Interactions of amphotericin B derivatives with lipid membranes--a molecular dynamics study

Amphotericin B (AmB) is a well-known polyene macrolide antibiotic used to treat systemic fungal infections. AmB targets more efficiently fungal than animal membranes. However, there are only minor differences in the mode of action of AmB against both types of membranes, which is a source of AmB toxicity. In this work, we analyzed interactions of two low toxic derivatives of AmB (SAmE and PAmE), synthesized in our laboratory, with lipid membranes. Molecular dynamics simulations of the lipid bilayers containing ergosterol (fungal cells) or cholesterol (animal cells) and the studied antibiotic molecules were performed to compare the structural and dynamic properties of AmB derivatives and the parent drug inside the membrane. A number of differences was found for AmB and its derivatives' behavior in cholesterol- and ergosterol-containing membranes. We found that PAmE and SAmE can penetrate deeper into the hydrophobic region of the membrane compared to AmB. Modification of the amino and carboxyl group of AmB also resulted in the conformational transition within the antibiotic's polar head. Wobbling dynamics differentiation, depending on the sterol present, was discovered for the AmB derivatives. These differences may be interpreted as molecular factors responsible for the improved selectivity observed macroscopically for the studied AmB derivatives.     

Interactions of amphotericin B derivatives with lipid membranes—A molecular dynamics study

Amphotericin B (AmB) is a well-known polyene macrolide antibiotic used to treat systemic fungal infections. AmB targets more efficiently fungal than animal membranes. However, there are only minor differences in the mode of action of AmB against both types of membranes, which is a source of AmB toxicity. In this work, we analyzed interactions of two low toxic derivatives of AmB (SAmE and PAmE), synthesized in our laboratory, with lipid membranes. Molecular dynamics simulations of the lipid bilayers containing ergosterol (fungal cells) or cholesterol (animal cells) and the studied antibiotic molecules were performed to compare the structural and dynamic properties of AmB derivatives and the parent drug inside the membrane. A number of differences was found for AmB and its derivatives' behavior in cholesterol- and ergosterol-containing membranes. We found that PAmE and SAmE can penetrate deeper into the hydrophobic region of the membrane compared to AmB. Modification of the amino and carboxyl group of AmB also resulted in the conformational transition within the antibiotic's polar head. Wobbling dynamics differentiation, depending on the sterol present, was discovered for the AmB derivatives. These differences may be interpreted as molecular factors responsible for the improved selectivity observed macroscopically for the studied AmB derivatives.     

Binding of antibiotic amphotericin B to lipid membranes: monomolecular layer technique and linear dichroism-FTIR studies

Amphotericin B (AmB) is one of the main antibiotics applied in treatment of deep-seated mycotic infections. Tensiometric technique has been applied to monitor binding of AmB, from the water subphase, to the lipid monomolecular layers, formed with dipalmitoylphosphatidylcholine at the air-water interface. Time dependencies of surface pressure in the monolayers demonstrate strong enhancement of AmB binding to monolayers brought about by sterols present in the membranes. The monolayers have been deposited to a solid support and examined by means of FTIR spectroscopy. FTIR measurements show that majority of the AmB molecules which bind to the membranes are localized in the polar headgroup region. The results of the linear dichroism-FTIR measurements are consistent with the microscopic picture according to which the molecules of the membrane-bound AmB are distributed among two orientational fractions: one horizontal and one vertical with respect to the plane of the membrane (59% versus 41% respectively, in the case of the membrane formed with the pure lipid without sterols). The presence of cholesterol in the membranes (50 mol% with respect to lipid) slightly affects such a distribution (53% horizontal versus 47% vertical) but the presence of ergosterol has a pronounced effect in the increase in population of the fraction of horizontally bound AmB (85% horizontal vs. 15% vertical). The results of the measurements indicate that mode of action of the AmB consists in disruption of the polar headgroup region of biomembranes, brought about by the AmB molecules bound horizontally with respect to the plane of the membrane.     

Binding of antibiotic amphotericin B to lipid membranes: monomolecular layer technique and linear dichroism-FTIR studies

Amphotericin B (AmB) is one of the main antibiotics applied in treatment of deep-seated mycotic infections. Tensiometric technique has been applied to monitor binding of AmB, from the water subphase, to the lipid monomolecular layers, formed with dipalmitoylphosphatidylcholine at the air-water interface. Time dependencies of surface pressure in the monolayers demonstrate strong enhancement of AmB binding to monolayers brought about by sterols present in the membranes. The monolayers have been deposited to a solid support and examined by means of FTIR spectroscopy. FTIR measurements show that majority of the AmB molecules which bind to the membranes are localized in the polar headgroup region. The results of the linear dichroism-FTIR measurements are consistent with the microscopic picture according to which the molecules of the membrane-bound AmB are distributed among two orientational fractions: one horizontal and one vertical with respect to the plane of the membrane (59% versus 41% respectively, in the case of the membrane formed with the pure lipid without sterols). The presence of cholesterol in the membranes (50 mol% with respect to lipid) slightly affects such a distribution (53% horizontal versus 47% vertical) but the presence of ergosterol has a pronounced effect in the increase in population of the fraction of horizontally bound AmB (85% horizontal vs. 15% vertical). The results of the measurements indicate that mode of action of the AmB consists in disruption of the polar headgroup region of biomembranes, brought about by the AmB molecules bound horizontally with respect to the plane of the membrane.     

Molecular aspects of the interaction between amphotericin B and a phospholipid bilayer: molecular dynamics studies

Amphotericin B (AmB) is a polyene macrolide antibiotic used to treat systemic fungal infections. The molecular mechanism of AmB action is still only partly characterized. AmB interacts with cell-membrane components and forms membrane channels that eventually lead to cell death. The interaction between AmB and the membrane surface can be regarded as the first (presumably crucial) step on the way to channel formation. In this study molecular dynamics simulations were performed for an AmB–lipid bilayer model in order to characterize the molecular aspects of AmB–membrane interactions. The system studied contained a box of 200 dimyristoylphosphatidylcholine (DMPC) molecules, a single AmB molecule placed on the surface of the lipid bilayer and 8,065 water molecules. Two molecular dynamics simulations (NVT ensemble), each lasting 1 ns, were performed for the model studied. Two different programs, CHARMM and NAMD2, were used in order to test simulation conditions. The analysis of MD trajectories brought interesting information concerning interactions between polar groups of AmB and both DMPC and water molecules. Our studies show that AmB preferentially took a vertical position, perpendicular to the membrane surface, with no propensity to enter the membrane. Our finding may suggest that a single AmB molecule entering the membrane is very unlikely.Figure The figure presents the whole structure of the system simulated—starting point. AmB is presented as a space-filling model, DMPC molecules—green sticks, water molecules—red sticks     

The effect of surfactants on the aggregation state of amphotericin B

We have studied the effect of two surfactants, one non-ionic, lauryl sucrose (LS) and the other ionic, sodium deoxycholate (DOC), on the aggregation state of amphotericin B (AmB) and its selectivity towards ergosterol and cholesterol. It is shown that the addition of these surfactants has very similar effects on the AmB micelles. Below the critical micellar concentration of the surfactants, mixed micelles with AmB are first formed as a result of the penetration of the surfactant molecules into the AmB micelles. At higher concentrations of the surfactant molecules, the micellar structure is completely destroyed and AmB is found as monomers in solution. When the concentration of the surfactant is further increased, micelles of the surfactant molecules are built up, AmB remaining in monomeric form. However, the critical micellar concentration of LS is modified by the presence of AmB in solution, while that of DOC is not affected, thereby indicating that the interactions of AmB with LS are stronger than those of DOC with AmB. We also show that both surfactants enhance the selectivity of the AmB binding to sterols at exactly the concentrations of the surfactants which induce the monomerization of the antibiotic. It is observed that the maximal selectivity is found at a concentration of the surfactants corresponding to their particular CMC in presence of the antibiotic.     

Spectrophotometric analysis of organisation of dipalmitoylphosphatidylcholine bilayers containing the polyene antibiotic amphotericin B

Amphotericin B (AmB) is a polyene antibiotic widely used in the treatment of deep-seated fungal infections. The mode of action of AmB is directly related to the effect of the drug on the lipid phase of biomembranes. In the present work the effect of AmB on the properties of lipid bilayers formed with dipalmitoylphosphatidylcholine (DPPC) and the effect of the lipid phase on the molecular organisation of AmB were studied with application of spectrophotometry in the UV-Vis region. The absorption spectra of AmB in lipid membranes display a complex structure with hypsochromically and bathochromically shifted bands indicative of formation of molecular aggregates of the drug. Formation of molecular aggregates was analysed at different concentrations of the drug in the lipid phase in the range 0.05--5 mol% and at different temperatures in the range 5--55 degrees C. The aggregation level of AmB in the ordered phase of DPPC displayed a minimum corresponding to a concentration of 1 mol% with respect to the lipid. An increase in the aggregation level was observed in the temperature region corresponding to the main phase transition. The structure of molecular aggregates of AmB is analysed on the basis of spectroscopic effects in terms of the exciton splitting model. Analysis of the position of the absorption maximum of AmB in the lipid phase of DPPC in terms of the theory of solvatochromc effects makes it possible to ascribe the refractive indices n=1.40 and n=1.49 to the hydrophobic core of the membrane in the L(alpha) and the P(beta)' phase respectively. Analysis of the aggregation of AmB in the lipid phase in relation to the physical state of the membrane reveals that the temperature range of the main phase transition of a lipid cluster in the immediate vicinity of AmB depends on its concentration. The termination of the phase transition temperature, as read from the AmB aggregation, varies between 42 degrees C at 1 mol% AmB in DPPC and 49 degrees C at 5 mol% AmB in DPPC. The exciton splitting theory applied to the analysis of the spectroscopic data makes it possible to calculate the diameter of the AmB pore as 2.8 A in the gel phase and 3.6 A in the fluid phase of the DPPC membrane, on the assumption that the pore is formed by nine AmB molecules.     

Organization of polyene antibiotic amphotericin B at the argon-water interface

Amphotericin B (AmB) is a life-saving antibiotic, used to treat deep-seated mycotic infections. Both the therapeutic and toxic side effects of AmB are directly dependent on its molecular organization. Organization of AmB was studied in monocomponent monomolecular layers formed at the argon-water interface, by means of polarized and non-polarized electronic absorption spectroscopy and analyzed in terms of the exciton splitting theory. The results provide direct indication that AmB forms spontaneously dimers that can be assembled into molecular structures characterized by homogeneous orientational distribution in the monolayer, interpreted as cylindrical pores. The structures are not stable at surface pressures higher than 20 mN/m and therefore dimers are concluded as abundant molecular organization forms of AmB in biomembranes. Possibility of stabilization of the cylindrical structures, at higher surface pressures, by other molecules, e.g. sterols, is also discussed.     

Organization of antibiotic amphotericin B in model lipid membranes. A mini review

Amphotericin B (AmB) is a polyene antibiotic frequently applied in the treatment of fungal infections. According to the general understanding, the mode of action of AmB is directly related to the molecular organization of the drug in the lipid environment, in particular to the formation of pore-like molecular aggregates. Electronic absorption and fluorescence techniques were applied to investigate formation of molecular aggregates of AmB in the lipid environment of liposomes and monomolecular layers formed at the argon-water interface. It appears that AmB dimers, stabilized by van der Waals interactions, are present in the membrane environment along with the aggregates formed by a greater number of molecules. Linear dichroism measurements reveal that AmB is distributed between two fractions of molecules, differently oriented with respect to the bilayer. Molecules in one fraction remain parallel to the plane of the membrane and molecules in the other one are perpendicular. Scanning Force Microscopy imaging of the surface topography of the monolayers formed with AmB in the presence of lipids reveals formation of pore-like structures characterized by the external diameter close to 17 A and the internal diameter close to 6 A. All the findings are discussed in terms of importance of the molecular organization of AmB in the pharmacological action, as well as of the toxic side effects of the drug.     

FTIR spectroscopic study of molecular organization of the antibiotic amphotericin B in aqueous solution and in DPPC lipid monolayers containing the sterols cholesterol and ergosterol

Langmuir monolayers of amphotericin B (AmB) were investigated by recording π-A isotherms under different pH conditions. To gain a better insight into antibiotic-membrane interactions they were monitored by use of the ATR-FTIR spectroscopy. It was observed for AmB monolayers that the limiting molecular area was larger at high than at neutral pH. Analysis of FTIR spectra at different pH revealed substantial differences, depending on ionic state, for different orientations of AmB molecules. These results enable better understanding of the participation of functional groups in the interactions between AmB and sterol-containing DPPC membranes. AmB molecules incorporated into two-component lipid monolayers bind strongly to the ergosterol-rich membrane (maximum penetration surface pressures ca 35?mN/m). The FTIR spectra revealed that the ionic state of AmB and the presence of sterols led to changes in membrane fluidity and molecular packing of the AmB molecules in the lipid membranes. These investigations should be further investigated to discover the molecular mechanism responsible for the mode of action AmB in biological systems.     

Analysis of amphotericin B-induced cell signaling with chemical inhibitors of signaling molecules

Although amphotericin B (AmB) is a major polyene antibiotic against invasive fungal infection, administration to patients sometimes causes inflammatory side effects, which limits the usage of the antibiotic. We studied the intracellular signaling that was induced by AmB. p65 (RelA) of nuclear factor‐κB (NF‐κB), a well‐known signaling molecule as an inducer of proinflammatory cytokines, was phosphorylated by AmB in RAW264.7 cells, a monocyte‐like cell line. Among chemical inhibitors of signaling molecules, U‐73122 (phospholipase C (PLC) inhibitor), Gö6976 (protein kinase C (PKC) inhibitor), BAPTA‐AM (calcium chelator), LFM‐A13 (Bruton's tyrosine kinase (Btk)‐specific inhibitor), and PP2 (c‐Src kinase inhibitor) suppressed AmB‐induced phosphorylation of p65 and translocation of p65 into the nucleus. U‐73122 and Gö6976 reduced AmB‐mediated induction of proinflammatory cytokines (tumor necrosis factor (TNF)‐α and interleukin (IL)‐6) in RAW264.7 cells. Furthermore, AmB‐induced activation of NF‐ κ B was observed in toll‐like receptor (TLR) 2‐expressed cells, and the activation of NF‐κB was inhibited by U‐73122, whereas peptidoglycan‐induced NF‐κB activation, which was also dependent on TLR2, was not inhibited by U‐73122. Finally, U‐73122 partially suppressed in vivo production of TNF‐α and IL‐6 induced by AmB administration in BALB/c mice. These results suggested that the signaling from AmB stimulation to proinflammatory cytokine production is mediated by TLR2, Btk, PLC, PKC, c‐Src and NF‐κB. These signaling molecules may become a target for chemotherapy suppressing AmB‐induced proinflammatory cytokine production.     

Spectroscopic studies of molecular organization of antibiotic amphotericin B in monolayers and dipalmitoylphosphatidylcholine lipid multibilayers

Amphotericin B (AmB) is considered the gold-standard in the treatment of serious systemic mycoses despite its numerous adverse effects. Both the mechanism of antifungal action and the toxicity of this drug are dependent on its molecular organization. The effect of AmB on the organization of lipid membranes formed with dipalmitoylphosphatidylcholine (DPPC) was studied with application of the Langmuir-Blodgett technique and ATR-FTIR spectroscopy. The aim of this research was to analyze the physical interactions leading to the formation of aggregated forms of AmB molecules in one-component monolayers and lipid multibilayers. Analysis of FTIR spectra of two-component multibilayers suggests the possibility the mutual reorientation of the amino-sugar moiety (mycosamine) and macrolide ring. This effect may be significant in the explanation of the aggregation processes of AmB in biological systems.     

Comparative molecular dynamics simulations of amphotericin B-cholesterol/ergosterol membrane channels

Amphotericin B (AmB) is a very effective anti-fungal polyene macrolide antibiotic whose usage is limited by its toxicity. Lack of a complete understanding of AmB's molecular mechanism has impeded attempts to design less toxic AmB derivatives. The antibiotic is known to interact with sterols present in the cell membrane to form ion channels that disrupt membrane function. The slightly higher affinity of AmB toward ergosterol (dominant sterol in fungal cells) than cholesterol (mammalian sterol) is regarded as the most essential factor on which antifungal chemotherapy is based. To study these differences at the molecular level, two realistic model membrane channels containing molecules of AmB, sterol (cholesterol or ergosterol), phospholipid, and water were studied by molecular dynamics (MD) simulations. Comparative analysis of the simulation data revealed that the sterol type has noticeable effect on the properties of AmB membrane channels. In addition to having a larger size, the AmB channel in the ergosterol-containing membrane has a more pronounced pattern of intermolecular hydrogen bonds. The interaction between the antibiotic and ergosterol is more specific than between the antibiotic and cholesterol. These observed differences suggest that the channel in the ergosterol-containing membrane is more stable and, due to its larger size, would have a higher ion conductance. These observations are in agreement with experiments.     

Binding of antibiotic amphotericin B to lipid membranes: a 1H NMR study

The (1)H NMR technique was applied to study binding of AmB, an antifungal drug, to lipid membranes formed with egg yolk phosphatidylcholine. The analysis of (1)H NMR spectra of liposomes, containing also cholesterol and ergosterol (at 40 mol%), shows that AmB binds preferentially to the polar headgroups. Such a binding restricts molecular motion of the choline fragment in the hydrophilic region at the surface of liposomes but increases the segmental motional freedom in the hydrophobic core. The same effects are also observed in the sterol-containing membranes, except that the effect on the hydrophobic core was exclusively observed in the membranes containing ergosterol.     

Optimizing efficacy of amphotericin B through nanomodification

Fungal infections and leishmaniasis are an important cause of morbidity and mortality in immunocompromised patients. The macrolide polyene antibiotic amphotericin B (AmB) has long been recognized as a powerful fungicidal and leishmanicidal drug. A conventional intravenous dosage form of AmB, AmB- deoxycholate (Fungizone or D-AmB), is the most effective clinically available for treating fungal and parasitic (leishmaniasis) infections. However, the clinical efficacy of AmB is limited by its adverse effects mainly nephrotoxicity. Efforts to lower the toxicity are based on synthesis of AmB analogues such as AmB esters or preparation of AmB-lipid associations in the forms of liposomal AmB (L-AmB or AmBisome), AmB lipid complex (Abelcet or ABLC), AmB colloidal dispersion (Amphocil or ABCD), and intralipid AmB. These newer formulations are substantially more expensive, but allow patients to receive higher doses for longer periods of time with decreased renal toxicity than conventional AmB. Modifications of liposomal surface in order to avoid RES uptake, thus increased targetability has been attempted. Emulsomes and other nanoparticles are special carrier systems for intracellular localization in macrophage rich organs like liver and spleen. Injectable nano-carriers have important potential applications as in site-specific drug delivery.     

Membrane effects of the polyene antibiotic amphotericin B and of some of its derivatives on lymphocytes

Amphotericin B (AmB) exhibits immunomodulating properties in mice.In vitro studies on lymphocytes, in relation with these properties, are reported here with AmB and two of its derivatives: the N-Fructosyl (N-Fru AmB) and the N-thiopropionyl (AmBSH) derivatives. Interactions of these molecules with thymocytes, a sensitive cell type, demonstrated that the extent of binding is not a toxicity parameter. In contrast, membrane fludity changes have been observed and appeared to be related to toxicity.Experiments performed with normal B lymphocytes have shown that Amphotericin B derivatives were more potent polyclonal B cell activators than the parent compound. To go further in the understanding of these events, we have investigated in a B cell line WEHI 231, the changes in intracellular Ca²⁺ and membrane potential induced by AmB and AmBSH. The two polyenes were shown to induce membrane depolarization but no intracellular Ca²⁺ increase.     

Influence of a lipid bilayer on the conformational behavior of amphotericin B derivatives — A molecular dynamics study

Amphotericin B (AmB) is an effective but very toxic antifungal antibiotic. In our laboratory a series of AmB derivatives of improved selectivity of action was synthesized and tested. To understand molecular basis of this improvement, comparative conformational studies of amphotericin B and its two more selective derivatives were carried out in an aqueous solution and in a lipid membrane. These molecular simulation studies revealed that within a membrane environment the conformational behavior of the derivatives differs significantly from the one observed for the parent molecule. Possible reasons for such a difference are analyzed. Furthermore, we hypothesize that the observed conformational transition within the polar head of AmB derivatives may lead to destabilization of antibiotic-induced transmembrane channels. Consequently, the selective toxicity of the derivatives should increase as ergosterol-rich liquid-ordered domains are more rigid and conformationally ordered than their cholesterol-containing counterparts, and as such may better support less stable channel structure.     

Physico-chemical properties of the heat-induced 'superaggregates' of amphotericin B

The aggregation state of amphotericin B (AmB) was previously reported to modulate its therapeutic efficiency. As a preliminary study to test the biological effects of 'superaggregates' generated by heat treatment, we present spectroscopic data related to their formation in aqueous solutions. Drastic changes in the AmB aggregation state in water were shown to occur on heating at 50-60 degrees C. The concentration of the aggregates formed at high (A(t)) or room (A) temperature, and the concentration of the monomeric form (M) of AmB were calculated by processing absorption data. The thermally induced conversion from A to A(t) depends on the AmB concentration. Rayleigh scattering measurements suggest that the A(t) aggregates are larger than the A aggregates. At room temperature, the condensation rate of A with M-leading to the 'superaggregated' form A(t)-was slower and depended on the concentration of M. The superaggregated species A(t) was shown to be the most chemically stable species. Physico-chemical properties of these superaggregates are discussed as a potential new solution to improve the therapeutic efficacy of AmB.     

Aggregation of amphotericin B in the presence of gamma-cyclodextrin

The macrolide antibiotic amphotericin B (AmB) forms an inclusion complex with gamma-cyclodextrin (gamma-CDx), resulting in a molecularly dispersed state of the drug. The state of aggregation of AmB in different solvents has been studied by absorption (uv-vis) and CD spectroscopy. While in aqueous solutions AmB forms colloid-like multimolecular aggregates, in the presence of gamma-CDx true solutions can be prepared, which show similar spectral properties as AmB dissolved in organic solvents. The AmB-gamma-CDx complex can be isolated as an amorphous, stable, water-soluble powder, indicating that gamma-CDx is a good carrier for the solubilization of this antibiotic. Using gamma-CDx as a carrier, the danger of precipitation of the drug during parenteral or intravenous administration can be largely reduced.     

Circular dichroism for the determination of amphotericin B binding to liposomes

Circular dichroism (CD) of the antifungal antibiotic amphotericin B (AmB) can be used to characterize the liposomal preparations of the drug with regard to the levels of drug bound to the lipids. The very intense dichroic doublet centered around 340 nm of free amphotericin B in water or the dichroism observed above 435 nm can be used to determine the percentages of bound AmB and free AmB in preparations containing high antibiotic/lipid ratios (ranging from 10(-2) to 10(-1] used in these carrier systems. Examples are given for AmB in the presence of small unilamellar vesicles prepared from four saturated fatty acyl chain phosphatidylcholines of different chain lengths, with or without cholesterol. The transfer of AmB from vesicles to two blood components, serum albumin, and lipoproteins can also be monitored by CD under particular conditions.