Inhibition of erythrocyte ghost ATPase by polyene antibiotics

The effect of micromolar concentrations of polyene antibiotics on erythrocyte ghost ATPase activities has been studied. (Mg2+)-ATPase is inhibited by amphotericin B and amphotericin B methyl ester, whereas (Na+ + K+ + Mg2+)-ATPase is inhibited by amphotericin B and lucensomycin. (Ca2+ + Mg2+)-ATPase is only slightly affected by polyene antibiotics.     

The water and ionic permeability induced by polyene antibiotics across plasma membrane vesicles from Leishmania sp

An osmotic method has been used to study the effect of the polyene antibiotics amphotericin B, nystatin and candicidin on the water permeability of plasma membranes prepared from Leishmania sp. The effect of amphotericin B on the permeability of Leishmania membranes to a salt such as potassium nitrate was also investigated. A non-linear and saturable enhancement of water and salt permeability was measured with increasing polyene concentrations, which could be adjusted to Hill cooperativity equation. The antibiotic concentrations that induce at 30 degrees C half-maximal effects on the water permeability of Leishmania vesicles were 0.021 microM for candicidin, 0.21 microM for amphotericin B and 1.4 microM for nystatin. At 30 degrees C, the concentration of amphotericin B required to induce half of the maximal effect on the permeability of Leishmania vesicles to potassium nitrate was 1.8 microM. The temperature dependence for amphotericin B, nystatin and candicidin enhancement of the water permeability of Leishmania vesicles was determined by using Q10 data at 20 and 30 degrees C. The estimated activation energies at increasing polyene concentrations display the same general pattern for all three polyene antibiotics investigated, that is, a maximal positive value at about the polyene concentrations required for half-maximal effect. The significance of these results for understanding the mechanisms of action of polyene antibiotics on natural membranes is discussed.     

Effects of polyene antibiotics on the membranes of dog kidney isolated nuclei]

The effects of amphotericin B and nistatin on the membranes of dog kidney isolated nuclei after their incubation with the antibiotics in question, have been studied. It is found that the polyene antibiotics, though they are superficially-active compounds, have no solubilizing effect on nuclear membranes and do not change their chemical composition. Electrophoretic study has revealed that nuclear membrane proteins, besides high- and low-molecular protein components, also contain a large amount of histones. The incubation of the nuclei with the polyene antibiotics results in marked changes in the fractional composition of nuclear membrane proteins, the most significant changes being induced by amphotericin B. It is assumed that polyene antibiotics induce proteolytic degradation of nuclear membrane proteins.     

The synthesis and characterisation of polyene complexes with divalent metal ions: Mg(II), Ca(II), Ni(II), Cu(II) and Zn(II)

Metal ion (Mg(II), Ca(II), Zn(II), Cu(II), Ni(II)) complexes of nystatin and amphotericin B (polyene antibiotics) have been prepared as solids. The stoichiometry of the complexes has been established. IR, ESR investigation indicates the metal-ligating sites in the polyene molecules. The existence of such complexes is discussed in the light of polyene mode- of-action theories.     

How do the polyene macrolide antibiotics affect the cellular membrane properties?

In the 1970's great strides were made in understanding the mechanism of action of amphotericin B and nystatin: the formation of transmembrane pores was clearly demonstrated in planar lipid monolayers, in multilamellar phospholipid vesicles and in Acholeplasma laidlawii cells and the importance of the presence and of the nature of the membrane sterol was analyzed. For polyene antibiotics with shorter chains, a mechanism of membrane disruption was proposed. However, recently obtained data on unilamellar vesicles have complicated the situation. It has been shown that: membranes in the gel state (which is not common in cells), even if they do not contain sterols may be made permeable by polyene antibiotics, several mechanisms may operate, simultaneously or sequentially, depending on the antibiotic/lipid ratio, the time elapsed after mixing and the mode of addition of the antibiotic, there is a rapid exchange of the antibiotic molecules between the vesicles. Although pore formation is apparently involved in the toxicity of amphotericin B and nystatin, it is not the sole factor which contributes to cell death, since K+ leakage induced by these antibiotics is separate from their lethal action. The peroxidation of membrane lipids, which has been demonstrated for erythrocytes and Candida albicans cells in the presence of amphotericin B, may play a determining role in toxicity concurrently with colloid osmotic effect. On the other hand, it has been shown that the action of polyene antibiotics on cells is not always detrimental: at sub-lethal concentrations these drugs stimulate either the activity of some membrane enzymes or cellular metabolism. In particular, some cells of the immune system are stimulated. Furthermore, polyene antibiotics may act synergistically with other drugs, such as antitumor or antifungal compounds. This may occur either by an increased incorporation of the drug, under the influence of a polyene antibiotic-induced change of membrane potential, for example, or by a direct interaction of both drugs. That fungal membranes contain ergosterol while mammalian cell membranes contain cholesterol, has generally been considered the basis for the selective toxicity of amphotericin B and nystatin for fungi. Actually, in vitro studies have not always borne out this assumption, thereby casting doubt on the use of polyene antibiotics as antifungal agents in mammalian cell culture media.(ABSTRACT TRUNCATED AT 400 WORDS)     

The change of template activity of dog kidney chromtin by polyene antibiotics in vivo and in vitro]

Effect of amphotericin B and nistatin on template activity of nuclear membrane-bound (DNPm) and free (DNPo) dog kidney chromatin after intravenous injections of antibiotics and after the incubation of isolated kidney cell nuclei with the antibiotics is studied. It is found that injections of amphotericin B and nistatin resulted in the increase of DNPo template activity in RNA polymerase system, the stimulating effect of nistatin being higher than that of amphotericin B. Injections of nistatine stimulated also template activity of DNPm, while amphotericin B produced no effect on DNPm. When studing the effect of polyene antibiotics on template activity of DNPo and DNPm in vitro, it is found that the intensity of RNA synthesis after incubation of isolated nuclei with antibiotics is considerably increased, and stimulating effect of nistatin is higher than of amphotericin B. Both antibiotics produced no effect on template activity of DNP in vitro. Thus, comparative analysis of changes in template activity of dog kidney chromatin under the effect of polyene antibiotics in vivo and in vitro has revealed the similarity of these drugs and draws to the conclusion that nistatin and amphotericin B produce a direct effect on template activity of chromatin.     

Lipid-based antifungal agents: a concise overview

The development of lipid formulations of antifungal drugs has been a remarkable progress in the systemic antifungal arena. The lipid-based amphotericin B formulations; amphotericin B lipid complex (ABLC), amphotericin B colloidal dispersion (ABCD), and liposomal amphotericin B (L-AMB) have been in clinical use since the 1990s. They are significantly less nephrotoxic than the parent compound and can be safely used at higher doses. The primary cost of these formulations is significantly high and the extent of data related to their head-to-head comparison remains limited. The lipid formulation of nystatin, liposomal nystatin, is another lipid-based polyene under development. Available data concerning the in vitro activity, pharmacokinetic profile, in vivo efficacy, and safety of these formulations are summarized in this overview.     

Polyene antibiotic action on lecithin liposomes: effect of cholesterol and fatty acyl chains

The effect of cholesterol incorporation upon amphotericin B and nystatin susceptibility of lecithin liposome systems containing various fatty acids has been studied. Cholesterol was shown to: 1) confer sensitivity to low concentrations of amphotericin B in liposomes derived from egg lecithin, and 2) suppress the amphotericin B and nystatin-induced response in liposomes derived from dipalmitoyl or distearoyl lecithins. This clear cut difference cannot be explained by mechanisms of drug action so far presented. They are discussed in connection with the possibility that susceptibility to these polyene antibiotics is related to the over-all state of the membrane organization, in particular to the over-all conformation of membrane components.     

The Effect of Polyene Antibiotics on Photosynthetic Electron Transfer

The effect of four polyene antibiotics and digitonin on photosyntheticelectron transfer by maize mesophyll chloroplasts has been investigated.All five compounds, at concentrations between 0.1 mM and 20mM, inhibited photosystem 2 activity as measured by the photo-reductionof ferricyanide and 2,6-dichlorophenolindophenol from water.Etruscomycin, amphotericin B, and digitonin were more inhibitorythan filipin and nystatin. Photosystem 1 activity was inhibitedby 1 mM concentrations of etruscomycin, amphotericin B, anddigitonin but not by filipin and nystatin. In all cases whereinhibition occurred, it was temperature dependent. The inhibition of photosystem 1 activity could be relieved byplastocyanin. Etruscomycin and digitonin, at concentrationsof 0.5 mM and above, caused disintegration of the chloroplasts,and this disintegration was accompanied by a two- to three-foldincrease in photosystem 1 activity in the presence of plastocyanin.It is concluded that the action of polyene antibiotics resultsin the release of plastocyanin from its site in the photosyntheticelectron transfer chain. The results are discussed in termsof the abilities of polyene antibiotics and digitonin to formcomplexes with sterols.     

Synthetic studies of polyene macrolide antibiotics. 1. Synthesis of C1-C6- and C7-C12 fragments of amphotericin B from carbohydrate precursors

Strategy of synthesis of polyene macrolide antibiotic amphotericin B using carbohydrates as starting material is discussed. The C1-C6 and C7-C12 fragments of amphotericin B are synthesized.     

Interaction of the polyene antibiotics with lipid bilayer vesicles containing cholesterol

The interaction of the polyene antibiotics, amphotericin B, nystatin and filipin with cholesterol-containing single bilayer lipid vesicles has been characterized using gel permeation chromatography and proton magnetic resonance. All three antibiotics bind to vesicles at low concentrations without causing a large amount of vesicle destruction. The strength of binding as determined by gel permeation studies is greater for filipin and amphotericin than for nystatin. Nystatin and amphotericin B at these low concentrations induce a rapid loss of internal vesicle contents consistent with pore formation. Filipin induces no leakage beyond that expected from partial vesicle destruction or general detergent action.At antibiotic levels above 1 : 1 antibiotic : cholesterol ratios the NMR results show all three antibiotics to cause extensive vesicle destruction. The onset of this behavior, which appears to be independent of the total antibiotic concentration, indicates a well defined antibiotic : cholesterol interaction stoichiometry. Despite the fact that cholesterol is required for antibiotic activity, the NMR spectra prior to vesicle destruction show no changes indicative of an antibiotic-induced reversal of cholesterol restriction of phosphatidylcholine mobility. The contrast with polyene antibiotic behavior in more extended bilayers is discussed.     

Interaction of the polyene antibiotics with lipid bilayer vesicles containing cholesterol.

The interaction of the polyene antibiotics, amphotericin B, nystatin and filipin with cholesterol-containing single bilayer lipid vesicles has been characterized using gel permeation chromatography and proton magnetic resonance. All three antibiotics bind to vesicles at low concentrations without causing a large amount of vesicle destruction. The strength of binding as determined by gel permeation studies is greater for filipin and amphotericin than for nystatin. Nystatin and amphotericin B at these low concentrations induce a rapid loss of internal vesicle contents consistents consistent with pore formation. Filipin induces no leakage beyond that expected from partial vesicle destruction or general detergent action. At antibiotic levels above 1:1 antibiotic: cholesterol ratios the NMR results show all three antibiotics to cause extensive vesicle destruction. The onset of this behavior, which appears to be independent of the total antibiotic concentraion, indicates a well defined antibiotic : cholesterol interaction stoichiometry. Despite the fact that cholesterol is required for antibiotic activity, the NMR spectra prior to vesicle destruction show no changes indicative of an antibiotic-induced reversal of cholesterol restriction of phosphatidylcholine mobility. The contrast with polyene antibiotic behavior in more extended bilayers is discussed.     

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.     

Nitroxide spin-probe study of amphotericin B-ergosterol interaction in egg phosphatidylcholine multilayers

The interaction between the polyene macrolide antibiotic, amphotericin B, and ergosterol in egg phosphatidylcholine multilayers was investigated using head group and acyl chain nitroxide spin-labelled phosphatidylcholine as probes. At physiological concentrations of less than 15 mol% sterol in egg phosphatidylcholine multilayers amphotericin B accumulates near the head group region until an amphotericin B : ergosterol ratio of approximately 0.7 is achieved. As the proportion of amphotericin B is increased above this value, formation of an acyl chain disordering complex occurs which has an approximate antibiotic:sterol ratio of unity. Dicetyl phosphate was used to increase the solubility of ergosterol past its normal limit in pure egg phosphatidylcholine (approximately 15 mol%). At concentrations of ergosterol higher than 15 mol% a complex of two ergosterol molecules and one amphotericin B was postulated when there was insufficient antibiotic to form a 1:1 complex.     

Influence of net charge on the aggregation and solubility behaviour of amphotericin B and its derivatives in aqueous media

The poor solubility of polyene antibiotics in aqueous media limits their application in the therapy of systemic fungal infections. In the present paper we have demonstrated that the ionic state (net electrical charge) of the antibiotic molecule is an important factor in determining the aggregation and solubility properties of amphotericin B and its derivatives. A multi-step model of polyene self-association in aqueous media has been proposed as an explanation for the fact that some major differences are observed when aggregation is monitored by different techniques. Offprint requests to: J. Mazerski     

Growth characteristics and polyene sensitivity of a fatty acid auxotroph of Candida albicans.

A fatty acid auxotroph of Candida albicans 6406, designated A' 44 and originally isolated as an oleic acid requiring strain, has been shown to be a delta9 desaturase mutant. Although lacking this step in fatty acid biosynthesis, it appears to retain the ability to desaturate monounsaturated fatty acids. The polyene sensitivity of the organism grown on different fatty acid supplements varied between 0-08 +/- 0-02 and 1-20 +/- 0-30 microgram amphotericin B methyl ester ml-1 for exponentially growing cells. In spite of this variation, the sterol composition remained fairly constant, the major differences lying in fatty acid composition. Stationary-phase cells were more resistant to amphotericin B methyl ester, although again this change was not associated with changes in sterol content. The organism was most resistant when grown in the presence of oleic or linoleic acid. Protoplasts derived from resistant organisms grown on these two fatty acids were also resistant, indicating that the structure of the cell wall was less important than that of the plasma membrane in determining polyene sensitivity under these conditions.     

Polyene macrolide antibiotic cytotoxicity and membrane permeability alterations I. Comparative effects of four classes of polyene macrolides on mammalian cells

The relationship between polyene macrolide-induced early membrane damage and cytotoxicity in B1 (hamster), B82 (mouse), and RAG (mouse) cells has been investigated. Filipin (FIL) induced the greatest immediate damage, as monitored by ⁵¹Cr release, followed by mediocidin (MED), amphotericin B-deoxycholate (Fungizone®) (FZ) and pimaricin (PIM). For long term effect, PIM was the least toxic followed by MED, FZ, and FIL as indicated by 24-hour survival, 72-hour viability, and growth rate of cells. In evaluating polyene macrolide-induced permeability alterations and cytotoxicity two types of interactions with mammalian cells were found: (1) cell toxicity at polyene macrolide levels not eliciting immediate membrane permeability changes; and (2) immediate membrane damage without long range toxicity.     

Interaction of local anesthetics with bilayer phospholipid membranes

Local anesthetics--cocaine, lidocaine, novocaine were tested for conductivity of bilayer lipid membranes containing anion-selective channels formed by polyene antibiotic amphotericin B. It has been shown that 5 X 10(-4) M cocaine doubles the membrane conductance. The line of efficiency of the tested anesthetics is: cocaine greater than lidocaine greater than novocaine. Possible molecular mechanism of the discovered effect is discussed.     

Molecular Modeling Studies on Amphotericin B and its Complex with Phospholipid

Abstract

Molecular dynamics simulation studies on polyene antifungal antibiotic amphotericin B, its head-to-tail dimeric structure and lipid - amphotericin B complex demonstrate interesting features of the flexibilities within the molecule and define the optimal interactions for the formation of a stable dimeric structure and complex with phospholipid.     

The site of inhibition of photosynthetic electron transfer by amphotericin B.

The inhibitory effect of the polyene antibiotic, amphotericin B, on photosynthetic electron transfer has been investigated. Treatment of chloroplasts with the inhibitor results in the release of plastocyanin from its site in the chloroplast membrane. This release is accompanied by a shift in the pH curve for ferricyanide photoreduction from water, which is similar to that observed when chloroplasts are treated by sonication or passage through a French press. Delayed light emission from photosystem 2 is not destroyed by amphotericin B treatment, indicating that photosystem 2 is not damaged. Amphotericin B does not inhibit photoreduction of ferricyanide from water by chloroplast preparations which are deficient in plastocyanin, such as maize bundle-sheath chloroplast fragments, Euglena chloroplasts, or maize mesophyll chloroplasts passed through a French press. Chloroplasts treated with amphotericin B are not able to photooxidize plastocyanin. This result demonstrates that little structural damage occurs to the membrane during treatment with the antibiotic as a capacity to photooxidize plastocyanin is observed only in damaged chloroplast membranes.