Amphotericin B channel conductance inactivation

Effects induced in bilayer lipid membranes by amphotericin B and its alkyl derivatives was analysed. Inactivation of the antibiotic-dependent multichannel membrane conductance was discovered. Kinetics of membrane conductivity was shown to depend on the antibiotic concentration in the membrane. At concentrations between 10(-8) and 10(-7) M, the resulting conductance appeared to the transient. We suggest that the phenomenon of biphasic kinetics of membrane conductance is the result of a consecutive transformation of polyene channels in the membrane: half-pores are assembled on either side of membrane-nonconducting 1; two half-pores combine to build up a conducting channels-conducting 2, and the conducting channels are disassemled to monomers and nonconducting self-associated forms inside the membrane-disassembled state (nonconducting 3). To explain the transient characteristics of the induced conductance, it is proposed that the antibiotic, present in the solution under self-associated form, binds the membrane and forms pores, then dissociates in the bilayer in a non-active monomeric form. The existence of definite monomers and nonconducting self-associated forms of amphotericin B molecules inside the membrane was estimated from the dependence of kinetic conductance of lipid membranes of amphotericin B and its alkyl derivatives, when the antibiotics are washed out from aqueous medium. Equilibrium between different antibiotic assemblies inside the membrane was demonstrated by the kinetics of conductance decrease following washing the antibiotic. Using circular dichroism measurements, we observed that amphotericin B alkyl derivatives were in self-associated form being susceptible to form pores across cholesterol-containing membranes. The phenomenon of biophasic kinetics was observed only in the cholesterol-containing membrane. The substitution of membrane cholesterol for ergosterol provides monotonic kinetics of membrane conductance at any antibiotic concentration.     

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.     

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.     

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.     

Molecular modelling of membrane activity of amphotericin B, a polyene macrolide antifungal antibiotic

Amphotericin B (AmB) is a well known polyene macrolide antibiotic used to treat systemic fungal infections. Despite its toxicity AmB is still regarded as a life-saving drug. The lack of adequate knowledge of the AmB mechanism of action is a serious obstacle to efficient development of new less toxic derivatives. Complementary to various experimental approaches, computational chemistry methods were used to study AmB mechanism of action. A programme lasting for a decade, that was run by our group covered studies of: i) molecular properties of AmB and its membrane targets, ii) structure and properties of AmB membrane channels, and iii) interaction of AmB with the membrane.     

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.     

Interactions of amphotericin B derivative of low toxicity with biological membrane components--the Langmuir monolayer approach

Amphotericin B (AmB)--a polyene macrolide antibiotic--exhibits strong antifungal activity, however, is known to be very toxic to mammalian cells. In order to decrease AmB toxicity, a number of its derivatives have been synthesized. Basing on in vitro and in vivo research, it was evidenced that one of AmB derivatives, namely N-methyl-N-D-fructopyranosylamphotericin B methyl ester (in short MF-AME) retained most of the antifungal activity of the parent antibiotic, however, exhibited dramatically lower animal toxicity. Therefore, MF-AME seems to be a very promising modification product of AmB. However, further development of this derivative as potential new antifungal drug requires the elucidation of its molecular mechanism of reduced toxicity, which was the aim of the present investigations. Our studies were based on examining the binding energies by determining the strength of interaction between MF-AME and membrane sterols (ergosterol-fungi sterol, and cholesterol-mammalian sterol) and DPPC (model membrane phospholipid) using the Langmuir monolayer technique, which serves as a model of cellular membrane. Our results revealed that at low concentration the affinity of MF-AME to ergosterol is considerably stronger as compared to cholesterol, which correlates with the improved selective toxicity of this drug. It is of importance that the presence of phospholipids is essential since--due to very strong interactions between MF-AME and DPPC--the antibiotic used in higher concentration is "immobilized" by DPPC molecules, which reduces the concentration of free antibiotic, thus enabling it to selectively interact with both sterols.     

An aromatic heptaene antibiotic levorin and its derivatives in muscle activity]

The review is concerned with the outlooks for the use of levorin, a membrane active and channel forming polyene antibiotic, and its alkyl derivatives in muscle activity. In complex with cholesterol and ergosterol, the aromatic heptaene antibiotic levorin forms structural ionic channels of the molecular size in the lipid and cell membranes. Levorin increases the membrane permeability for monosucrose and other neutral molecules as follows: H2O > urea > acetamide > glycerine > ribose > arabinose > glucose > saccharose. As a channel forming compound, levorin is able to induce in the cell membranes of the muscle fibres formation of additional channels permeable for the cations and to increase the flow of the energy dependent substrates to the cells and the outburst of the metabolites from them during intensive muscle activity. Levorin several times decreases the surface tension of aqueous solutions. In some models of experimental animals levorin promoted an increase of the blood fluidity and accelerated the blood stream in the blood vessels both in rest and in muscle activity. Physical load in a high power zone increases the intensity of lipid peroxidation that results in fatigue and lower physical efficiency. Possible prevention of an increase of the rate of free radical reactions by levorin and its alkyl derivatives providing higher antioxidant protection is discussed.     

N-Methyl-N-D-fructosyl amphotericin B methyl ester (MF-AME), a novel antifungal agent of low toxicity: monomer/micelle control over selective toxicity

Rational chemical modification of amphotericin B (AMB) led to the synthesis of sterically hindered AMB derivatives. The selected optimal compound, N-methyl-N-D-fructosyl amphotericin B methyl ester (MF-AME) retains the broad spectrum of antifungal activity of the parent antibiotic, and exhibits a two orders of magnitude lower toxicity in vivo and in vitro against mammalian cells. Comparative studies of MF-AME and AMB comprising the determination of the spectroscopic properties of monomeric and self-associated forms of the antibiotics, the investigation of the influence of self-association on toxicity to human red blood cells, and of the antibiotic-sterol interaction were performed. On the basis of the results obtained it can be assumed that the improvement of the selective toxicity of MF-AME could in part be a consequence of the diminished concentration of water soluble oligomers in aqueous medium, and the better ability to differentiate between cholesterol and ergosterol.     

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.     

Effects of new amphotericin analogues on the scrapie isoform of the prion protein

Prion diseases or Transmissible Spongiform Encephalopathies (TSEs) are a group of neurodegenerative disorders associated with the conversion of a normal host prion protein (PrP(C)) into a pathogenic isoform (PrP(Sc)). Despite years of research, there is still no known cure for TSEs. Amphotericin B (AmB), an anti-fungal antibiotic, has antiprion activity but its usage is limited by its toxicity. This study assessed the antiprion properties of new amphotericin analogues in which the exocyclic carboxyl groups were replaced by methyl groups. These analogues reduced levels of the abnormal PrP(Sc) isoform of the mouse prion protein in cultured cells. 16-descarboxyl-16-methyl-amphotericin B (16B) had antiprion activity equivalent to that of amphotericin B and was significantly less toxic to cells as determined by a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide dye reduction assay. A non-anti-fungal analogue, 16-descarboxyl-16-methyl-19-O-(6-deoxyhexosyl)-19-O-desmycosaminyl-amphotericin (16-19B) had higher antiprion activity and significantly lower toxicity than AmB. Some of the new amphotericin analogues may have potential as antiprion drugs.     

Activity and selectivity of histidine-containing lytic peptides to antibiotic-resistant bacteria

Lytic peptides are a group of membrane-acting peptides that are active to antibiotic-resistant bacteria but demonstrate high toxicity to tissue cells. Here, we reported the construction of new lytic peptide derivatives through the replacement of corresponding lysine/arginine residues in lytic peptide templates with histidines. Resulting lytic peptides had the same lethality to antibiotic-resistant bacteria, including methicillin-resistant Staphylococcus aureus, but showed greatly improved selectivity to bacteria. When incubated with co-cultured bacteria and tissue cells, these histidine-containing lytic peptide derivatives killed bacteria selectively but spared co-cultured human cells. Membrane insertion and peptide-quenching studies revealed that histidine protonation controlled peptide interactions with cell membranes determined the bacterial selectivity of lytic peptide derivatives. Compared with parent peptides, lytic peptide derivatives bound to bacteria strongly and inserted deeply into the bacterial cell membrane. Therefore, histidine-containing lytic peptides represent a new group of antimicrobial peptides for bacterial infections in which the antibiotic resistance has developed.     

Aminoglycoside antibiotic derivatives: preparation and evaluation of toxicity on cochlea and vestibular tissues and antimicrobial activity

Aminoglycoside antibiotic derivatives such as neamine, methyl neobiosaminide B, 2-deoxystreptamine, tetra-azidoneamine, tetra-N-acetylneamine, tetra-N-carboxy-benzylneamine, tetra-N-carboxy-methylneamine and tetra-p-methoxy-benzyliminoneamine were prepared and evaluated as to their cochlear and vestibular toxicity. Methyl neobiosaminide B, the most promising derivative in the series showed selective, cochlea-dissociated vestibulotoxic activity and was considered to be a potential lead compound for the treatment of Ménière's disease. Antimicrobial properties of the compounds, qualitatively evaluated against a group of pathogenic bacteria, indicated that neomycin B sulfate, neamine as a free base and methyl-neobiosaminide B dihydrochloride show a broader range of activity when compared to the other derivatives.     

Modulation of polyene antibiotics self-association by ions from the Hofmeister series

The toxicity of the antifungal polyene antibiotic amphotericin B (AMB) has been related to its low solubility, more specifically to a self-associated form termed toxic aggregate. In addition, AMB in aqueous medium gives rise to concentration, ionic strength, and time-dependent polydisperse systems. For this reason different approaches, including the use of several lipid aggregates, have been used in attempts to improve the drug's solubility and increase its therapeutic index. In this context, understanding AMB's self-association properties should help in the preparation of less toxic formulations. Ions from the Hofmeister series alter water properties: while kosmotropes (water structure makers-sulfate, citrate, phosphate) decrease solute solubility, chaotropes (water structure breakers-perchlorate, thiocyanate, trichloroacetate, and the neutral molecule urea) have opposite effects. This work reports a study of the effect of Hofmeister ions and urea on the self-aggregation of AMB and some of its derivatives. Optical absorption and circular dichroism spectra were used to monitor monomeric and aggregated antibiotic. While kosmotropes increased aggregation in a concentration-dependent manner, the opposite was observed for chaotropes. It is shown, for the first time, that thiocyanate and trichloroacetate can induce complete AMB monomerization. The understanding of these processes at the physicochemical and molecular levels and the possibility of modulating the aggregation state of AMB and its derivatives should contribute to elucidate the mechanisms of action and toxicity of this widely used antibiotic and to develop more efficient and less toxic preparations.     

One-sided action of amphotericin B on cholesterol-containing membranes is determined by its self-association in the medium

The inducement of K+ permeability through membranes by the polyene antibiotic amphotericin B (AmB) has been analyzed as a measure of the antibiotic activity. Dose-response curves have been obtained with cholesterol- and ergosterol-containing egg yolk phosphatidylcholine large unilamellar vesicles (LUVs), human erythrocytes, and Saccharomyces cerevisiae cells. Conductance changes induced by AmB in sterol-containing planar bilayer membranes have also been studied. AmB self-association in aqueous buffer was determined by circular dichroism (CD) as a function of the antibiotic concentration. Electronic absorption and CD spectra of AmB were recorded in the presence of LUVs. For given AmB concentrations, the extent of permeability inducement is dependent on the lipid concentration. On the other hand, for cholesterol-containing LUVs or erythrocytes, a critical AmB concentration had to be reached before any permeability is observed. Independent of lipid concentration, this concentration was directly related to antibiotic self-association in the aqueous buffer. The same observation was made for erythrocytes and nystatin. The AmB absorption and CD spectra were totally different for ergosterol- and cholesterol-containing LUVs. Formation of single channels by one-sided addition of AmB could be observed only in ergosterol-containing membranes. These data lead us to propose that the permeability pathways induced by amphotericin B or nystatin, in ergosterol- and in cholesterol-containing membranes, are of different natures. In the latter case the antibiotics are only active, by single-sided addition, in the self-associated form. These findings offer important clues for the design of less toxic derivatives of AmB: they should have a low degree of self-association in water.     

The role of the carboxyl and amino groups of polyene macrolides in their interactions with sterols and their selective toxicity. A P-NMR study

The permeability induced by amphotericin B and vacidin A derivatives in large unilamellar lipidic vesicles containing various sterols has been studied using the proton-cation exchange method and ³¹P-NMR spectroscopy. Derivatives which have a free ionizable carboxyl group induce biphasic ‘all or none’ permeability typical of channel-forming ionophores, whatever the sterol present. In sterol-free membranes, they have no significant activity. Derivatives which lack a free ionizable carboxyl group exhibit this channel-like mode of action only in membranes containing ergosterol or sterols with an alkyl side like that of ergosterol. In membranes containing cholesterol or sterol whose side-chain is alike, a slow and progressive permeability is observed at high concentrations. This activity is observed in sterol-free membranes as well. Derivatives containing sugars with substituted amino groups always have lower ionophoric activity than those which are unsubstituted. The greatest decrease in activity was observed for N-acetyl derivatives. Substitution of the amino groups has no effect on the mode of action. A model of interaction of polyenes with sterols is presented accounting for the data obtained on vesicles and the observed selective toxicity of polyene derivatives in biological membranes.     

Amphotericin B channels in the bacterial membrane: role of sterol and temperature

Amphotericin B is an antibiotic that forms ion channels in the membrane of a host cell. The change in permeability produced by these channels is greatly improved by sterols; nevertheless, the single channel conductivity remains invariant. Hence, it is proposed that sterols do not act directly, but rather through the modulation of the membrane phase. We look at the formation of these channels in the bacterial membrane to determine the mechanism of its known antibiotic resistance. We found that channels can indeed be formed in this membrane, but a substantial amount of amphotericin B is required. We also study the effects of the antibiotic concentration needed for channel expression as well as the dynamics of channels affected by both sterol and temperature in phosphatidylcholine membranes. The results support the idea that membrane structure is a determining factor in the action of the antibiotic.     

Chemical modification of an antineoplastic antibiotic bleomycetin by the C-end fragment]

Bleomycetin, an antitumor antibiotic, was subjected to chemical modification by the C-end fragment i.e. the residue of 3-[(4-aminobutyl)amino]propylamine (spermidine++) with acylation, carbamoylation and reducing alkylation, which yielded its new semisynthetic derivatives. The use of physicochemical methods showed that the chemical modification involved the primary and secondary amino groups++ of spermidine++ and gave rise to N,N'-diacyl, N,N'-dicarbamoyl and N,N'-dialkyl bleomycetins. The biological properties of the derivatives, i.e. their cytotoxic activity, acute and pulmonary toxicities were studied. The transformation of bleomycetin by the C-end fragment lowered the antibiotic toxicity and was believed to be a promising approach to modifying its molecule.     

How do ionic channel properties depend on the structure of polyene antibiotic molecules?

A study has been made of the properties of ionic channels formed in phospholipid-cholesterol bilayers by polyene antibiotics of various molecular structures. Properties of channels created by natural antibiotics with different structures of the lactone ring (amphotericin B-nystatin-mycoheptin) as well as by some derivatives of amphotericin B modified with respect to the amino and carboxyl groups are compared. Neutralization of one or both charges of the amphotericin B molecule (both by chemical modification and by pH shift) increases the probability of the channel to be in a nonconducting state. An increase of cholesterol concentration in the membrane produces an opposite effect. It is assumed that the electrostatic interaction of the amino group of an antibiotic molecule with the carboxyl group of an adjacent one stabilized the channel. Conductance and selectivity of an open channel are not influenced by changes in the charged groups. These properties strongly depend on the structure of the polar chain of the lactone ring. For example, the appearance of one more carbonyl group in the mycoheptin molecule results in a sharply decreasing anion permeability of channels. An antibiotic concentration which is necessary to observe single channels depends on the polyene chain structure: this is about 10(-7) M for tetraene nystatin and 2.10(-8) M for heptaene amphotericin B an mycoheptin.