Non hemolytic short peptidomimetics as a new class of potent and broad-spectrum antimicrobial agents

Article abstract of DOI:10.1016/j.bmcl.2013.06.016

Since the bacterial resistance to antibiotics is increasing rapidly, numerous studies have contributed to the design and synthesis of potent synthetic mimics of antimicrobial peptides (AMPs). In an attempt to find the pharmacophore of short antimicrobial

Full text of DOI:10.1016/j.bmcl.2013.06.016

Bioorganic & Medicinal Chemistry Letters 23 (2013) 4633–4636
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Bioorganic & Medicinal Chemistry Letters
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Non hemolytic short peptidomimetics as a new class of potent
and broad-spectrum antimicrobial agents
Ravichandran N. Murugan a, , Binu Jacob b, , Eun-Hee Kim a, , Mija Ahn a, Hoik Sohn c, Ji-Hyung Seo a,
Chaejoon Cheong a, Jae-Kyung Hyun d, Kyung S. Lee e, Song Yub Shin b, , Jeong Kyu Bang a,
⇑
⇑
a Division of Magnetic Resonance, Korea Basic Science Institute, Ochang, Chung-Buk 363-883, Republic of Korea
b Department of Bio-Materials, Graduate School and Department of Cellular & Molecular Medicine, School of Medicine, Chosun University, Gwangju 501-759, Republic of Korea
c Department of Chemisty and Biochemistry, College of Natural Science, University of Texas at Austin, Austin, TX 78712, USA
d Division of Electron Micorscopic Research, Korea Basic Science Institute, 113 Gwahanno, Daejeon 305-333, Republic of Korea
e Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Since the bacterial resistance to antibiotics is increasing rapidly, numerous studies have contributed to
the design and synthesis of potent synthetic mimics of antimicrobial peptides (AMPs). In an attempt
to find the pharmacophore of short antimicrobial peptidomimetics through systematic tuning of hydro-
phobic and hydrophilic patterns, we have identified a set of short histidine-derived antimicrobial pep-
tides (SAMPs) with potent and broad-spectrum activity. A combination of high antimicrobial activity
against methicillin-resistant Staphylococcus aureus (MRSA), without hemolytic activity and proteolytic
stability makes these molecules promising candidates for novel antimicrobial therapeutics.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 12 April 2013
Revised 4 June 2013
Accepted 7 June 2013
Available online 14 June 2013
Keywords:
Short antimicrobial peptidomimetics
Methicillin-resistance Staphylococcus aureus
Proteolytic stability
During the last few decades, there has been an urgent need for
the development of novel antibiotics due to emergence of multi-
drug resistant microorganisms, combined with the decline of
new classes of antibacterial agents for clinical treatments. Antimi-
crobial peptides (AMPs) are secreted by innate immune system of
many organisms, such as plants, insects, amphibians, and mam-
mals in response to infection by foreign bacteria, viruses, or fun-
gi.1–3 They are considered as a promising new generation of
antibiotics because of their rapid and broad-spectrum antimicro-
bial properties, ability to kill multidrug-resistant bacteria, and a
low propensity for the development of resistance.4,5 Despite some
potential advantages over conventional antibiotics, pharmaceutical
development into therapeutic agents has been limited by the large
size of these peptides and the corresponding high production cost
to synthesize peptides in bulk, vulnerability to protease degrada-
tion, and cytotoxicity to red blood cells. To address this issue, a
study was undertaken to identify the potent short antimicrobial
peptides using natural amino acids like RWR or WRWR (R: Argi-
nine, W: Tryptophan), without losing the cationic amphiphilic
structure but it did not exhibit promising antimicrobial activity.6
Therefore, it is necessary to develop short antimicrobial peptidom-
imetics using unnatural amino acids that capture the essential bio-
logical properties of AMPs. Keeping this in mind, we envisioned the
use of simple histidine (His) derivative for modulating the amphi-
pathicity and also capable of positioning the amino acids in a prop-
er manner. To test this hypothesis, we utilized the two
nonequivalent but similarly reactive nitrogen atoms on histidine
(His), designed N(p) and N(s), for the stepwise design and optimi-
zation of short histidine-derived antimicrobial peptidomimetics
(SAMPs) having pendant dialkylated tail which was an effective
method to obtain potent antimicrobial activity.
In the present work, we focused on developing the short AMPs
(2- or 3-residues) having potent, nonhemolytic antimicrobial activ-
ity by modulating the peptide parameters, such as charge, hydro-
phobicity, and amphipathicity. A series of SAMPs composed of
one His derivative with hydrophobic alkyl tails and one or two
Arg residues were designed and synthesized. The solid phase syn-
thesis of SAMPs is straightforward using our His derived amino
acids, and their potential diversification is easy at relatively low
cost. In order to identify the most promising, cost-effective ultra-
short AMPs as a drug candidate, the designed SAMPs were com-
pared in terms of (i) broad spectrum of antimicrobial activity, (ii)
prokaryotic selectivity, (iii) antiMRSA activity, and (iv) proteolytic
stability. To the best of our knowledge, this is the first study that
demonstrates an effective SAMPs (2- or 3-residues) having potent
antimicrobial activity against Gram-positive and Gram-negative
bacteria including methicillin-resistant Staphylococcus aureus
⇑
Corresponding authors. Tel.: +82 62 230 6769; fax: +82 62 233 6337 (S.Y.S.);
tel.: +82 43 240 5023; fax: +82 43 240 5059 (J.K.B.).
E-mail addresses: syshin@chosun.ac.kr (S.Y. Shin), bangjk@kbsi.re.kr (J.K. Bang).
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.06.016

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R. N. Murugan et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4633–4636
(MRSA) and without hemolytic activity. Briefly, the synthesis of
Fmoc-protected His derivatives was preceded by three-step syn-
thesis method using commercially available Fmoc-His(Trt)-OH as
measuring their hemolytic activity toward human red blood cells
(hRBCs) (Table1).
The therapeutic potential of antimicrobial agents lies in their
prokaryotic selectivity to effectively kill bacterial cells without
exhibiting significant cytotoxicity toward mammalian cells. The
prokaryotic selectivity of our designed SAMPs is defined by the
concept of the therapeutic index (TI) used widely as a parameter
to represent the specificity of antimicrobial agents. It is a measure
of peptide’s capability to differentiate any pathogen against host
cells. The TI value of each peptide was calculated as the ratio of
the HC10 (the peptide concentration that produces 10% hemolysis
toward human red blood cells) to the GM (geometric mean of MICs
against four selected bacterial strains). Larger values of TI corre-
spond to greater prokaryotic selectivity.
a
starting material. Firstly, the esterification of acid, Fmoc-
His(Trt)-OH was efficiently carried out using DCC and HOBt as a
coupling agents in the presence of methanol. Secondly, the dialky-
lation at N(p)- and N(s)- positions on Fmoc-His(Trt)-OMe, was pro-
ceeded using 2 equiv of corresponding alcohol in the presence of
triflic anhydride and DIEA. Finally, the acid hydrolysis of ester pro-
duced the expected Fmoc-protected dialkylated His derivatives in
good yield (Supplementary Scheme S1). Next, the above synthe-
sized N(p)-alkyl, N(s)-alkyl bis-adducted His monomers were suc-
cessfully used to generate five SAMPs using Rink amide resin by
the conventional solid phase peptide synthesis (Supplementary
Scheme S1 and Fig. 1).
The MICs obtained for the (WR)3-NH2 (4–16 lg/ml) was lower
Recently, a range of very short cationic peptides containing only
Arg and Trp residues [(WR)3-NH2, (WR)2-NH2 and RWR-NH2], re-
ported as short and potent antimicrobial peptides were used as se-
quence templates and compared their antimicrobial and hemolytic
activities with five SAMPs (Supplementary Fig. S1 and Fig. 1).6 Hu-
man cathelicidin LL-37, a potent antimicrobial peptide was served
as a positive control for the comparison of antimicrobial and hemo-
lytic activities. The hydrophobicity of all of the peptides was mea-
sured with the use of reverse phase (RP)-HPLC retention time (Rt)
(Supplementary Table S1).
All of the peptides were assayed for their antimicrobial
activities towards two Gram-negative bacteria (Escherichia coli
and Pseudomonas aeruginosa), and two Gram-positive bacteria
(Staphylococcus epidermidis and S. aureus). The prokaryotic selectiv-
ity of the peptides to mammalian cells was determined by
than LL-37, whereas the (WR)2-NH2, having two repeating WR
units showed eight times decreased in the antimicrobial activity
and finally, the removal of one Trp unit from the (WR)2-NH2
(MIC: >64
lg/ml) at the N-terminal completely lost its antimicro-
bial activity (Table 1). In contrary, all of Trp/Arg-rich AMPs showed
no hemolytic activity even at the highest concentration (256
ml) tested (Table 1 and Supplementary Fig. S2).
lg/
Based on the above results, we designed and synthesized tri-
meric SAMP-1 [Arg–His((CH2)3Cyclohexyl)2-Arg-NH2] to probe
the effect of hydrophobicity on inactive short RWR-NH2 peptide
using our Fmoc-His-derived amino acid monomer (Supplementary
Fig. S1 and Fig. 1) and tested the antimicrobial activity.
Surprisingly, the shorter trimeric SAMP-1 displayed the potent
antimicrobial activity with the MIC value around 4–8
lg/ml
against Gram-positive and Gram-negative bacteria and without
Figure 1. The structures of SAMPs tested for their antimicrobial activity.

R. N. Murugan et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4633–4636
4635
Table 1
Antimicrobial and hemolytic activities and therapeutic index (prokaryotic selectivity) of the peptides
c
Peptides
Minimal inhibitory concentration (MIC)a
GMb
HC10
TId (HC10/GM)
E. coli [KCTC 1682]
P. aeruginosa [KCTC 1637]
S. epidermidis [KCTC 1917]
S. aureus [KCTC 1621]
(WR)3-NH2
(WR)2-NH2
RWR- NH2
SAMP-1
SAMP-2
SAMP-3
SAMP-4
SAMP-5
LL-37
4 (3.8)
16 (15.3)
32 (45.6)
>64 (>124)
8 (11.2)
64 (108.2)
32 (57.2)
8 (11.2)
8 (7.7)
8 (7.7)
9 (8.6)
26 (37)
>64(>124)
5 (7)
36 (60.9)
44 (78.6)
4 (5.6)
5 (7)
>256 (>245.2)
>256 (>364.5)
>256 (>495.8)
>256 (>357.8)
>256 (>432.9)
>256 (>457.6)
>256 (>357.7)
>256 (357.7)
165 (36.7)
56.9
19.7
4.0
102.4
14.2
11.6
128.0
102.4
4.6
32 (45.6)
>64 (>124)
4 (5.6)
64 (108.2)
>64 (>114.4)
4 (5.6)
8 (11.4)
>64 (>124)
4 (5.6)
8 (13.5)
8 (14.3)
2 (2.8)
32 (45.6)
>64 (>124)
4 (5.6)
8 (13.5)
8 (14.3)
2 (2.8)
4 (5.6)
32 (7.1)
8 (11.2)
32 (7.1)
4 (5.6)
64 (14.2)
4 (5.6)
16 (3.6)
36 (8)
a
Minimal inhibitory concentration of peptides in
l
g/ml; values in parentheses are in
The geometric mean (GM) of the MIC values for four bacterial strains in g/ml; values in parentheses are in
g/ml), the value of twice maximum concentration (128 g/ml) was used for calculation of the therapeutic index.
Peptide concentration that induces 10% hemolysis of human erythrocytes, in g/ml; values in parentheses are in M. When no detectable hemolytic activity was observed
at the maximum concentration (256 g/ml), the value of twice maximum concentration (512 g/ml) was used for calculation of the therapeutic index.
lM.
b
l
lM. When no detectable antimicrobial activity was observed
at the maximum concentration (64
l
l
c
l
l
l
l
d
Therapeutic index (TI) is the ratio of the HC10 value to the GM.
hemolytic activity (Table 1 and Supplementary Fig. S2). Particu-
larly, the activity of SAMP-1 against P. aeruginosa, S. epidermidis,
and S. aureus was two times higher than Trp/Arg-rich peptide,
(WR)3-NH2 peptide. Moreover, this assay also confirmed that the
SAMP-1 covered the broad spectrum of antimicrobial activity in
comparison to at least Trp/Arg-rich AMPs. Furthermore, the
SAMP-1, the most potent analog showed 22 times higher in thera-
peutic index (TI = 102.4) in comparison to LL-37 (TI = 4.6, Table 1).
The above results warrant further investigation on SAMP-1 via
Table 2
Antimicrobial activity of designed SAMPs against MRSA strains
Peptides Minimal inhibitory concentration (MIC)a
MRSA 1 [CCARM
3089]
MRSA 2 [CCARM
3090]
MRSA 3 [CCARM
3095]
SAMP-1
SAMP-2
SAMP-3
SAMP-4
SAMP-5
LL-37
4 (5.2)
4 (5.2)
4 (5.2)
32 (54.1)
8 (14.3)
4 (5.6)
4 (5.6)
>32 (>7.1)
>32 (>54.1)
16 (28.6)
4 (5.6)
8 (11.2)
>32 (>7.1)
32 (54.1)
8 (14.3)
2 (5.6)
4 (5.6)
>32 (>7.1)
systematic tuning of hydrophobicity (N(p)-monoalkylated SAMP-
2), charge (SAMP-3 lacking one Arg residue), and relative position
of His derivative (SAMPs -4 and-5). The effect of hydrophobicity on
SAMP-2 showed a 2- and 16-fold decreased in activity against
Gram-negative and Gram-positive bacteria. This result suggests
that by reducing the hydrophobicity of the peptide, the antimicro-
bial activity was reduced as expected (Table 1). On the other hand,
Arg deletion surprisingly retained the nonhemolytic property on
the dimeric SAMP-3, but displayed a significant reduction in anti-
microbial activity (2- or 32-fold) against Gram-negative and
Gram-positive bacteria when compared to SAMP-1 (Table 1).
Overall, as a result of removing either pendant alkyl tail or Arg,
SAMP-2 and SAMP-3 were significantly less active than the most
potent SAMP-1. Thus, the higher activity of SAMP-1 depends on
the existence of delicate balance between the charge density and
hydrophobicity. Finally, the generation of terminal, rather than fa-
cial segregation of hydrophobic residues through the spatial posi-
tioning of the hydrophobic alkyl tail either at the N-terminus
(SAMP-4) or at the C-terminus (SAMP-5), showed that there was
no appreciable changes on the antimicrobial activity when com-
pared to the SAMP-1 (Fig. 1 and Table 1). Interestingly, all of the
SAMPs were nonhemolytic and possessed higher therapeutic index
values than the LL-37. The structure–activity relationship (SAR) in
this series of short peptides revealed that the maximum potencies
and spectra of the antimicrobial activities correlated with the pres-
ence of one His having pendant alkyl tail (hydrophobicity) and two
Arg residues (cationicity) (Table 1).
a
Minimal inhibitory concentration of peptides in
are in M.
l
g/ml; values in parentheses
l
that abound in the body and are selective for basic residues. Tryp-
sin specifically catalyzes the hydrolysis of C-terminal amide bonds
of lysine and arginine. Since our SAMPs possess one or two arginine
residues, their proteolytic stability was examined by tryptic degra-
dation. The peptides were pretreated with trypsin and their resid-
ual antimicrobial activity was assayed by the broth microdilution
assay. Percentage of remaining peptides at each degradation time
was also examined using analytical RP-HPLC chromatograms after
the trypsin treatment. All of our SAMPs except SAMP-2 preserved
completely their antimicrobial activity against E. coli and S. aureus
after trypsin treatment, while melittin, a honey bee-derived AMP,
completely abolished their antimicrobial activity (Fig. 2). Unlike
melittin, SAMP-4 and SAMP-5 retained the antimicrobial activity
by forming dimeric peptides after the C-terminal Arg deletion,
whereas the SAMP-1 and SAMP-3 were highly stable to trypsin
treatment (Fig. 2 and Supplementary Fig. S3).
Morphological changes in bacterial strains, E. coli and S. aureus,
upon the addition of designed SAMPs (SAMP-1 and SAMP-4) were
examined using transmission electron microscopy (TEM). In addi-
tion, LL-37 was also tested for bacterial lysis as a positive control.
In comparison to untreated control, obvious disruption of cellular
structure was observed when SAMPs and LL-37 were added
(Fig. 3). Clear rupturing of bacterial cells and release of internal
materials were clearly visible, whereas the structures of untreated
cells were preserved without any indication of cell damage.
Among the various structural determinants such as conforma-
tion, charge, hydrophobicity, amphipathicity, and polar angle, the
antimicrobial activity is known to be strongly influenced by the
hydrophobicity of the antimicrobial peptides. In conclusion, we
demonstrated the first synthesis of SAMPs having diverse
structural scaffolds obtained by the facile chemical tailoring of
hydrophobic cyclohexyl group and some of our designed SAMPs
(SAMP-1, -4 and -5) represent promising templates for alternative
MRSA strains, which have become resistant to most antibiotics,
are most often found associated with institutions such as hospitals,
but they are also becoming increasingly prevalent in community-
acquired infections.7 Most of our SAMPs except SAMP-2 were
found to retain good activity with the MIC range of 2–8
against three MRSA strains tested, whereas LL-37 did not show
antimicrobial activity even at 32.0 g/ml (Table 2).
lg/ml
l
A major limitation to the development of cationic AMPs as hu-
man therapeutics is their inactivation by endogenous proteases.
Poor proteolytic stability severely limits the clinical use of many
therapeutic peptides.8 Of great concern are trypsin-like proteases

4636
R. N. Murugan et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4633–4636
Figure 2. Inhibition of antimicrobial activity of the peptides by trypsin assessed using the broth microdilution method.
Figure 3. Electron micrographs of negatively stained E. coli and S. aureus untreated or treated with SAMPs and LL-37. All images are scaled, and the scale bar represents 1 lm.
antimicrobial drug design according to its overall advantageous
features such as no hemolytic activity, broad-spectrum antimicro-
bial properties, proteolytic stability, and most notably, potent
activity against MRSA strains.
containing MRSA, hemolytic activity, proteolytic stability, and syn-
thesis of peptide analogs) associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.06.
016.
Acknowledgments
References and notes
This work was supported in part by the Korea Basic Science
Institute’s research grant T33418 (J.K.B.), T33518 (J-K.H.), the Na-
tional Cancer Institute’s Intramural Research grant (K.S.L.) and
the Korea Research Foundation funded by the Korean Government
(KRF-2011-0009039 to S.Y.S.).
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Supplementary data
Supplementary data (detailed experimental methods followed
to determine antimicrobial activity including bacterial strains