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.     

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.     

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     

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.     

Effect of antibiotic amphotericin B on structural and dynamic properties of lipid membranes formed with egg yolk phosphatidylcholine

Amphotericin B (AmB) is a popular antibiotic applied in treatment of deep-seated mycotic infections. The mode of action of AmB is based upon interactions with biomembranes but exact binding properties of the antibiotic to the lipid membranes still remain obscure. Effect of incorporation of AmB into egg yolk phosphatidylcholine membranes in the concentration range from 0.01 to 5 mol% on structural and dynamic properties of lipid bilayers was studied with application of small-angle neutron scattering, X-ray diffractometry and Fourier-transform infrared spectroscopy (FTIR). The results of the experiments show that AmB is located predominantly in the headgroup region of the membranes at concentrations below 1 mol%. The process of AmB aggregation, at concentrations above 1 mol%, is associated with ordering effect within the acyl chain region and therefore indicates incorporation of AmB into the hydrophobic membrane core.     

The chain conformational order of ergosterol- or cholesterol-containing DPPC bilayers as modulated by Amphotericin B: a FTIR study

Amphotericin B (AmB) is the most widely used antibiotic to treat systemic fungal infections. However, the molecular mechanism of its activity is still not completely understood. In the present work we have used FTIR spectroscopy to investigate the conformational state of the aliphatic chains of DPPC liposomes using the 2850 cm(-1) band, associated with the methylene symmetric stretching mode. The liposomes were either binary mixtures of the lipid with AmB, cholesterol or ergosterol, or ternary systems of these constituents. The two sterols contribute to an ordering of the aliphatic chains of the lipid, this ordering being slightly more important with ergosterol. In the gel state, AmB does not change the conformational order of DPPC even at high concentration. In the fluid phase, however, the drug clearly structures its lipid environment. Our results show that AmB can initiate a redistribution of the ergosterol in the plane of the membrane, but not of the cholesterol molecules, which might constitute an additional mechanism to explain the activity of the antibiotic.     

In vitro and in vivo antifungal activities of liposomal amphotericin B,and amphotericin B lipid complex

The in vitro and in vivo antifungal activities of liposomal amphotericin B (L-AMPH) and amphotericin B lipid complex (ABLC), which is composed of amphotericin B and the phospholipids dimyristoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol, were compared with those of conventional amphotericin B (Fungizone®, AMPH). The acute intravenous toxicity was markedly lower in BALB/c mice; 50% lethal doses (LD_50s) were 2.75 mg/kg in AMPH, 32.9 mg/kg in L-AMPH and >75 mg/kg in ABLC. In vitro antifungal activities againstCandida albicans, C. parapsilosis, C. tropicalis, C. glabrata, andC. krusei were evaluated by the agar plate dilution method. The activities were unchanged againstC. albicans, but MICs increased more than four fold in 18 of the 20 strains other thanC. albicans in L-AMPH and in 9 of the 20 in ABLC. L-AMPH and ABLC were as efficacious as AMPH in the treatment of mice infected withC. albicans, and at a dose of 0.5 and 1.0 mg/kg of body weight, ABLC was more efficacious on survival. A ten-times larger dose (10 mg/kg) of L-AMPH and ABLC was administered to mice with 100% survival, suggesting improved tolerability as compared to amphotericin B.     

Determination of amphotericin B, liposomal amphotericin B, and amphotericin B colloidal dispersion in plasma by high-performance liquid chromatography

Amphotericin B is a potent polyene antifungal drug for intravenous treatment of severe infections. It is used as amphotericin B-deoxycholate and in order to reduce amphotericin B toxicity as lipid-formulated complex (liposomal or colloidal dispersion). A sensitive and specific analytical method is presented for the separation of lipid-complexed and plasma protein-bound amphotericin B in human heparinized plasma. This separation, which is required for pharmacokinetic studies, is achieved by solid-phase extraction (SPE) via Bond Elut C₁₈. The protein-bound amphotericin B has a higher affinity to the SPE material and is therefore retained, whereas the lipid-complexed amphotericin B is eluted in the first step. The recovery of the SPE was >75% for high concentrations and >95% for low concentrations. Quantification was performed by reversed-phase HPLC using a LiChrosorb-RP-8 column, UV detection (λ=405 nm) and a mixture of acetonitrile–methanol–0.010 M NaH₂PO₄ buffer (41:10:49, v/v) as mobile phase. The retention time for amphotericin B under the given conditions was 6.7 min. The calibration curves were found to be linear (r≥0.999) in two different ranges (5.0–0.50 μg/ml and 0.50–0.005 μg/ml). Intra- and inter-day precision and accuracy fulfilled the international requirements. No interference from other drugs (typical broad medication for intensive-care patients) or common plasma components was detected in >400 samples analyzed.     

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.     

Effect of magnesium sulfate in oxidized lipid bilayers properties by using molecular dynamics

Magnesium sulfate (MgSO₄) has been used as a protector agent for many diseases related to oxidative stress. The effect of MgSO₄ on the oxidized lipid bilayer has not yet been studied using molecular dynamics calculations. In this work, the effects of oxidation were evaluated by using a POPC membrane model at different concentrations of its aldehyde (-CHO) and hydroperoxide (-OOH) derivatives with and without MgSO₄. Several quantitative and qualitative properties were evaluated, such as membrane thickness, area per lipid, area compressibility modulus, snapshots after simulation finish, density distributions, time evolutions of oxidized group positions, and radial distributions of oxidized group concerning Mg. Results indicate that in the absence of MgSO₄ the mobility of oxidized groups, particularly –CHO, toward the surface interface is high. At a low oxidation level of the bilayer there is an increase in the compressibility modulus as compared to the unoxidized bilayer. MgSO₄, at a low oxidation level, tends to lessen the oxidation effects by lowering the dispersion in the distribution of oxidized species toward the membrane surface and the water region. However, MgSO₄ does not change the trends of decreasing membrane thickness and area compressibility modulus and increasing area per lipid upon oxidation. In this regard, MgSO₄ diminishes the electrostatic long-distance attractive interactions between the oxidized groups and the charged headgroups of the interface, owing to the Mg⁺² and SO₄^-2 screening effects and an electrostatic stabilization of the headgroups, preventing the pore formation, which is well-known to occur in oxidized membranes.     

Transient permeability induced by alkyl derivatives of amphotericin B in lipid membranes

Individual ionic channels were shown to be formed in the brain cholesterol containing phospholipid membranes by two-sided addition of the amphotericin B alkyl derivatives. At concentrations between 10⁻⁸ and 10⁻⁷ M, the resulting conductance appeared to be transient. Existence of different antibiotic assemblies was justified by the kinetic analysis of the membrane conductance decline following the antibiotic washing out. In order to account for the transient characteristics of the induced conductance, it was proposed that the antibiotic oligomers incorporate into the membrane from the aqueous phase, form channels aggregating with cholesterol, and then dissociate in the bilayer into non-active degraded oligomeric or monomeric forms.     

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.     

A systematic molecular dynamics simulation study of temperature dependent bilayer structural properties

Although lipid force fields (FFs) used in molecular dynamics (MD) simulations have proved to be accurate, there has not been a systematic study on their accuracy over a range of temperatures. Motivated by the X-ray and neutron scattering measurements of common phosphatidylcholine (PC) bilayers (Ku?erka et al. BBA. 1808: 2761, 2011), the CHARMM36 (C36) FF accuracy is tested in this work with MD simulations of six common PC lipid bilayers over a wide range of temperatures. The calculated scattering form factors and deuterium order parameters from the C36 MD simulations agree well with the X-ray, neutron, and NMR experimental data. There is excellent agreement between MD simulations and experimental estimates for the surface area per lipid, bilayer thickness (D_B), hydrophobic thickness (D_C), and lipid volume (V_L). The only minor discrepancy between simulation and experiment is a measure of (D_B − D_HH) / 2 where D_HH is the distance between the maxima in the electron density profile along the bilayer normal. Additional MD simulations with pure water and heptane over a range of temperatures provide explanations of possible reasons causing the minor deviation. Overall, the C36 FF is accurate for use with liquid crystalline PC bilayers of varying chain types and over biologically relevant temperatures.     

Structural dynamics of the cell wall precursor lipid II in the presence and absence of the lantibiotic nisin

Representing a physiological “Achilles' heel”, the cell wall precursor lipid II (L_II) is a prime target for various classes of antibiotics. Over the years L_II-binding agents have been recognized as promising candidates and templates in the search for new antibacterial compounds to complement or replace existing drugs. To elucidate the molecular structural basis underlying L_II functional mechanism and to better understand if and how lantibiotic binding alters the molecular behavior of L_II, we performed molecular dynamics (MD) simulations of phospholipid membrane-embedded L_II in the absence and presence of the L_II-binding lantibiotic nisin. In a series of 2 × 4 independent, unbiased 100 ns MD simulations we sampled the conformational dynamics of nine L_II as well as nine L_II–nisin complexes embedded in an aqueous 150 mM NaCl/POPC phospholipid membrane environment. We found that nisin binding to L_II induces a reduction of L_II mobility and flexibility, an outward shift of the L_II pentapeptide, an inward movement of the L_II disaccharide section, and an overall deeper insertion of the L_II tail group into the membrane. The latter effect might indicate an initial step in adopting a stabilizing, scaffold-like structure in the process of nisin-induced membrane leakage. At the same time nisin conformation and L_II interaction remain similar to the 1WCO L_II–nisin NMR solution structure.     

Structure and dynamics of the antifungal molecules Syringotoxin-B and Syringopeptin-25A from molecular dynamics simulation

The phytopathogen Pseudomonas syringae pv. syringae produces toxic cyclic lipodepsipeptides (CLPs): nona-peptides and syringopeptins. All CLPs inhibit the growth of many fungal species, including human pathogens, although different fungi display different degrees of sensitivity. The best studied CLPs are Syringomycin-E (SR-E), Syringotoxin-B (ST-B) and Syringopeptin-25A (SP-25A). Their biological activity is affected by membrane composition and their structural differences. We previously (Matyus et al. in Eur Biophys J 35:459–467, 2006) reported the molecular features and structural preferences of SR-E in water and octane environments. Here we investigate in atomic detail the molecular features of the two other main CLP components, ST-B and SP-25A, in water and octane by 200 ns molecular dynamics simulations (MD), using distance restraints derived from NMR NOE data (Ballio et al. in Eur J Biochem 234:747–758, 1995). We have obtained three-dimensional models of ST-B and SP-25A CLPs in different environments. These models can now be used as a basis to investigate the interactions of ST-B and SP-25A with lipid membranes an important further step towards a better understanding of the antifungal and antibacterial activity of these peptides. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Atom-scale molecular interactions in lipid raft mixtures

We review the relationship between molecular interactions and the properties of lipid environments. A specific focus is given on bilayers which contain sphingomyelin (SM) and sterols due to their essential role for the formation of lipid rafts. The discussion is based on recent atom-scale molecular dynamics simulations, complemented by extensive comparison to experimental data. The discussion is divided into four sections. The first part investigates the properties of one-component SM bilayers and compares them to bilayers with phosphatidylcholine (PC), the focus being on a detailed analysis of the hydrogen bonding network in the two bilayers. The second part deals with binary mixtures of sterols with either SM or PC. The results show how the membrane properties may vary substantially depending on the sterol and SM type available, the membrane order and interdigitation being just two of the many examples of this issue. The third part concentrates on the specificity of intermolecular interactions in three-component mixtures of SM, PC and cholesterol (CHOL) under conditions where the concentrations of SM and CHOL are dilute with respect to that of PC. The results show how SM and CHOL favor one another, thus acting as nucleation sites for the formation of highly ordered nanosized domains. Finally, the fourth part discusses the large-scale properties of raft-like membrane environments and compares them to the properties of non-raft membranes. The differences turn out to be substantial. As a particularly intriguing example of this, the lateral pressure profiles of raft-like and non-raft systems indicate that the lipid composition of membrane domains may have a major impact on membrane protein activation.     

Conjugated double bonds in lipid bilayers: a molecular dynamics simulation study

Conjugated linoleic acids (CLA) are found naturally in dairy products. Two isomers of CLA, that differ only in the location of cis and trans double bonds, are found to have distinct and different biological effects. The cis 9 trans 11 (C9T11) isomer is attributed to have the anti-carcinogenic effects, while the trans 10 cis 12 (T10C12) isomer is believed to be responsible for the anti-obesity effects. Since dietary CLA are incorporated into membrane phospholipids, we have used Molecular Dynamics (MD) simulations to investigate the comparative effects of the two isomers on lipid bilayer structure. Specifically, simulations of phosphatidylcholine lipid bilayers in which the sn-2 chains contained one of the two isomers of CLA were performed. Force field parameters for the torsional potential of double bonds were obtained from ab initio calculations. From the MD trajectories we calculated and compared structural properties of the two lipid bilayers, including areas per molecule, density profiles, thickness of bilayers, tilt angle of tail chains, order parameters profiles, radial distribution function (RDF) and lateral pressure profiles. The main differences found between bilayers of the two CLA isomers, are (1) the order parameter profile for C9T11 has a dip in the middle of sn-2 chain while the profile for T10C12 has a deeper dip close to terminal of sn-2 chain, and (2) the lateral pressure profiles show differences between the two isomers. Our simulation results reveal localized physical structural differences between bilayers of the two CLA isomers that may contribute to different biological effects through differential interactions with membrane proteins or cholesterol.     

A database of 32 DNA triplets to study triple helices by molecular mechanics and dynamics

We present here a database of 32 deoxyribonucleotide triplets, that can be used as building blocks of triple helix forming deoxyribonucleotides on a computer. This database is made of all the pairing schemes of the triplets ATT, GCC⁺, ATA and GCG where the third base forms two hydrogen bonds with the purine of the first two Watson-Crick strands. The essential features of the known triple helices were preserved in the resulting structures. A triple helix can be easily built from any combination of these basic triplets. Four homogeneous and alternate triple helices thus obtained were studied by molecular mechanics and dynamics in vacuo. The results are in agreement with known experimental observations for ATT and suggest a possible structure for the GCG triple helix. In order to characterize the geometry of the structures obtained, the definitions of nucleic acid structure parameters (R.E. Dickerson et al., EMBO J. 8 (1989) 1–4) have been extended to triple helical polynucleotides.     

Ocular amphotericin B delivery by chitosan-modified nanostructured lipid carriers for fungal keratitis-targeted therapy

Abstract

Context: Fungal keratitis, a corneal fungal infection of the eye caused mainly by Candida species, has become the leading cause of blindness resulting from corneal disease in China. Present limitations in the management of ophthalmic fungal infections include the inability to provide long-term extraocular drug delivery without compromising intraocular structures and/or systemic drug exposure.

Objective: The aim of this study was to construct amphotericin B (AmB) loaded, chitosan-modified, nanostructured lipid carriers (AmB-CH-NLC) for prolonged ocular application and for the improvement of the targeted delivery of AmB to the ocular mucosa.

Materials and methods: The AmB-CH-NLC was produced by the method of emulsion evaporation-solidification at low temperature. The particle size, zeta potential, and encapsulation efficiency, drug-release behavior, and corneal penetration ability were performed in vitro and in vivo.

Results and discussion: The prepared AmB-CH-NLC nanoparticles exhibited a measured size of 185.4?nm, a zeta potential of 27.1?mV, and an entrapment efficiency of 90.9%. Sustained drug release behavior was observed in vitro. The in vivo ocular pharmacokinetic study indicated improved bioavailability of AmB-CH-NLC. The corneal penetration study showed that the AmB-CH-NLC could successfully penetrate into the cornea with no obvious irritation to the rabbits’ eyes.

Conclusion: The results support that this novel nanomedicine could be a promising system for effective ocular delivery of amphotericin B for fungal keratitis-targeted therapy.     

Cholesterol markedly reduces ion permeability induced by membrane-bound amphotericin B

It is widely accepted that amphotericin B (AmB) together with sterol makes a mixed molecular assemblage in phospholipid membrane. By adding AmB to lipids prior to preparation of large unilamellar vesicles (LUV), we directly measured the effect of cholesterol on assemblage formation by AmB without a step of drug's binding to phospholipid bilayers. Potassium ion flux assays based on ³¹P-nuclear magnetic resonance (NMR) clearly demonstrated that cholesterol markedly inhibits ion permeability induced by membrane-bound AmB. This could be accounted for by a membrane-thickening effect of cholesterol since AmB actions are known to be markedly affected by the thickness of membrane. Upon addition of AmB to an LUV suspension, the ion flux gradually increased with increasing molar ratios of cholesterol up to 20 mol%. These biphasic effects of cholesterol could be accounted for, at least in part, by the ordering effect of cholesterol.