Mimicking an antimicrobial peptide polymyxin B by use of cyclodextrin

Article abstract of DOI:10.1039/c1cc16369h

Cyclodextrin derivatives prepared to mimic a membrane active antibacterial peptide polymyxin B strongly permeabilized bacterial membrane and inhibited bacterial proliferation.

Full text of DOI:10.1039/c1cc16369h

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COMMUNICATION
Mimicking an antimicrobial peptide polymyxin B by use of cyclodextrinw
Hatsuo Yamamura,*a Ken Suzuki,a Kazuma Uchibori,a Atsushi Miyagawa,a Masao Kawai,a
Chie Ohmizob and Takashi Katsub
Received 13th October 2011, Accepted 17th November 2011
DOI: 10.1039/c1cc16369h
Cyclodextrin derivatives prepared to mimic a membrane active
antibacterial peptide polymyxin B strongly permeabilized bacterial
membrane and inhibited bacterial proliferation.
In this study we used a cyclic oligosaccharide called cyclodextrin
(CD) for this purpose.
The oligosaccharide consists of a D-glucose residue, with
numbers six, seven or eight called a-, b-, or g-CD, respectively.
It has a truncated cone structure in which one side of the cone
is rimmed by primary 6-OH groups of glucose residues and the
other by secondary 2-and 3-OH groups. Its size (diameter) is
ca. 1nm, almost the same as that of polymyxin B. We believed
that a regiospecific chemical modification of the CD molecule4
would allow the CD to realize a polymyxin B-like structure with
arranged functional groups on its cyclic structure. An additional
advantage of the CD to mimic polymyxin B is the rigidity of its
cyclic structure due to hydrogen bonds between the residues. In
analogue peptide studies, substitution and insertion of amino
acid residue(s) often causes changes in conformation of the
peptide that resulted in less antibacterial activity.5
Polymyxin B is a membrane active cyclic peptide that strongly
permeabilizes both the outer and cytoplasmic membranes of
gram-negative bacteria (Fig. 1) and has been recognized as a
promising agent against multidrug-resistant bacteria.1 The peptide
contains five diaminobutanoic acid residues whose side chain
amino groups form a cationic region of an amphiphilic peptide
structure. In order to disrupt the bacterial membrane, the
polycationic region is considered to interact with the anionic
phosphate moiety of the membrane, while the hydrophobic
fatty acid tail interacts with the fatty acid moiety of the lipid
membrane.2 Polymyxin B nonapeptide, a proteolytic product of
polymyxin B lacking the fatty acid tail, possesses no antibacterial
activity but retains the high outer membrane-permeabilizing
activity, and is expected to be of clinical importance for the
development of bacterial outer membrane permeabilizers.3
Studies to mimic the antibiotic peptide may lead not only to
understanding of the relationship between their structures and
activities but also to the development of new antimicrobial drugs.
Firstly, as an amino-group assembly on the CD mimicking
the polycationic region of polymyxin B to interact with an
anionic bacterial membrane, we prepared polyamino CD
derivatives possessing amino groups on C-6 and whose number
of amino groups varied from six to eight, namely hexamino-a-
CD 1, heptaamino-b-CD 2 and octaamino-g-CD 3, respectively
(Fig. 2).6 Each was derived from conversion of the corresponding
poly-6-O-sulfonyl-CD7 to an azide followed by reduction to the
amine according the method of Lehn et al.8
Activity of the amino CDs 1–3 to permeabilize a bacterial
membrane was examined. The CD acted on bacteria and K+
efflux from bacterial cell cytosol was measured by a K+
electrode (Fig. 3).9 The presence of CDs did not interfere with
the measurement of the electrode. The compound 3 possessing
eight amino groups caused the most K+ efflux from both
Staphylococcus aureus FDA 209P and Escherichia coli K12
strain W3110. Membrane activity of the heptaamino derivative
2 was moderate and that of hex-amino derivative 1 was the
Fig. 1 Structure of polymyxin B.
a Graduate School of Engineering, Nagoya Institute of Technology,
Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan.
E-mail: yamamura.hatsuo@nitech.ac.jp; Fax: +81 52 7355246;
Tel: +81 52 7355246
b Graduate School of Medicine, Dentistry and Pharmaceutical
Sciences, Okayama University, 1-1-1 Tsushima-naka, Kita-ku,
Okayama 700-8530, Japan
w Electronic supplementary information (ESI) available. See DOI:
10.1039/c1cc16369h
Fig. 2 Structures of cyclodextrins 1–3.
c
892 Chem. Commun., 2012, 48, 892–894
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the importance of the number of amino groups on the molecule
to effectively interact with a bacterial membrane.z
The observed K+ efflux from E. coli (gram negative) was
smaller than that from S. aureus (gram positive), which may be
due to the differences in membrane structure between the
bacteria. Presumably the outer membrane in a gram negative
strain may obstruct CD. However the K+ efflux from E. coli
suggested that not only the inner cytoplasmic membrane of the
bacteria but also its outer membrane were permeabilized. This
was confirmed by the synergistically high antimicrobial activity
of the combination of the CD and the probe antibiotics (Fig. 4).
The probes, novobiocin and erythromycin, exhibited low activity
when dosed alone due to the outer membrane acting as a barrier.
Upon addition of the amino CDs, both antibiotics inhibited the
growth of E. coli. In particular, the effect of octaamino 3 almost
equaled to that of polymyxin B nonapeptide as a positive control,
while none of the amino CDs 1–3 themselves showed significant
antimicrobial activity (MIC >128 mg mLꢀ1). Also, none of them
showed hemolytic toxicity (B0%, CD 100 mM). These results are
also similar to the effects of polymyxin B nonapeptide.
Fig. 3 K+ efflux from S. aureus (a) and E. coli (b) caused by
polyamino CDs 1–3.
least, while other derivatives with less than five amino groups
were ineffective (data not shown). The observed dependence on
the number of amino groups of the CD molecule demonstrated
Next we added to the amino CDs benzyl groups as hydrophobic
moieties, creating an amphiphilic property, because appropriate
amphiphilicity is important to enhance antimicrobial activity.10
Substitution of the corresponding CD poly-6-O-sulfonates by
benzylamine gave polybenzylamino derivatives 4–6.y
Their values of MIC against E. coli were moderate
(32–64 mg mLꢀ1), while those against S. aureus were strong
(4–8 mg mLꢀ1) (Table 1). These values were similar to those of
gramicidin S, an antibacterial peptide against gram-positive
bacteria.11 Furthermore, we observed that 4–6 caused complete
loss of K+ in S. aureus (Fig. 5), whose concentrations giving
50% K+ efflux (5–20 mM) were similar to that of gramicidin S
(5–10 mM),11 demonstrating that the benzylamino CDs caused
higher disruption of the bacterial membrane than polyamino
derivatives 1–3, with the prime target being the cytoplasmic
membrane. Hemolytic toxicity of 6 at 100 mM was 94% while
that of gramicidin S was 100%. It is interesting that the addition
of hydrophobic benzyl groups to polymyxin B nonapeptide-like
amino CDs led to gramicidin S-like membrane activity.
Table 1 MIC of polybenzylamino CDs 4–6
MIC/mg mLꢀ1
Compounds
S. aureus
E. coli.
4
5
6
4
4
8
4
64
64
64
32
32
0.5
gramicidin Sa
polymyxin Bb
Fig. 4 Synergistically antimicrobial activity of the combination of
polyamino CDs 1–3 with novobiocin (a) and erythromycin (b). None:
the antibiotics only. PMBN: polymyxin B nonapeptide.
a
b
See ref. 11. See ref. 5a.
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 892–894 893

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J = 12.5 Hz, benzyl CH2), 3.73 (6H, d, J = 12.5, benzyl CH2), 3.91
(6H, t, J = 9.0 Hz, H3), 4.25 (6H, m, H5), 5.01 (6H, br s, H1),
7.10–7.30 (30H, phenyl CH), 5; 1H NMR (300 MHz, D2O) d 3.07–3.17
(14H, H6), 3.43 (7H, t, J = 9.0 Hz, H4), 3.51 (7H, d, J = 12.5 Hz,
benzyl CH2), 3.55 (7H, dd, J = 9.5, 3.0 Hz, H2), 3.75 (7H, d, J = 12.5,
benzyl CH2), 3.87 (7H, t, J = 9.5, H3), 4.23 (7H, m, H5), 5.03 (7H, d,
J = 3.0 Hz, H1), 7.10–7.17 (35H, phenyl CH), 6; 1H NMR (300 MHz,
D2O) d 3.10–3.20 (16H, H6), 3.57 (8H, t, J = 9.5Hz, H4), 3.69
(8H, dd, J = 10.0, 3.0 Hz, H2), 3.75 (16H, br s, benzyl CH2), 3.98
(8H, t, J = 9.5, H3), 4.35(8H, m, H5), 5.22 (8H, d, J = 3.5 Hz, H1),
7.25–7.31 (40H, phenyl CH).
1 J. Li, R. L. Nation, R. W. Milne, J. D. Turnidge and K. Coulthard,
Int. J. Antimicrob. Agents, 2005, 25, 11; A. P. Zavascki, L. Z. Goldani
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4 For example, C. J. Easton and S. F. Lincoln, Modified Cyclodextrins.
Scaffolds and Templates for Supramolecular Chemistry, Imperial
College Press, 1999; M. V. Rekharsky, H. Yamamura, M. Kawai
and Y. Inoue, J. Am. Chem. Soc., 2001, 123, 5360; H. Yamamura,
M. V. Rekharsky, Y. Ishihara, M. Kawai and Y. Inoue, J. Am. Chem.
Soc., 2004, 126, 14224.
5 (a) H. Urakawa, K. Yamada, K. Komagoe, S. Ando, H. Oku, T. Katsu
and I. Matsuo, Bioorg. Med. Chem. Lett., 2010, 20, 1771;
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A. J. de Neeling, P. H. Nibbering, R. H. Mars-Groenendijk, D. Noort,
J. M. Otero, A. L. Llamas-Saiz, M. J. van Raaj, G. A. van der Marel,
H. S. Overkleeft and M. Overhand, Chem.–Eur. J., 2010, 16, 12174;
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M. Kiricsi, M. Jelokhani-Niaraki, R. N. A. H. Lewis, R. S. Hodges
and R. N. McElhaney, J. Biol. Chem., 2005, 280, 2002.
Fig. 5 K+ efflux from S. aureus caused by polybenzylamino CDs 4–6.
In conclusion, we prepared amino group-containing CD
derivatives to mimic membrane active antibacterial peptide
polymyxin B. The derivatives effectively disrupted bacterial
membranes. It is noteworthy that the CD derivatives with
relatively simple substituents realized similar activities to those
of peptides with relatively complex structures. Preliminary
experiments showed that both the MIC values of 6 against
methicillin-resistant S. aureus (MASA) and vancomycin-resistant
Enterococci (VRE) were 12.5 mg mLꢀ1. All the results shown here
demonstrated that our chemistry to mimic antibacterial peptides
by CD molecules is promising. Also, the method to construct a
nano-scale antibacterial site may lead to novel antimicrobial
modifications for material surfaces. Further studies are on the
way. The authors wish to thank Japan Maize Products Co. Ltd
for the generous gift of CD.
6 K. Kano, S. Arimoto and T. Ishimura, J. Chem. Soc., Perkin
Trans. 2, 1995, 1661.
7 K. Fujita, H. Yamamura, A. Matsunaga, T. Imoto, K. Mihashi and
T. Fujioka, J. Am. Chem. Soc., 1986, 108, 4509; H. Yamamura and
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8 J. Boger, R. L. Corcornan and J.-M. Lehn, Helv. Chim. Acta, 1978,
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Notes and references
9 T. Katsu, H. Kobayashi and Y. Fujita, Biochim. Biophys. Acta,
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10 S. Nakao, K. Komagoe, T. Inoue and T. Katsu, Biochim. Biophys.
Acta, 2011, 1808, 490.
z The observed higher activity of octaamino g-CD 3 than those of
hexaamino a-CD 1 and heptaamino b-CD 2 may be due not only to
the larger number of amino groups but also to its ring structure.
Additional experiments by use of not-fully-aminated g-CD and
aminated linear oligosaccharides would be performed to address it.
y 4; 1H NMR (300 MHz, D2O) d 3.05–3.30 (12 H, H6), 3.44 (6H, t,
J = 8.5 Hz, H4), 3.53 (6H, br d, J = 10.5 Hz, H2), 3.60 (6H, d,
11 M. Kawai, R. Tanaka, H. Yamamura, K. Yasuda, S. Nnarita,
H. Umemoto, S. Ando and T. Katsu, Chem. Commun., 2003, 1264.
c
894 Chem. Commun., 2012, 48, 892–894
This journal is The Royal Society of Chemistry 2012