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MOLLIQ-02847; No of Pages 4
Journal of Molecular Liquids xxx (2008) xxx–xxx

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Journal of Molecular Liquids

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m o l l i q

1 The characterizing of the interaction of amphotericin B with cholesteryl esters

2 Loredana Elena Vijan ⁎, Carmen Topala
3 University of Pitesti, Faculty of Science, Pitesti, Tg. Vale Street No.1, 110040, Romania
4

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a r t i c l e i n f o a b s t r a c t

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6
7 Available online xxxx Amphotericin B has been widely applied as a probe for sterol location in biological membrane since an
8 alteration of its adsorption spectrum is induced by formation of sterol–polyene antibiotic complex. The
Keywords: ^^
9 binding of amphotericin B to cholesterol, cholesteryl oleate and cholesteryl linoleate has been investigated
10 Amphotericin B using absorbance measurements and the results have been rationalized assuming a system 1:1 drug —
Cholesterol ^
11 steroid. The binding constant of amphotericin B to cholesterol, cholesteryl oleate and cholesteryl linoleate
Cholesteryl oleate

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12 has been determined using different methods: Benesi–Hildebrand, Scott and Scatchard. The differences in
Cholesteryl linoleate ^
13 UV–Vis spectroscopy values of binding parameters are due to the small structural differences between cholesterol, cholesteryl
14 ^ oleate and cholesteryl linoleate molecules.
15 © 2008 Published by Elsevier B.V.
16
18
19 1. Introduction with a view to remarks of the influences on the interaction 48
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determined by substituent of C-3 sterolic from the steroids. Using 49
20 Polyene macrolide antibiotics comprise over 200 natural com- ^
absorbance measurements, we indicate how the small structural 50
21 pounds produced usually by Streptomyces spp. Most of the polyene differences between Ch, Ch-Ol and Ch-Lin molecules result in varied 51
22 macrolides exhibit antifungal activity. Some of them are also active ^ ^
interaction with AmB. 52
23 against parasites. Only few of polyene macrolides are sufficiently non-
CT

24 toxic to be used as antifungal drugs. Among them the most important

^ 2. Experimental 53
25 one is amphotericin B (AmB), which is used to treat systemic fungal
26 infections (as fungizone or as liposomal formulations) [1].
Amphotericin B from Streptomyces sp. was Sigma-Aldrich product. 54
27 Generally accepted mode of action of polyene macrolide antibiotics is ^
The stock solutions of AmB were prepared in ethanol and their 55
28 the interaction of their molecules with cellular membranes. Polyene
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concentration was determined using the molar absorption coefficient 56

29 macrolide antibiotics cause an impairment of membrane function of
value: ε408 nm = 160,000M− 1cm− 1 [3]. 57
30 sensitive cells, usually leakage of cellular constituents and eventually cell ^ ^
Linoleic acid, oleic acid and cholesterol were Merck products. 58
31 death. Detergent type of action or channel formation in cellular
Cholesteryl linoleate and cholesteryl oleate were synthesized from 59
32 membrane is responsible for the disturbance of membrane barrier
cholesterol and linoleic acid, oleic acid respectively in the presence of 60
33 function. Studies on molecular action of polyene macrolide antibiotics,
OR

the p-toluensulphonic acid, according to the indications in the 61

34 especially for AmB, carried out within last 50 years resulted in huge ^
literature [4]. The purity of these two esters was checked by thin- 62
35 collection of data. Nevertheless, the mechanism responsible for AmB
layer chromatography. Also, the cholesteryl esters solutions used in 63
36 chemotherapeutic selectivity (pathogen cells versus host cells) is still not ^
the spectral study were prepared in anhydrous ethanol. 64
37 well understood. Chemotherapeutic application of AmB is based on
The structures of the compounds were built within the 65
38 higher sensitivity of ergosterol-containing fungal cells to the antibiotic
^ HyperChem Pro 6 program and optimised by the semiempirical 66
C

39 compared to cholesterol-containing mammalian cells. Therefore, it is

^ AM1 method (parameters: SCF control of 0.01, RHF spin pairing, 67
40 postulated that sterol molecules are necessary for AmB channel
Polak–Ribiere optimizer, RMS gradient 0.01 kcal/mol.Å). 68
41 formation and participate in the channel structure. Due to the complex ^
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The absorption spectra were recorded on a Perkin–Elmer Lambda 69

42 mode of action of polyene macrolide antibiotics on membranes of living ^
25 UV–Vis spectrophotometer using the 1 cm optical path length 70
43 organisms many simpler models were worked out, namely, lipid ^
quartz cell, at room temperature. All readings of absorbance were 71
44 monolayer, bilayer and liposomes and recently in silico models [2].
made at 408 nm, the wavelength that corresponds to maximum of 72
45 In our spectral study, we followed the determination of the binding
absorption of the monomer form of the amphotericin B. 73
46 parameters of AmB to cholesterol (Ch) and two cholesteryl esters,
47 namely cholesteryl oleate (Ch-Ol) and cholesteryl linoleate (Ch-Lin)
^ ^ 3. Results and discussion 74

⁎ Corresponding author. Fax: +40 0248216448. Amphotericin B (Fig. 1) is an amphoteric compound composed of a 75
E-mail address: loredana.vijan@upit.ro (L.E. Vijan). hydrophilic polyhydroxil chain along one side and lipophilic polyene 76

0167-7322/$ – see front matter © 2008 Published by Elsevier B.V.

doi:10.1016/j.molliq.2008.10.005

Please cite this article as: L.E. Vijan, C. Topala, J Mol Liq. (2008), doi:10.1016/j.molliq.2008.10.005
 ARTICLE IN PRESS
2 L.E. Vijan, C. Topala / Journal of Molecular Liquids xxx (2008) xxx–xxx

Fig. 1. The structure of AmB.

77 hydrocarbon chain on the other side. AmB binds to sterols,

78 preferentially to the primary fungal cell membrane sterol. This

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79 binding disrupts osmotic integrity of the fungal membrane, resulting
80 in the leakage of intracellular potassium, magnesium, sugars and
81 metabolites and then cellular death. The mechanism of action is due to
82 the intrinsic antifungal activity of AmB [1].
83 Due to amphiphilic and amphoteric properties, AmB is very poorly

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84 soluble in water but also in pure non-polar solvents. Solubility and the
^ Fig. 3. The structure of Ch (a), Ch-Lin (b) and Ch-Ol (c).
85 state of AmB in aqueous media are well characterized [5]. In aqueous
86 media, the optical absorption spectrum of AmB is concentration
87 dependent [6,7]. AmB is fully soluble in water only below concentra- properties was extensively studied by both experimental and 109
88 tion 10− 7M (monomeric forms). When the concentration increases simulation methods. Linoleic acid is a phospholipids and cholesteryl 110
^
above 10− 7M, AmB undergoes complicated processes of self-
89
90
91
92
93
^
^
association and formation of dimers, soluble oligomers and at
concentrations higher than 10− 5M, insoluble aggregates are observed.
^
The associated forms of AmB are characterized by electronic
absorption and circular dichroism spectroscopies [8–11]. It has been
DP esters specific constituent. Approximately 50% from acyl rest from
cholesteryl esters are linoleyl radicals.
Cholesteryl oleate (Ch-Ol) and cholesteryl linoleate (Ch-Lin) are two
^ ^
mesogens lipid derivatives, which posses a hydrophobic moiety: a rigid
111
112
113
114
skeleton and two lateral chains at C-17 and C-3 sterolic and a linker 115
94 established that the state of aggregation of AmB determines ^ ^
functional group such as carboxylate at C-3 sterolic. These two steroids 116
95 ergosterol/cholesterol selectivity in antibiotic sterol suspensions as ^
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(Fig. 3) are very similar molecules, the main structural difference 117
96 well as in of model and cell membranes [12]. resulting from the presence in Ch-Lin of an additional double bound on 118
97 AmB in polar organic solvents (e.g. methanol, ethanol, DMF and ^
the side chain at C-3. They are the major cholesteryl esters in plasma and 119
98 DMSO) exists as a monomolecular dispersion and its optical absorp- ^
are response for atherosclerosis. One demonstrated that alterations in 120
99 tion spectra are not concentration dependent. Fig. 2 presents the dietary fatty acid composition can effectively alter the fatty acid 121
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100 absorption spectra of AmB, at three concentrations of drug: 6.77 µM distribution of LDL and HDL in hypercholesterolemic subjects, that 122
101 (1), 4.14 µM (2) and 2.45 µM (3). These absorption spectra presents a susceptibility to LDL oxidation is altered by these changes and that the 123
102 vibronic structure and four bands are observed in the region 350– substitution of monounsaturated (rather than polyunsaturated) fatty 124
103 450 nm. It may be observed that these spectra are independent on
^ acids for saturated fatty acids in the diet might be preferable for the 125
104 concentration and absorbency increased with concentration in accord prevention of atherosclerosis [13]. 126
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105 with Lambert–Beer's law.

^ The structure of compounds, AmB, Ch, Ch-Ol and Ch-Lin, was built 127
106 Sterol molecules are essential for maintaining the proper structure ^ ^
and optimised by semiempirical AM1 method. The charge distribu- 128
107 and function of eukaryotic cell membranes. The influence of tions in these compounds were determined. It is noticeable that the 129
108 cholesterol (the principal sterol of higher animals) on the lipid bilayer probably positions for the electrophil attack in the steroids are the 130
centres with the higher charge, as oxygen at C-1/ from carbonyl group, 131
^
CO

oxygen at C-3 sterolic, C-6 from B-ring sterolic and carbon atoms from 132
^ ^ ^
the rest of the fatty acids implicated in the double bonds, respectively 133
the atoms C-9/, C-10/ for both esters and C-12/, C-13/ from Ch-Lin. The 134
^ ^ ^ ^ ^
smaller charge for steroid nucleus at C-3 sterolic is observed (this 135
^
position is probably an indication for a nucleophil attack). 136
For all compounds, the frontier molecular orbitals are π orbitals.
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137
For AmB, the highest occupied molecular orbital (εhomo ~ − 8.46 eV) and 138
^
the first vacant orbital (εlemo ~ − 0.41 eV) are preferentially localized on 139
^
the polyenic chain, ensuring a high superposition of the π-system and 140
^
explaining thus the strong interaction tendency of this drug with 141
sterols. For all steroids, the frontier orbitals are preferentially localized 142
on the carboxylic structure from C-3 sterolic, the steroid A and B- 143
^
rings; the rest of the steroid nucleus and lateral chain from C-17 are 144
^ ^
non-implication on these orbitals. 145
^
One determined the electrostatic potential for each compounds, 146
the minimum and maximum values being presented in Table 1. Due to 147
Fig. 2. Absorption spectra of AmB at following concentrations of drug: 6.77 µM (1), the presence of the lone pairs, the oxygen atoms are characterized by 148
4.14 µM (2) and 2.45 µM (3). negative regions of the electrostatic potential, while the hydroxyl 149

Please cite this article as: L.E. Vijan, C. Topala, J Mol Liq. (2008), doi:10.1016/j.molliq.2008.10.005
 ARTICLE IN PRESS
L.E. Vijan, C. Topala / Journal of Molecular Liquids xxx (2008) xxx–xxx 3

t1:1 Table 1
Results of AM1 calculations
t1:2
t1:3 Compound AmB Ch Ch-Ol Ch-Lin
t1:4 Parameter
t1:5 εhomo, eV −8.46 −9.27 −9.43 −9.43
t1:6 εlumo, eV −0.67 1.22 1.05 1.05
t1:7 Vmin, Kcal/mol − 36.74 −19.86 −4.31 −22.84
t1:8 Vmax, Kcal/mol 2664.88 230.05 3675.09 208.19
t1:9 ΔHf, Kcal/mol −769.21 −121.65 −234.15 −205.07
t1:10 µ, D 5.38 1.71 4.75 4.81

150 groups are characterized by positive electrostatic potential. So,

151 possible centres of interaction with AmB are oxygens from CO group
152 and from C-3, carbons 1/, 5 and 6 implicated in double bound from
^
153 steroidic ring, 9/, 10/ for both cholesteryl esters. In addition, are
154 implicated carbons 12/ and 13/ for Ch-Lin from the double bounds of
^
155 the rest of fatty acids from C-3.

F
^ Fig. 5. Benesi–Hildebrand plot for AmB–Ch-Ol system.
156 Conformational analysis of isolated AmB [14–17] was performed
157 and revealed that mutual location of lactone ring and aminosugar

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158 moiety (defined by two dihedral angles) takes only two positions conformation. In addition, the theoretical studies [14–21] were 166
159 called “open” and “closed” conformation. In “closed” conformation, showed that AmB preferentially took a vertical position, perpendicular 167
160 amino group of AmB tends to form intramolecular hydrogen bond, via to the membrane surface, with no propensity to enter the membrane. 168
161 water molecule, with carboxyl group. On the other hand, “open” The influence of Ch, Ch-Lin or Ch-Ol on AmB was determined. In 169
^ ^
162 conformation of both fragments enable AmB to form intermolecular Fig. 4 are presented two family of curves obtained at the titration of 170
AmB' solutions with the two steroids. One observes that the AmB–Ch- 171

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163 hydrogen bonds with other partners. Dynamic molecular simulations ^
164 of AmB monomer and dimer (head-to-tail orientation) [18–20] also Lin or Ch-Ol complex is characterised by the decrease of the major 172
^ ^ ^ ^
165 showed that aminosugar moiety may take both “open” and “closed” bands from the region 350–450 nm. In addition, it may be noted that 173
^
at small Ch-Lin or Ch-Ol to drug ratios (p), the changes of the 174
^ ^
absorption spectra of AmB are similar to those observed on increasing 175
the drug' concentration. 176
Supposing a 1:1 binding ratio and do not account explicitly for 177
ED
either the self-association of the drug or cooperativity effects on the 178
^
binding, we have determined the binding constant by Benesi– 179
Hildebrand, Scott and Scatchard methods [22–24]. Thus, the binding 180
^
constant was obtained in terms of these theories, on basis of 181
equations: 182
CT

- Benesi–Hildebrand: 183
^
1 1 1 1
=
+ ð1Þ
ΔA C 0
K
Δe ½steroid C 0 Δe
RE

- Scott: 186

1
½steroid 1 1
= 0
½steroid + 0 ð2Þ
ΔA C
Δe C
K
Δe
OR

- Scatchard: 189

ΔA K
= −
ΔA + C 0
K
Δe ð3Þ
1
½steroid 1
C

where l is the path length, ΔA — the absorbance change, C0 — the 191

^ ^
concentration of drug, [steroid] — the steroid concentration, K — the 192
^ ^
binding constant, Δε — the molar absorptive difference (Δε = εB − εF), 193
UN

^ ^
εF — the free drug absorption coefficient, εB — the bound drug 194
^ ^
absorption coefficient.

Table 2 t2:1
Binding constant values of AmB-steroids interaction
t2:2
System K, M− 1 t2:3
Method AmB–Ch AmB–Ch-Ol AmB–Ch-Lin t2:4
Benesi–Hildebrand 0.76 (± 0.08)·103 6.22 (± 0.43)·103 10.12 (± 0.78)·103 t2:5
Fig. 4. a. Absorption spectra of AmB in the presence of Ch-Lin at following Ch-Lin to AmB
Scott 0.42 (± 0.06)·103 5.74 (± 0.32)·103 9.35 (± 0.67)·103 t2:6
ratios: 0 (1), 26 (2), 124 (3) and 255 (4). b. Absorption spectra of AmB in the presence of
Scatchard 0.44 (± 0.06)·103 5.91 (± 0.34)·103 7.88 (± 0.62)·103 t2:7
Ch-Ol at following Ch-Ol to AmB ratios: 0 (1), 53 (2), 106 (3) and 265 (4).

Please cite this article as: L.E. Vijan, C. Topala, J Mol Liq. (2008), doi:10.1016/j.molliq.2008.10.005
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196 For all three systems, the Benesi–Hildebrand, Scott and Scatchard [11] A.R. Balskrishnan, K.R.K. Easwaran, Biochim. Biophys. Acta 1148 (1993) 269, 219
^ doi:10.1016/0005-2736(93)90139-Q. 220
197 plots are linear. In Fig. 5 is presented a Benesi–Hildebrand plot for ^ ^
[12] J. Barwicz, P. Tancrede, Chem. Phys. Lipids 85 (1997) 145, doi:10.1016/S0009-3084 221
^ ^
198 AmB–Ch-Ol system. The results obtained are summarized in Table 2. (96)02652-7. 222
^ ^ ^
[13] P. Reaven, S. Parthasarathy, B.J. Grasse, E. Miller, D. Steinberg, J.L. Witztum, J. Clin. 223
199 The differences in values of binding parameters are due to the small
Invest 91 (1993) 668, doi:10.1172/JCI116247. 224
200 structural differences between Ch, Ch-Ol and Ch-Lin molecules. 225 Q1
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245

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DP
TE
EC
RR
CO
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Please cite this article as: L.E. Vijan, C. Topala, J Mol Liq. (2008), doi:10.1016/j.molliq.2008.10.005