrifice and normalized. The images are representative of one of three mice that showed comparable results.
SbmA, a secondary transporter with low-specificity for substrates
previously shown to translocate Bac, is also involved in the uptake
of BacE-PEG and BacA-PEG [5,10,12]. This protein is required for the
uptake of structurally quite different molecules including proline-
rich peptides, bleomycin, bacterial mycrocins and peptide nucleic
acids [42]. Our results suggest that SbmA's transport capacity ex-
tends to the uptake of even quite large molecules (z24 kDa), albeit
with a somewhat reduced efficiency, making it a potentially useful
for transporting cargo into bacteria that express it.
The reduced excretion of the peptide is one of the most
important effects of Bac7 PEGylation. A fluorescent derivative of
BacE-PEG was still detectable at 24 h post-injection while the un-
modified peptide disappeared within 3 h after injection [14].
Despite the molecular weight cutoff for glomerular filtration is
thought to be 30e50 kDa [43], the increased mass of Bac7(1e35)
to z 24 kDa in the PEGylated form strongly delays the excretion of
the peptide. The slower renal clearance increases peptide bio-
distribution compared with the unPEGylated form, which included
the liver and peritoneal cavity other than kidney in agreement with
previous observation. This result supports the possibility of
increasing the therapeutic potential of Bac7.
In conclusion we have demonstrated that the addition of a
20 kDa tail of PEG to Bac is a workable strategy to enhance the
in vivo properties of the peptide, improving its pharmacokinetics,
while only marginally affecting its antibacterial activity and not
substantially modifying its mode of action. PEG has already been
approved for use in the formulation of many drugs and cosmetics
[44] and the number of PEGylated products on the market is
continuously increasing [39]. For this reason this kind of modifi-
cation is promising for the future therapeutic application of anti-
infective agents based on Bac7, and may be applied also to a vast
repertoire of other AMPs with relative ease.
Author contributions
Monica Benincasa performed most of the experiments on the
antibacterial activities of the peptides and wrote the initial draft of
the manuscript; Sotir Zahariev designed, synthesized and purified

M. Benincasa et al. / European Journal of Medicinal Chemistry 95 (2015) 210e219
(2002) 389e395.
219
all the compounds some of which with the help of Chiara Pelillo;
Chiara Pelillo and Annalisa Milan performed part of the in vitro
assays and data analysis; Renato Gennaro contributed to concep-
tual advices and to the writing of the manuscript; Marco Scocchi
supervised the experiments and edited the manuscript with the
contribution of all the other authors. All authors have given
approval to the final version of the manuscript.
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