Improved bioactivity of antimicrobial peptides by addition of amino-terminal copper and nickel (ATCUN) binding motifs

Article abstract of DOI:10.1002/cmdc.201402033

Antimicrobial peptides (AMPs) are promising candidates to help circumvent antibiotic resistance, which is an increasing clinical problem. Amino-terminal copper and nickel (ATCUN) binding motifs are known to actively form reactive oxygen species (ROS) upon

Full text of DOI:10.1002/cmdc.201402033

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DOI: 10.1002/cmdc.201402033
Improved Bioactivity of Antimicrobial Peptides by
Addition of Amino-Terminal Copper and Nickel (ATCUN)
Binding Motifs
M. Daben Libardo,[a] Jorge L. Cervantes,[b] Juan C. Salazar,[b] and Alfredo M. Angeles-Boza*[a]
Antimicrobial peptides (AMPs) are promising candidates to
help circumvent antibiotic resistance, which is an increasing
clinical problem. Amino-terminal copper and nickel (ATCUN)
binding motifs are known to actively form reactive oxygen
species (ROS) upon metal binding. The combination of these
two peptidic constructs could lead to a novel class of dual-
acting antimicrobial agents. To test this hypothesis, a set of
ATCUN binding motifs were screened for their ability to induce
ROS formation, and the most potent were then used to modify
AMPs with different modes of action. ATCUN binding motif-
containing derivatives of anoplin (GLLKRIKTLL-NH2), pro-apop-
totic peptide (PAP; KLAKLAKKLAKLAK-NH2), and sh-buforin
(RAGLQFPVGRVHRLLRK-NH2) were synthesized and found to be
more active than the parent AMPs against a panel of clinically
relevant bacteria. The lower minimum inhibitory concentration
(MIC) values for the ATCUN–anoplin peptides are attributed to
the higher pore-forming activity along with their ability to
cause ROS-induced membrane damage. The addition of the
ATCUN motifs to PAP also increases its ability to disrupt mem-
branes. DNA damage is the major contributor to the activity of
the ATCUN–sh-buforin peptides. Our findings indicate that the
addition of ATCUN motifs to AMPs is a simple strategy that
leads to AMPs with higher antibacterial activity and possibly to
more potent, usable antibacterial agents.
Introduction
The emergence of bacterial strains resistant to small-molecule
antibiotics drives the need to develop agents with a unique
mode of action. Due to their broad-spectrum antimicrobial po-
tency and low propensity to induce drug resistance, antimicro-
bial peptides (AMPs) constitute an important model for the
design of novel antibiotics.[1,2] AMPs differ in sequence but are
usually short, amphipathic, and contain an overall positive
charge.[1–3] They serve as natural antibiotics to certain multicel-
lular organisms and exert their action against pathogens either
by immune modulation or by direct interaction with patho-
gens, via membrane solubilization or by binding to internal mi-
crobial targets.[1,3] Because of their diverse modes of action,
a large effort has been made to develop new classes of anti-in-
fective agents based on naturally occurring AMPs.[4–8]
and indolicidin kill bacteria by inducing ROS formation.[10,11]
The ROS formed can result in an increase in the susceptibility
to antimicrobial agents.[10–13] Specifically, the antimicrobial
action of histatins, histidine-rich peptides present in the saliva
at micromolar concentrations, has been related to their ability
to generate ROS after binding copper and iron ions.[14] In the
case of indolicidin, it has been recently reported that electron
donors, such as NADH from the tricarboxylic acid cycle play an
important role in the generation of superoxides. Thus, it is
clear that the synthesis of bactericidal agents with dual action
could lead to the development of clinically useful mole-
cules.[11,15] Notably, peptides have been combined with ROS
production to achieve antimicrobial defense. Conjugation of
photosensitizers to AMPs have led to improved antimicrobial
action due to the production of ROS upon irradiation with visi-
ble light.[16–18]
The formation of reactive oxygen species (ROS) has been
argued to play a role in the action of the naturally occurring
AMPs histatins and indolicidin.[9–11] The ROS hypothesis propos-
es that along with the classical antimicrobial action, histatins
The amino-terminal copper and nickel (ATCUN) binding
unit,[19] H2N-AA1-AA2-His, is a naturally occurring structural fea-
ture present in peptides and proteins that binds certain metal
ions through a free NH2 terminus, a histidine residue in the
third position, and two backbone amide groups from residue
AA2 and histidine (Figure 1).[19–21] It is commonly found in pro-
teins that interact with metal ions, such as albumins and prota-
mins.[19] Interestingly, the ATCUN motif is also found in AMPs
histatin-3 and histatin-5.[14] Decades of investigations have
shown that ATCUN sequences can interact with copper ions to
form metal complexes with nuclease and protein cleavage ac-
tivity.[22–27] This lytic activity is the result of oxidative damage
caused by ROS formed from the CuII/CuIII cycling that arises
from interaction of the bound copper ions to O2 or H2O2.[26,27]
[a] M. D. Libardo, Prof. A. M. Angeles-Boza
Department of Chemistry, University of Connecticut
Unit 3060, 55 North Eagleville Rd, Storrs, CT 06269 (USA)
E-mail: alfredo.angeles-boza@uconn.edu
[b] Dr. J. L. Cervantes, Prof. J. C. Salazar
Department of Pediatrics, University of Connecticut Health Center
263 Farmington Ave, MC 6203, Farmington, CT 06030
and
Division of Pediatric Infectious Diseases, Connecticut Children’s Medical
Center
282 Washington St., Hartford, CT 06106 (USA)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201402033.
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Table 1. Summary of initial rates for ascorbic acid consumption promot-
ed by CuII–ATCUN complexes.
[a]
Complex
Source protein
Initial rate [mmminꢀ1
]
with H2O2
without H2O2
Cu–DAH
Cu–DMH
Cu–DSH
Cu–DTH
Cu–EAH
Cu–GGH
Cu–GKH
Cu–GNH
Cu–LKH
Cu–NGH
Cu–RTH
Cu–SMH
Cu–VIH
Human serum albumin
Neuromedin K
Histatin 5
Bovine serum albumin
Rat serum albumin
Simplest ATCUN
–
Neuromedin C
Xnf7
Met Lyase
Human protamine 2
1.45ꢁ0.07
1.36ꢁ0.02
1.31ꢁ0.03
2.89ꢁ0.07
1.14ꢁ0.04
52.54ꢁ0.76
1.21ꢁ0.09
1.15ꢁ0.04
10.8ꢁ0.27
1.29ꢁ0.09
13.12ꢁ0.27
1.39ꢁ0.04
39.21ꢁ1.08
57.21ꢁ0.81
1.13ꢁ0.03
0.20ꢁ0.02
0.16ꢁ0.01
0.13ꢁ0.01
1.28ꢁ0.06
0.12ꢁ0.02
19.35ꢁ0.43
0.13ꢁ0.01
0.096ꢁ0.002
4.52ꢁ0.17
0.18ꢁ0.01
5.67ꢁ0.20
0.11ꢁ0.01
15.5ꢁ0.07
20.03ꢁ0.37
0.19ꢁ0.02
Figure 1. Schematic representation of the ATCUN sequence bound to
a Cu2+ ion.
The ROS-forming ability of the ATCUN motif has been exploit-
ed to design catalytic metallodrugs.[24,25,28]
In the present work, several ATCUN sequences were assayed
for their ability to form ROS when bound to Cu2+ ions. Three
ATCUN binding motifs were selected, the two most active se-
quences and a weakly active ATCUN sequence as a control,
and these were added to the amino terminus of three AMPs
with different modes of action: 1) the membrane-disrupting
peptides, anoplin (GLLKRIKTLL-NH2)[29–31] and PAP (KLAKLAK-
KLAKLAK-NH2),[32–34] as well as 2) the non-membrane-active
peptide, sh-buforin (RAGLQFPVGRVHRLLRK-NH2).[35–37] The goal
was to develop dual-activity compounds that combine the se-
lectivity and potency of AMPs with the oxidative stress
brought about by the reactivity of the ATCUN motif to yield
a new class of metal binding AMPs with improved efficacies.
Human TBX3
–
–
–
Cu2+ only
No Cu–XXH
[a] Data represent the meanꢁstandard deviation of three independent
experiments.
Antimicrobial activity of ATCUN–AMPs
The efficacy of the ATCUN–AMPs was assessed by determining
their minimum inhibitory concentration (MIC) against four dif-
ferent bacterial strains: Gram-positive Bacillus subtilis and
Staphylococcus epidermidis and Gram-negative Escherichia coli
and Enterobacter aerogenes. Data obtained using standard anti-
microbial susceptibility testing are shown in Table 2.[38]
Results
The microbiological characterization showed that the
ATCUN–AMPs were generally more effective against the Gram-
positive bacteria used in this study. Incorporation of the
ATCUN motif to anoplin, PAP, and sh-buforin largely resulted in
Design and preparation of ATCUN–AMPs
A 13-membered ATCUN library was prepared by established
methods using solid-phase peptide synthesis (SPPS) and stan-
dard fluorenylmethyloxycarbonyl (Fmoc) protocols. Most of the
ATCUN sequences occur naturally in a variety of proteins
(Table 1). We first assessed the ability of these ATCUN sequen-
ces to produce ROS after binding copper ions by using an
assay that measures the rate at which the Cu–ATCUN com-
plexes undergo multiple turnover redox process (Table 1).[24]
The rate at which reduced ascorbic acid is consumed in the
presence and absence of hydrogen peroxide was monitored to
measure the turnover rate of the Cu–ATCUN complexes using
either dioxygen or hydrogen peroxide as a co-reactant. Initial
rates in the presence of hydrogen peroxide are higher than in
its absence—a behavior that has been observed in other
copper peptide complexes.[24] Cu–GGH and Cu–VIH gave the
highest rates for ascorbic acid consumption under both condi-
tions, indicating rapid production of ROS.
Table 2. Minimum inhibitory concentration (MIC) values of synthesized
peptides. The MIC is defined as the lowest concentration required to in-
hibit visual growth of bacteria.
Peptide
MIC [mm][a]
B. subtilis
S. epidermidis
E. coli
E. aerogenes
Anoplin
4
2
2
0.5
8
16
8
16
8
4
>32
DAH–anoplin
GGH–anoplin
VIH–anoplin
>32[b]
>32[b]
32[b]
2
2
PAP
0.5
0.25
0.125
0.125
4
4
1
1
>32
DAH–PAP
GGH–PAP
VIH–PAP
0.25
0.125
0.25
>32[b]
>32[b]
32[b]
0.25
Since the most efficient catalysts for ROS formation were the
copper complexes of GGH and VIH, both sequences were se-
lected for addition to the AMPs. DAH showed slower rates of
ROS formation when compared to GGH and VIH, thus, it was
selected to serve as a low ROS-producing control sequence.
The ATCUN sequences were incorporated at the N terminus of
the AMPs. For instance, GGH–anoplin corresponds to the
amino acid sequence: GGHGLLKRIKTLL-NH2. All of the ATCUN–
AMPs were synthesized by SPPS using Fmoc protocols.
sh-Buforin
8
2
4
2
8
4
16
8
32
16
16
8
>32
DAH–sh-buforin
GGH–sh-buforin
VIH–sh-buforin
>32[b]
>32[b]
>32[b]
[a] Data are the mode of three independent experiments. Mann–Whitney
test was used to compared these results with those of the parental AMP;
numbers in bold correspond to Pꢂ0.05; all other results are not statisti-
cally significant. [b] Chi-square (C2) test was performed due to presence
of ties.
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an increase in their activity, particularly when the ATCUN se-
quence was VIH. For instance, against E. coli, VIH–anoplin was
eight-times more active than the parent peptide, whereas VIH–
sh-buforin as well as VIH–PAP were four-times more active
than sh-buforin and PAP, respectively. GGH was the second
most active ATCUN sequence when bound to the membrane-
active peptides anoplin and PAP. In the case of sh-buforin, DAH
was the second most active sequence.
Since the ATCUN–AMPs bind metal ions, we tested whether
the addition of copper ions could affect the antimicrobial activ-
ity. When medium was supplemented with Cu2+ ions, no de-
crease in the activity of the peptides against B. subtilis and
E. coli was observed (Table 3). These results suggest that this
family of peptides do not induce starvation for copper ions.
Table 3. Minimum inhibitory concentration (MIC) values of ATCUN–AMPs
in the presence of CuII ions.
Figure 2. ATCUN motif increases the membrane-permeabilizing activity of
anoplin, PAP, and sh-buforin. Absorbance at 405 nm obtained from leakage
of b-galactosidase induced by ATCUN–AMPs. E. coli cells were incubated
with 16 mm anoplin and ATCUN–anoplin; 1 mm PAP and ATCUN–PAP; and
32 mm sh-buforin and ATCUN–sh-buforin. Background leakage was subtract-
ed from absorbance of the experimental runs. The meanꢁstandard devia-
tion of three independent experiments is shown (*, P ꢂ0.05; N.S.=not sig-
nificant).
Cu–Peptide
MIC [mm][a]
B. subtilis
E. coli
Cu–Anoplin
4
2
2
0.5
16
8
4
Cu–DAH–Anoplin
Cu–GGH–Anoplin
Cu–VIH–Anoplin
2
Cu–PAP
0.25
0.5
0.06
0.03
1
Cu–DAH–PAP
Cu–GGH–PAP
Cu–VIH–PAP
0.25
0.06
0.125
ATCUN–AMPs bound to copper ions can peroxidate lipids
Since ROS formation can induce damage to bacterial mem-
branes,[16,40] we then evaluated whether the diffusible and
short-lived ROS generated by the ATCUN–AMPs bound to
copper ions (Cu–ATCUN–AMP) could potentially destabilize the
bacterial membrane through oxidative stress. The oxidative
damage brought about by ROS production was assessed by
measuring lipid peroxidation in small unilamellar vesicles
(SUVs) that mimic the E. coli outer membrane. The Cu–ATCUN–
AMP complexes were prepared by mixing 1.5 equivalents of
peptide with 1 equivalent of Cu2+ and pre-incubating at room
temperature for 30–45 min. The slight excess of ATCUN–AMP is
used to ensure no free copper ions are present, as suggested
by Cowan et al.[25] Cu–ATCUN–AMPs were incubated with
80:20 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)/
1,2-dioleoyl-sn-glycero-3-phospho-1’-rac-glycerol sodium salt
(DOPG) SUVs in the presence of hydrogen peroxide and
sodium ascorbate. The malonyldialdehyde formed was tested
via a standard thiobarbituric acid test.
Cu–sh-Buforin
8
2
4
1
16
16
8
Cu–DAH–sh-Buforin
Cu–GGH–sh-Buforin
Cu–VIH–sh-Buforin
4
[a] Cu2+ ions (32 mm) were added to the culture media; data are the
mode of three independent experiments.
ATCUN motif increases the membrane-permeabilizing activi-
ty of an AMP
Since the addition of the ATCUN motif could affect the ability
of the AMPs to turn the bacterial membranes permeable and
allow the cellular contents to leak out,[39] we assayed mem-
brane permeabilization in E. coli by measuring the amount of
b-galactosidase leakage from the bacterial cytoplasm after ex-
posure to the ATCUN–AMPs. After isolating the leaked b-galac-
tosidase, its activity was monitored by adding 2-nitrophenyl-b-
d-galactopyranoside and measuring the increase in absorbance
at 405 nm. Upon addition of the DAH motif to anoplin, we
found that the membrane lytic activity of the AMP was im-
proved (Figure 2). The addition of GGH and VIH to anoplin did
not result in an enhancement of the membrane-permeabilizing
property of the peptide. In contrast to the ATCUN–anoplin
peptides, the addition of the GGH and VIH motifs to PAP in-
creased its membrane-permeabilizing activity, while DAH con-
jugation to PAP did not result in increased lytic activity. A four-
and fivefold increase in activity was observed for GGH–sh-bu-
forin and VIH–sh-buforin, respectively.
As can be seen in Figure 3, the Cu–ATCUN–PAP and –sh-bu-
forin complexes showed no differences among themselves in
the amount of peroxidized lipid formed. Interestingly, the Cu–
ATCUN–anoplin peptides show a trend that trails the observed
MIC values of the ATCUN–AMPs; that is, Cu–VIH–anoplin and
Cu–GGH–anoplin being more active than Cu–DAH–anoplin. To-
gether, these results suggest that the increased antimicrobial
action of the ATCUN derivatives of the membrane-disrupting
peptide anoplin is largely influenced by the oxidative stress on
the microbial membrane caused by the peptides.
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Fluo-Anoplin, Fluo-PAP and their VIH derivatives localized at
the bacterial outer membrane. PAP and VIH–PAP were also ob-
served bound to the flagellum (see movie available as Support-
ing Information). No differences were observed in the localiza-
tion of the Fluo-AMPs when compared to the VIH–Fluo-AMPs.
Fluo-sh-Buforin and VIH–Fluo-sh-buforin also localize in the cy-
toplasmic space and bind to bacterial DNA. The affinity of
Fluo-sh-buforin and its VIH derivative for DNA is nicely reflect-
ed in the images as the area where the nucleoid is localized
showed decreased fluorescence intensity. This phenomenon
originates from the fluorescence quenching of carboxyfluores-
cein by adenine and guanine bases through a photoinduced
electron-transfer process.[41]
ATCUN–sh-buforin peptides bound to copper ions generate
ROS inside E. coli
Considering the ability of the fluorescently labeled VIH–sh-bu-
forin to penetrate into bacteria, we next examined the poten-
tial ability of the ATCUN–sh-buforin derivatives to generate in-
tracellular ROS. The intracellular probe 2’,7’-dichlorofluorescein
diacetate (DCFDA) was used to measure the ability of the
ATCUN–sh-buforin peptides to produce ROS when combined
with copper ions in E. coli (Figure 5). DCFDA diffuses through
the bacterial cell membranes and is enzymatically deacetylated
by intracellular esterases to the nonfluorescent 2’,7’-dihydrodi-
chlorofluorescein.[42,43] This highly polar molecule cannot cross
the membrane and is effectively trapped inside the cell. Upon
reaction with ROS, it generates the fluorescent molecule di-
chlorofluorescein. VIH– and GGH–sh-buforin peptides pro-
duced the largest amounts of intracellular ROS as compared to
the DAH derivative. The intracellular ROS formed by VIH–sh-bu-
Figure 3. Lipid peroxidation promoted by copper complexes of the ATCUN–
AMPs. Percent lipid peroxidized were normalized against the activity of free
Cu2+, which was set to 100%. The meanꢁstandard deviation of three inde-
pendent experiments is shown (*, P ꢂ0.05; N.S.=not significant).
Cellular localization of ATCUN–AMPs
Since modifications in the amino acid sequence of the AMPs
could result in cellular localization changes, we then evaluated
the localization of the AMPs and ATCUN–AMPs. We utilized
laser confocal microscopy to visualize the fluorescently labeled
AMPs in E. coli (Figure 4). 5(6)-Carboxyfluorescein-labeled pep-
tides were synthesized by attaching the fluorophore (Fluo) to
the e-amino group of an additional lysine residue. The lysine
residue was added to the N-terminal residue of the parent
peptides anoplin (Fluo-anoplin), PAP (Fluo-PAP), and sh-buforin
(Fluo-sh-buforin). The fluorescently labeled VIH–AMPs were
synthesized by inserting a lysine residue after the VIH frag-
ment.
Figure 5. Cu–ATCUN–sh-buforin complexes promote formation of intracellu-
lar ROS in E. coli. Relative fluorescence units were used as a measure of gen-
eralized oxidative stress caused by exposing the cells to Cu–ATCUN–sh-bu-
forin complexes ([Cu–ATCUN–sh-buforin]=MIC/4), 10 mm H2O2 or 32 mm
Cu2+ ions. The meanꢁstandard deviation of three independent experiments
is shown (*, P ꢂ0.05; N.S.=not significant).
Figure 4. Laser confocal microscopy fluorescence images of live E. coli cells
exposed to 5(6)-carboxyfluorescein-labeled anoplin (4 mm), VIH–anoplin
(0.5 mm), PAP (0.25 mm), VIH–PAP (0.06 mm), sh-buforin (8 mm) and VIH–sh-bu-
forin (2 mm) for 60 min.
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forin is similar to the amount of ROS produced by 32 mm Cu2+
ions, although considerably less than the amount formed after
exposure to 10 mm hydrogen peroxide.
within the bacteria, generating DNA nicks that could be re-
sponsible for the antibacterial action of ATCUN–sh-buforin.
ATCUN–AMP peptides exhibited low cytotoxicity against
mammalian cells
ATCUN–sh-buforin peptides bound to copper ions can
cleave DNA
To assess the selectivity of the tested compounds towards bac-
teria over mammalian cells, their hemolytic activity was investi-
gated. Release of hemoglobin from packed human red blood
cells (RBCs) was measured spectrophotometrically, and hemoly-
sis of the peptides was normalized against the hemolytic activ-
ity of Triton X-100. As observed in Table 4, all of the synthe-
Given that our data shows internalization of the ATCUN–sh-bu-
forin AMPs, and it is well known that the parent buforin II pep-
tide targets nucleic acids,[36,37] we next investigated the effect
of the ROS generated by the copper complexes of the ATCUN–
sh-buforin peptides on DNA. DNA cleavage promoted by the
Cu–ATCUN–sh-buforin was measured electrophoretically in vi-
tro using pUC19 plasmid as a target. Damage to the DNA plas-
mid pUC19 by ROS was monitored after 30 min and 2 h of in-
cubation with the Cu–ATCUN–sh-buforin complexes. As ob-
served in Figure 6, a time-dependent cleavage of pUC19 was
observed when incubated with all of the Cu–ATCUN–sh-buforin
complexes. For the ATCUN–sh-buforin peptides, the percent-
age of supercoiled plasmid decreased during the 2 h incuba-
tion time, and inversely correlated with an increase in the line-
arized form. Altogether, this evidence points to a capability of
Cu–ATCUN–sh-buforin to induce generalized oxidative stress
Table 4. Hemolytic activity and therapeutic index of the synthesized pep-
tides.
Peptide
Hemolysis [%][a]
HD10 [mm][b]
TI[c]
Anoplin
6ꢁ1
7ꢁ3
6ꢁ2
7ꢁ1
>64
64
16
4
>4
8
4
DAH–anoplin
GGH–anoplin
VIH–anoplin
2
PAP
3 ꢁ1
4ꢁ1
64
8
4
64
32
32
DAH–PAP
GGH–PAP
VIH–PAP
4ꢁ1
13ꢁ5
<1
<1
sh-Buforin
8ꢁ1
8ꢁ2
7ꢁ2
7ꢁ2
64
32
64
64
2
2
4
8
DAH–sh-buforin
GGH–sh-buforin
VIH–sh-buforin
[a] % Hemolysis observed when treated with test compound at the MIC
value against E. coli; data represent the meanꢁstandard deviation of
four independent experiments. [b] Peptide concentration that results in
lysis of 10% red blood cells (HD10); data are representative of 4 independ-
ent determinations. [b] The therapeutic index (TI) was calculated as HD10/
MIC(E. coli).
sized peptides had low hemolytic activity at the observed MIC
value for E. coli as all of the peptides had less than 10% hemol-
ysis (with the exception of VIH–PAP with 13%), suggesting a se-
lectivity of these peptides to lyse bacterial cells instead of
RBCs. The data also showed that, upon addition of the ATCUN
motif to anoplin and PAP, general membrane lytic activity in-
creased.
Discussion
This study presents the development of bifunctional antimicro-
bial agents that combine the bactericidal/bacteriostatic action
of AMPs with the ROS-forming ability of a copper(II)–ATCUN
complex. We hypothesize that the new ATCUN–AMPs will take
advantage of the presence of the existing pool of labile
copper ions in bacteria that flux in response to environmental
stimuli.[44,45] The large affinity of the ATCUN motif for copper
ions (logK~14–15) would allow successful competition for this
endogenous pool of copper.[19] For this purpose, the ability of
certain ATCUN sequences to form ROS was initially assessed by
Figure 6. ATCUN–sh-buforin peptides bound to copper ions cleave DNA in
a time-dependent fashion: a) agarose gel electrophoresis experiments show-
ing time-dependent conversion of pUC19 from supercoiled (S) form to
nicked (N) and linearized (L) forms; b) normalized DNA cleavage activity pro-
moted by Cu–ATCUN–sh-buforin complexes.
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monitoring consumption of reduced ascorbic acid. In the pres-
ence of the oxidant hydrogen peroxide and reductant ascorbic
acid, hydroxyl radicals (HO·) and other ROS are produced via
the Fenton reaction,[22–26,46] as the bound copper cycles back
and forth between its +2 and +3 oxidation states. Our experi-
ments showed that both Cu–GGH and Cu–VIH produced ROS
at the highest rates, and as such were chosen to be added at
the N terminus of anoplin, PAP, and sh-buforin. DAH only ex-
hibited weak ROS forming ability, and as such, the correspond-
ing DAH–AMPs were used as negative controls.
oxygen with the lipid radical [reaction (2)]. The radical can
then be propagated via reaction (3).
RH þ HOꢃ! R ꢃ þHOH
R ꢃ þO2! ROOꢃ
ð1Þ
ð2Þ
ð3Þ
RH þ ROOꢃ! R ꢃ þROOH
The de novo peptide PAP was designed to be amphipathic
in its helical conformation and strongly interact with bacterial
membranes.[32–34,50] Initial studies have established that PAP is
a highly active AMP with low toxicity towards mammalian
cells.[32,51] Overall, the addition of the ATCUN motif increased
the antimicrobial activity of PAP. The confocal microscopy
images corroborate the strong interaction of PAP towards the
microbial membrane. The addition of both GGH and VIH dou-
bled the membrane-permeabilizing activity of PAP, whereas no
significant difference was observed with the DAH derivative.
The increased ability to disrupt the membrane structure by the
VIH and GGH derivatives can be explained by analyzing the
helical wheel diagram of the three peptides (Figure 7). If a pep-
tide adopts an a-helical conformation, as expected for PAP, the
helical wheel diagram provides a simple tool for the analysis of
amphipathicity.[52] As observed in Figure 7, VIH–PAP can poten-
tially have the highest amphipathicity, followed by GGH–PAP.
In DAH–PAP, the Asp residue is detrimental to the amphipatici-
ty of the peptide, since it would appear surrounded by hydro-
phobic residues. The close correlation between the mem-
brane-permeabilizing activity and antimicrobial activity is an in-
dication that the main mechanism of action of the ATCUN–PAP
peptides is the disruption of the prokaryotic cell membrane.
When the ATCUN–AMPs were tested for their potency
against a panel of four different bacteria, the ATCUN motif was
found to increase the activity of anoplin, PAP and sh-buforin
up to a maximum of 16-fold (in the case of VIH–PAP against
S. Epidermis). Some AMPs use metal chelation as their mecha-
nism of action and do not involve the production of ROS.[47] In
those cases, the addition of metal ions such as Cu2+ to the
medium negatively affects their antimicrobial activity.[47] This is
not the case for the ATCUN–AMPs presented in this study,
since when 32 mm of Cu2+ ions were deliberately added to the
growth media, the antibacterial activity of the ATCUN deriva-
tives of anoplin, PAP, and sh-Buforin was retained.[48] This clear-
ly indicates that metal depletion due to the chelating ability of
the ATCUN–AMPs synthesized in this work is not the mecha-
nism of action used by these peptides. The ATCUN–AMPs have
a low toxicity towards mammalian cells, as tested by an assay
that measures their hemolytic activity against RBCs. Although
the ATCUN derivatives are slightly more hemolytic than their
parent peptides, none of them cause more than 33% hemoly-
sis at a concentration as high as 32 mm.
ATCUN–anoplin and ATCUN–PAP peptides
ATCUN–sh-buforin peptides
Anoplin is a membrane-acting AMP, isolated from the venom
of the wasp Anoplius samariensis, with broad-spectrum antimi-
crobial activity.[29–31] Fluorescently labeled anoplin and VIH–
anoplin localize within E. coli at the bacterial membrane, indi-
cating that the addition of the ATCUN motif does not change
to a major extent the affinity of the anoplin sequence for its
target. The highest antibacterial activity against the four bacte-
rial strains tested was observed for VIH–anoplin. The pore-
forming ability of the ATCUN derivatives seems to be larger
than that of anoplin; however, no correlation between the
membrane-permeabilizing activity and the MIC values is found.
Importantly, ROS-mediated damage of lipids by the ATCUN–
anoplin peptides in combination with copper ions correlates
with the antimicrobial activity. Thus, it is reasonable to suggest
that the increased antimicrobial activity of the anoplin deriva-
tives has as the origin the nonenzymatic oxidative damage of
membrane phospholipids.[49] The process is likely initiated by
hydroxyl radicals since peroxyl radicals are unable to react with
fatty acids with rates that are biochemically important.[40] The
hydroxyl radicals produced via the ATCUN–CuIII/ATCUN–CuII cy-
cling can initiate membrane peroxidation through a hydrogen
atom abstraction from a CH2 group alpha to a carbon–carbon
double bond of an unsaturated fatty acid [reaction (1)]. Next,
a lipid peroxyl radical can be formed from the reaction of di-
sh-Buforin is
a
truncated analogue of buforin II
(TRSSRAGLQFPVGRVHRLLRK), a potent AMP that kills bacteria
without cell lysis and has a strong affinity for oligonucleo-
tides.[35–37] The N-terminal random structure (residues 1–4:
TRSS) does not appear to be important to the antibacterial ac-
tivity of buforin II,[37] thus, the addition of the ATCUN motif to
the N-terminal region of sh-buforin was not expected to per-
turb its mode of action. The presence of the ATCUN sequences
increases the membrane-permeabilizing activity of sh-buforin
in E. coli. We can evaluate the amphipathicity of the sh-buforin
derivatives using a helical wheel diagram. However, in this
family of peptides, we only need to analyze up to the proline
residue, since this residue would act as a hinge if the a-helix
were to extend throughout all the residues.[55] As observed in
Figure 8, the VIH and GGH derivatives have five hydrophobic
residues on one side of the helix, whereas the DAH derivative
has only four. The parent peptide, sh-buforin, has only three
hydrophobic residues (not shown). This indicates that the VIH–
and GGH–sh-buforin peptides are more amphipathic than
DAH–sh-buforin and the parent sh-buforin, resulting in an in-
crease in the membrane-disrupting activity.
Despite the increase in membrane-permeabilization activity,
it is unlikely that this property is the sole contributor to the
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Figure 7. Helical wheel diagrams of ATCUN–PAP peptides. Eisenberg consensus hydrophobicity scale was used.[52]
Figure 8. Helical wheel diagrams of ATCUN–sh-buforin peptides up to the Pro residue. Eisenberg consensus hydrophobicity scale was used.[52]
mode of action since the increase of the antibacterial activity
does not trace the lytic properties of the sh-buforin derivatives.
The microscopy results reveal that both sh-buforin and VIH–sh-
buforin are present in the cytoplasmic space of E. coli and bind
to DNA after incubation for 1 h. In addition, the GGH and VIH
derivatives produced the largest amounts of intracellular ROS
as expected from the initial assay of the ATCUN motifs; howev-
er, the intracellular ROS production in E. coli does not correlate
with the antimicrobial activity against this microorganism.
Against E. coli, VIH–sh-buforin was the most active peptide,
while DAH–sh-buforin was as active as GGH–sh-buforin. This
activity correlates with the observed DNA cleavage results, in
which the activity follows the order: VIH–sh-buforin > DAH–
sh-buforin ꢄ GGH–sh-buforin. Altogether, the results indicate
that ROS reacting with the bacterial DNA is more important
than the total production of intracellular ROS. Interestingly,
similar nuclease activity has been recently observed and sug-
gested as the main contributor to the increase in antibacterial
activity in a metallopeptide containing the GGH motif bound
to the DNA-targeting antibacterial sequence WRWYCR.[53,54]
depends largely on the parental peptide. The addition of the
ATCUN motif to anoplin and PAP results in an increase of their
membrane-permeabilizing activity. In the case of anoplin, the
membrane-permeabilization is caused by ROS-mediated
damage to the lipids; whereas the activity of the PAP deriva-
tives is related to the increase in amphipathicity. On the other
hand, the ATCUN–sh-buforin peptides damage bacterial DNA
due to the production of intracytoplasmic ROS and the known
affinity of the sh-buforin sequence for DNA. In the field of drug
design, it is well accepted that the ability to attack microbes
with multiple mechanisms of activity may have better chances
of generating new antibiotics with broader-spectrum and en-
hanced antimicrobial activity. Therefore, agents such as the
ATCUN–AMPs described in this work are promising lead com-
pounds that deserve further studies to fully describe their
mechanism of action and their potential therapeutic applica-
tions.
Experimental Section
Peptide synthesis, purification, and quantification: Peptides in
their C-terminal amidated forms were synthesized manually on
a Rink amide resin (ChemPep Inc, Wellington, FL, USA) using stan-
dard fluorenylmethyloxycarbonyl (Fmoc)-protected l-amino acids
(Matrix Innovation, Quebec, Canada). Coupling reactions were
done with 4 equiv of amino acids and 4 equiv of 2-(1H-benzotria-
zole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU),
deprotection using 20% piperidine in DMF and cleavage using
Conclusions
The present study reveals that the addition of the ATCUN
motifs DAH, GGH, and VIH leads to an increase in antimicrobial
activity of the parental AMPs anoplin, PAP, and sh-buforin. Al-
though the ATCUN–AMPs can induce the formation of ROS,
the mechanistic mode of action differs among peptides and
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95:2.5:2.5 trifluoroacetic acid (TFA)/H2O/triisopropylsilane (TIS). Free
peptides were purified via reverse phase-HPLC (Schimadzu LC-
20AD) on a C18 semi-prep 250ꢁ10 mm column (Grace Davison,
Deerfield, IL, USA) using 0.1% TFA in H2O (Buffer A) and 0.1% TFA
in CH3CN (Buffer B). Tripeptides were subjected to a linear gradient
of 5–95% Buffer B over 20 min, and ATCUN–AMPs were purified
using a linear gradient of 30–60% Buffer B over 30 min.
with the Cu–ATCUN–AMP complexes, the same dilutions and bac-
terial manipulations were done using MHB containing Cu2+. A
small aliquot (62.5 mL) of a 20.5 mm Cu2+ solution was added to
40 mL of MHB to bring the final Cu2+ concentration to 32 mm (well
below the toxic level of Cu2+ in E. coli, which is 3.5 mm).[57] The
minimum inhibitory concentration (MIC) was defined as the con-
centration that prevented visual growth of bacteria, and results
were confirmed by matching to a plate reader OD600 nm measure-
ment. MIC values reported here are the average of three independ-
ent trials.
5(6)-Carboxyfluorescein was purchased from Sigma–Aldrich, and la-
beled peptides were synthesized by attaching the fluorophore to
the e-amino group of an additional Lysine residue and purified in
the same way as the ATCUN–AMPs. Pure fractions were collected,
and the identities confirmed by electrospray ionization-mass spec-
trometry (ESI-MS) run in the positive ionization mode. All purified
peptides were subsequently re-injected on a C18 analytical 250ꢁ
4.6 mm column (Grace Davison, Deerfield, IL, USA) and were found
to be ꢅ95% pure.
All peptides were quantified using a method suggested by Grup-
pen.[56] Briefly, lyophilized pure peptides were dissolved in nano-
pure H2O, and a small aliquot (10 mL) was diluted 1:40 in 80:20:0.1
H2O/CH3CN/formic acid, and the absorbance of this solution was
read using a Varian Cary 50Scan UV–Vis spectrometer at 214 nm.
Molar extinction coefficients for each peptide were calculated on
a sequence-specific manner based on the reported values for each
amino acid and the peptide bond.
b-Galactosidase leakage assay: The membrane disruption caused
by the peptides was assessed by measuring the amount of leaked
b-galactosidase. Overnight cultures of E. coli were inoculated in
fresh Luria–Bertani (LB) broth and were grown to OD600 nm ꢄ0.6.
Overexpression of b-galactosidase was induced for 1 h by addition
of isopropyl-b-d-thiogalactopyranoside (IPTG; Fisher) at 1 mm final
concentration. The cells were washed three times with PBS and re-
suspended in fresh LB broth. A 75 mL aliquot of the bacterial sus-
pension was mixed with 75 mL of two-fold serial dilutions of the
peptides (starting from 32 mm) in sterile microcentrifuge tubes. The
mixture was incubated at 378C for 1 h. After incubation, tubes
were spun down at 4400 rpm at 48C for 10 min, then 100 mL of
the supernatant was transferred to a clear 96-well plate. A 50 mL
aliquot of 2-nitrophenyl-b-d-galactopyranoside (ONPG; Thermo Sci-
entific) in PBS was added at 0.8 mgmLꢀ1 final concentration. The
b-galactosidase activity was monitored by measuring the increase
in absorbance at 405 nm every 5 min for a period of 1 h. Data
shown are the final absorbance readings for three independent
trials and are presented as the meanꢁstandard deviation.
Lipid peroxidation assay: The extent of oxidative damage to the
surface of the cell was measured by quantifying the peroxidation
products of unsaturated phospholipids. 1,2-Dioleoyl-sn-glycero-3-
phosphoethanolamine (DOPE) and 1,2-dioleoyl-sn-glycero-3-phos-
pho-1’-rac-glycerol sodium salt (DOPG) were purchased from
Avanti Polar Lipids (Alabaster, AL, USA), and an 80:20 mole percent
mixture of DOPE and DOPG in CHCl3 was used to make a model
E. coli membrane. The solvent was evaporated under a steady
stream of N2, and the lipid cake was dried further overnight on
a high vacuum line. The lipid cake was rehydrated with 20 mm
HEPES, 100 mm NaCl, at pH 7.40 for 1 h, vortexed five times and
sonicated for 20 min to generate small unilamellar vesicles (SUVs).
A 100 mm total lipid solution of SUVs was incubated with 10 mm of
CuII–ATCUN–AMP complexes with 1 mm H2O2 and 1 mm sodium as-
corbate in rehydration buffer for 1 h. Then, 50 mL of butylated hy-
droxytoluene was added followed by 1.5 mL of 0.44m H3PO4. The
mildly acidic mixture was incubated for 10 min prior to addition of
500 mL of 2-thiobarbituric acid (TBA). The mixture was then heated
on a dry block heater set at 908C for 30 min. After cooling to room
temperature, the amount of malonyldialdehyde–TBA adduct was
quantified by injecting 50 mL of the final reaction mixture in a C18
analytical RP-HPLC column. Elution was done using 35% MeOH
and 65% 50 mm KH2PO4/KOH buffer at pH 7.00, with monitoring at
532 nm. The peak at ~4.6 min corresponds to the pink MDA-TBA
adduct, and the area under the curve was used to quantify the
amount of adduct present. The average value obtained from three
independent trials are reported and presented as the meanꢁstan-
dard deviation.
Peptide handling and preparation: All water used in this study
was obtained from a Barnstead NANOpure Diamond filtration
system with a 0.2 mm pore size filter. Purified peptides dissolved in
nanopure water were stored at 48C, and were diluted to the re-
quired concentration on the day of use. Concentrations were regu-
larly checked via UV-Vis spectrophotometry. To form CuII–peptide
complexes, 1.5 equiv of peptide was mixed with 1 equiv of Cu2+
(excess peptide used to ensure no free metal ions present), and
the solutions were pre-incubated at room temperature for 30–
45 min.[25]
Determination of ascorbic acid consumption: The rate at which
ascorbic acid was consumed by the CuII–ATCUN complex was mea-
sured by the decrease in absorbance of reduced ascorbic acid. Re-
action mixture contained 10 mm CuII–XXH complex with 1 mm as-
corbic acid with and without 1 mm H2O2 in 20 mm 4-(2-hydroxye-
thyl)piperazine-1-ethanesulfonic acid (HEPES), 100 mm NaCl at
pH 7.40. Absorbance at 300 nm was measured on a clear 96-well
plate using a Molecular Devices FlexStation 3 plate reader. Absorb-
ance was plotted versus time, and the slope of the linear portion
of the curve was used to determine the rate constant of reaction.
Ascorbic acid molar absorptivity was used to convert units of rate
to mm ascorbic acid/min. Average values were taken from three
trials and presented as the mean ꢁstandard deviation.
Antimicrobial assay: Antimicrobial susceptibility testing were
done using the broth microdilution method as suggested by Han-
cock.[38] Gram-positive bacteria Bacillus subtilis (PS832), Staphylococ-
cus epidermidis (ATCC 12228) and Gram-negative bacteria Escheri-
chia coli (DL7), and Enterobacter aerogenes (ATCC 13048) were
grown in Mueller–Hinton broth (MHB; Difco) for 3–5 h until mid-
log phase was reached. Peptide stock solutions were diluted in
phosphate-buffered saline (PBS; Gibco), pH 7.40, and 50 mL aliquots
of two-fold serial dilutions (starting from 32 mm) were placed on
a sterile 96-well poplypropylene plate (Greiner). To each well, 50 mL
of a bacterial suspension was added, in a final inoculum of 5ꢁ
105 CFUmLꢀ1 per well. Ampicillin (Sigma–Aldrich) was used as
a positive control, and PBS as a negative control. Plates were incu-
bated at 378C (308C for E. aerogenes) for 18–20 h. For experiments
Measurement of intracellular oxidative damage: To measure the
extent of generalized intracellular oxidative stress brought about
by the Cu–ATCUN–AMP complexes, the fluorescence of dichloro-
fluorescein was quantified. E. coli cells in mid-logarithmic phase
were washed with fresh MHB and resuspended in M9 + glucose
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Supporting Information
minimal media containing 10 mm of the profluorescent compound
2’,7’-dichlorofluorescein diacetate. The dye was loaded into the
cells for 1 h before washing unloaded dye away. The loaded cells
were resuspended in fresh MHB and allowed to grow for an addi-
tional 30 min, after which the cells were incubated with the Cu–
ATCUN–AMP complexes at one-quarter the MIC value for 1 h. The
resulting fluorescence was measured using a Molecular Devices
FlexStation 3 plate reader set to a top read mode. H2O2 (10 mm)
and Cu2+ (32 mm) was used as a positive control, while loaded cells
without any peptide was used as a negative control to account for
oxidation of the dye by other cellular components. Media contain-
ing 10 mm of 2’,7’-dichlorofluorecein diacetate was used as back-
ground to account for autooxidation of extracellular dye by com-
ponents of the media. The average value obtained from three in-
dependent trials are shown as meanꢁstandard deviation.
Synthesis and characterization of peptides, and plots of ascorbic
acid consumption are provided in the Supporting Information
available via http://dx.doi.org/10.1002/cmdc.201402033.
Acknowledgements
A.M.A.-B. acknowledges the University of Connecticut (USA) for
start-up funds. The authors are grateful to Prof. Peter Setlow and
Prof. Ashis Basu for their generous gift of B. subtilis and E. coli, re-
spectively.
Keywords: antimicrobial peptides · amino-terminal copper &
nickel (ATCUN) binding motifs · bioinorganic chemistry ·
cytotoxicity
Confocal fluorescence microscopy imaging: Images were ac-
quired on Zeiss LSM-510 confocal microscope using a 63ꢁ (1.4
N.A.) oil immersion objective at a pixel resolution of 512ꢁ512, and
then processed utilizing ImageJ (US National Institutes of Health).
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DNA cleavage studies: In vitro DNA damage brought about by
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100 nm CuII–ATCUN–AMP with 1 mm H2O2 and 1 mm sodium ascor-
bate in 20 mm HEPES, 100 mm NaCl, at pH 7.40. Reaction was incu-
bated at room temperature and quenched by adding 3X loading
dye containing 1 mm EDTA at two time points—30 min and 2 h.
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a 75 mL aliquot of a two-fold serial dilution series of the peptides,
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Statistical analysis: Data were analyzed for statistical differences
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could not be assumed. Whenever ties were present, comparison
was done using a Chi-square test. Statistical significance for all
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Received: February 17, 2014
Published online on && &&, 0000
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 11
&10
&
ÞÞ
These are not the final page numbers!

FULL PAPERS
A one–two punch! Dual-acting antimi-
crobial peptides (AMPs) are described
that combine the antimicrobial potency
of AMPs with the reactive oxygen spe-
cies (ROS) inducing activity of amino-
terminal copper and nickel (ATCUN)
binding motifs. The novel peptide deriv-
atives show superior activities to the
parent peptides against a panel of clini-
cally relevant bacterial strains.
M. D. Libardo, J. L. Cervantes,
J. C. Salazar, A. M. Angeles-Boza*
&& – &&
Improved Bioactivity of Antimicrobial
Peptides by Addition of Amino-
Terminal Copper and Nickel (ATCUN)
Binding Motifs
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 11
&11
&
ÞÞ
These are not the final page numbers!