Amphotericin B covalent dimers forming sterol-dependent ion-permeable membrane channels

Article abstract of DOI:10.1021/ja012026b

Polyenemacrolides such as amphotericin B (AmB) were thought to assemble together and form an ion channel across plasma membranes. Their antimicrobial activity has been accounted for by this assemblage, whose stability and activity are dependent on sterol constituents of lipid bilayer membranes. The structure of this channel-like assemblage formed in biomembranes has been a target of extensive investigations for a long time. For the first step to this goal, we prepared several AmB dimers with various linkers and tested for their channel-forming activity. Among these, AmB dimers that bore an aminoalkyl-dicarboxylate tether covalently linked between amino groups of AmB showed potent hemolytic activity. Furthermore, K⁺ influx actions monitored by measuring the pH of the liposome lumen by ³¹P NMR revealed that the dimers formed the molecular assemblage similar to that of AmB in phospholipid membrane. Judging from changes in ³¹P NMR spectra, the dimers appeared to induce "all-or-none"-type ion flux across the liposome membrane in the presence of ergosterol, which suggested that the ion channel formed by ergosterol/dimer is similar to that of AmB. With these data in hand, we are now trying to elucidate the structure of the ion-channel complex by making the labeled conjugates of AmB for NMR measurements. Copyright

Full text of DOI:10.1021/ja012026b

Published on Web 03/26/2002
Amphotericin B Covalent Dimers Forming Sterol-Dependent Ion-Permeable
Membrane Channels
Nobuaki Matsumori, Nahoko Yamaji, Shigeru Matsuoka, Tohru Oishi, and Michio Murata*
Department of Chemistry, Graduate School of Science, Osaka UniVersity, 1-16 Machikaneyama, Toyonaka,
Osaka 560-0043, Japan
Received August 22, 2001; Revised Manuscript Received February 5, 2002
For over 30 years, the polyene antibiotic amphotericin B (AmB,
1) has been used for the treatment of systemic fungal infections.
Its antifungal action is generally accounted for by the formation of
an ion-permeable channel across the lipid bilayer, where the drug
has a higher affinity for ergosterol, an abundant sterol in fungal
membranes, than for cholesterol, a major constituent of mammalian
plasma membranes. Despite extensive investigations by UV/CD
spectroscopic methods1 and by molecular dynamics calculations,2
details of the molecular architecture of the ion-permeable channel
are not fully understood; a generally accepted model is a barrel-
stave assemblage comprising around eight pairs of AmB/sterol.3
The mechanism of bimolecular recognition by AmB/AmB or by
AmB/sterol is essential for the better understanding of the drug’s
action, which may lead to a reduction of serious side effects in
mammals. However, the molecular assemblage is thought to reach
a dissociation/association equilibrium, resulting in an exchange
between conducting and nonconducting states of the channel,
hampering spectroscopic observation of the active assemblage. As
suggested for channel-forming peptides,4 if dimers, instead of
monomers, participate in this process, the equilibrium should shift
to the associated state, thus stabilizing the assemblage long enough
for NMR measurements (Figure 1). We therefore attempted to
prepare covalently linked conjugates of AmB/AmB and AmB/sterol.
In this communication we report preparation of AmB dimers with
channel-forming ability that can be utilized in investigating some
of the details underlying bimolecular recognition.
in most solvents hampered product recovery during purification,
the coupling reaction itself proceeded in moderate yield of ca. 20%
for 4 and 5.
Scheme 1
It has been reported that cation current across a liposome
membrane can be monitored by pH-dependent changes in the 31P
NMR chemical shift of phosphate.8 In spectra c and d of AmB
and dimer 5 (Figure 2), new signals appearing at δ 3.1 were derived
from phosphate entrapped in liposomes on which AmB or dimer 5
formed ion-permeable channels and H+ leaked out at the expense
of K+ influx. This pH rise caused a downfield shift of a phosphate
Figure 1. Possible equilibrium shift in formation of molecular assemblages
by covalently linked dimers.
AmB dimers 2 and 3 were prepared by linking each amino group
of AmB with dicarboxylic acid. These dimers bearing an N-acyl
substitutent at their amino groups showed no significant bioactivity,5
as suggested by previous reports.6 We next prepared dimers that
retained amine functionality.7 After several trials, bioactive con-
jugates, which had an N-aminoalkylamide linkage via dicarboxylic
acid, were successfully furnished (Scheme 1). Among other dimers,
4, 5, and 6 exhibited the prominent hemolytic activity, exceeding
that of AmB.5 Although the extremely low solubility of these dimers
signal. AmB and dimer 5 elicited an all-or-none ion flux9 in
ergosterol-containing lipid membranes, where the conductance of
their channels was so large that, once the channel was formed, K+/
H+ exchange instantly reached equilibrium, thus leaving only two
kinds of liposomes. One kind, which was free of the channels,
retained an initial pH (δ 1.2), and the other, which had been
permeabilized, showed a higher pH (δ 3.1).8a The spectra in the
absence of sterol or in the presence of cholesterol (spectra a and b
in Figure 2) showed markedly different features from those in the
* To whom correspondence should be addressed. E-mail: murata@ch.wani.osaka-
u.ac.jp.
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C O M M U N I C A T I O N S
Figure 3. CD spectra of dimer 5 and AmB in phospholipid-ergosterol
liposomes and in buffer without liposomes. CD spectra of dimer 5 were
measured in MeOH, in buffer and in liposomes R ) 0.5 × 10-2 (R: sample/
lipid molar ratio), and a trace for AmB was obtained in the same conditions
except R ) 10-2 (the same concentration as monomer AmB). The liposomes
were prepared in the same manner as that for 31P NMR measurements except
for addition of FCCP.8 Concentrations of dimer 5 and AmB were kept at
2.5 and 5 µM, respectively. ∆ꢀ was normalized to the concentration of the
monomer part for dimer 5.
Figure 2. 31P NMR spectra of liposome-entrapped phosphate for AmB
and Dimer 5. The peak around δ 1.2 corresponds to H2PO-4 at pH 5.5 of
intact liposomes, and that around δ 3.1 corresponds to HPO24- at pH 7.5 of
the buffer. (a) No sterol was present in liposome membrane, R ) 10-3 (R:
sample/lipid molar ratio). (b) Phosphatidylcholine (PC)-cholesterol, R )
10-3. (c) PC-ergosterol, R ) 10-3. (d) PC-ergosterol, R ) 10-4. Control,
where no AmB or dimer was added, depicted the intensity of the peak at
δ 1.2 when liposomes stayed intact. *AmB at R ) 10-3 showed an
unexpected pattern, where the intensity of a phosphate signal due to
permeabilized liposomes was smaller than that in trace d (AmB, R ) 10-4).
This can be explained by disruption action of higher concentrations of AmB,
which should result in not only influx of K+ but efflux of phosphate.
References
(1) For recent examples: (a) Fujii, G.; Chang, J.-E.; Coley, T.; Steere, B.
Biochemistry 1997, 36, 4959-4968. (b) Millie´, P.; Langlet, J.; Berge`s,
J.; Caillet, J.; Demaret, J.-P. J. Phys. Chem. B 1999, 103, 10883-10891.
(2) (a) Bonilla-Marin, M.; Mereno-Bello, M.; Orgeta-Blake, I. Biochim,
Biophys. Acta 1991, 1061, 65-77. (b) Khutorsky, V. E. Biochim, Biophys.
Acta 1992, 1108, 123-127. (c) Baginski, M.; Resat, H.; McCammon, J.
A. Mol. Pharmacol. 1997, 52, 560-570.
(3) (a) De Kruijiff, B.; Demil, R. A. Biochim. Biophys. Acta 1974, 339, 57-
70. (b) Andreoli, T. E. Ann. N.Y. Acad. Sci. 1974, 235, 448-468. (c)
Bolard, J. Biochim. Biophys. Acta 1986, 864, 257-304. (d) Brajtburg, J.;
Powderly, W. G.; Kobayashi, G. S.; Medoff, G. Antimicrob. Agents
Chemother. 1990, 34, 183-188.
presence of ergosterol, indicating that ion conductance of these
channels was not high enough to induce the all-or-none flux
although reductions in the intensity of signals at δ 1.2 suggested
that liposome membranes were partly permeabilized by AmB or
dimer 5. These spectral characteristics of 5, particularly ergosterol-
dependent all-or-none type flux, are essentially similar to those of
AmB although the efficacy of the dimer was more or less reduced.
Dimers 4 and 6 also revealed a typical NMR change of the all-or-
none type flux in ergosterol-containing liposomes (see Supporting
Information), whereas the directly linked dimers 2 and 3 were found
to lack ion-conducting activity. Assemblage formation by dimer 5
was further examined by CD spectra. In ergosterol-containing
phospholipid liposomes, 5 showed a larger split Cotton effect than
that of AmB (Figure 3).10 This spectral difference appears to reflect
close vicinity of heptaene chromophores of the dimers in a
molecular assemblage.10
These findings suggest that dimers 4, 5, and 6 form a molecular
assemblage that is functionally similar to that of AmB. With
bioactive dimers in hand, regio-specific labeling with NMR nuclei
for each monomer part could facilitate the determination of an
interatomic distance even in highly symmetrical structures proposed
for AmB channels. For this purpose, we are trying to synthesize
dimers that are labeled with 13C and 19F nuclei for use in structure
(4) (a) Mutter, M.; Vuilleumier, S. Angew. Chem., Int. Ed. Engl. 1989, 28,
535-554. (b) You, S.; Shuyun Peng, S.; Lien, L.; Breed, J.; Sansom, M.
S. P.; Woolley, G. A. Biochemistry 1996, 35, 6225-6232.
(5) EC50 values of 4, 5, and 6 with regard to hemolytic activities were all 0.3
µM (against 1% human erythrocytes), while the potency of AmB was
1.5 µM. Those of directly linked dimers 2 and 3 were over 100 µM. The
antifungal activity of 4, 5, and 6 was not evident compared with that of
1. ED50 values of 5 and 6 in growth inhibition assays against Candia
albicans were 8 µM and 4 µM, respectively, which were significantly
weaker than that of AmB, 0.3 µM. This low activity may be due to their
low solubility or small critical micelle concentrations in aqueous media.
Details in the bioactivity of the analogues will be published elsewhere.
(6) For example: Mazerski, J.; Bolard, J.; Borowski, E. Biochim. Biophys.
Acta 1995, 1236, 170.
(7) Preparation of some of the AmB dimers was briefly reported in patents;
Griffin, J. H. Int. Appl. PCT/US99/12699, 1999.
(8) Liposomes were prepared from a mixture of egg phosphatidylcholine-
ergosterol (or cholesterol) 9:1 in phosphate buffer (pH 5.5). After
sonication, freezing, and thawing, the liposomes were passed through a
0.2 µm filter, and the medium was neutralized to pH 7.5. A sample and
FCCP (H+ carrier) were added to the liposomes and left for 6 h at 23 °C.
Upon NMR measurements, the concentration of lipids in the buffer was
9 mM. 31P NMR was measured at 202.35 MHz after addition of 1 mM
MnCl2. For experimental details, see Supporting Information and the
following reference: (a) Herve´, M.; Cybulska, B.; Gary-Bobo, C. M. Eur.
Biophys. J. 1985, 12, 121-128. (b) Herve´, M.; Debouzy, J. C.; Borowski,
E.; Cybulska, B.; Gary-Bobo, C. M. Biochim. Biophys. Acta 1989, 980,
261-272.
studies based on solid-state NMR.
Acknowledgment. We are grateful to Professor Yuzuru Mikami,
Research Center for Pathogenic Fungi and Microbial Toxicoses,
Chiba University, for antifungal assays. This work was supported
by a Grant-In-Aid for Scientific Research on Priority Area (A) from
the Ministry of Education, Sciences, Sports, Culture, and Technol-
ogy, Japan; by a grant from the CREST, Japan Science and
Technology Corporation, and by the Yamada Science Foundation.
Supporting Information Available: Experimental sections for
synthesis of AmB dimers, the preparation of liposomes, measurements
of NMR and CD/UV spectra; 31P NMR spectra of dimers 4/6, and UV
spectra of dimer 5 (PDF). This material is available free of charge via
the Internet at http://pubs.acs.org.
(9) An all-or-none ion flux is reported to occur with channel-forming
ionophores such as AmB and gramicidin D, which are reported to elicit
a biphasic evolution of phosphate NMR signals.8
(10) The increase in CD split of dimer 5 by binding to liposome membrane
(as seen in Figure 3) was confirmed by different experiments, see
Supporting Information. Bolard et al., however, reported that micelles of
AmB formed in water give rise to the Cotton effect at 330 nm as seen for
the trace for AmB in buffer (Figure 3). The large CD split for dimer 5,
therefore, may be partly due to micelles of dimer 5 in aqueous phase. (a)
Bolard, J.; Seigneuret, M.; Boudet, G. Biochim. Biophys. Acta 1980, 599,
280-293. (b) Bolard, J.; Legrand, P.; Heitz, F.; Cybulska, B. Biochemistry
1991, 30, 5707-5715.
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