Interaction between nystatin and natural membrane lipids in Langmuir monolayers--the role of a phospholipid in the mechanism of polyenes mode of action

Nystatin (NYS), a polyene antifungal antibiotic, has been investigated in Langmuir monolayers alone and in mixtures with mammalian and fungi membrane sterols (cholesterol and ergosterol, respectively) as well as with a model phospholipid (DPPC). The interactions between film molecules have been examined both in a qualitative and quantitative way with the excess area per molecule (AExc), excess free energy of mixing (DeltaGExc) and the interaction parameter (alpha). The obtained results have been compared with those previously reported for another polyene antimycotic: amphotericin B (AmB) mixed with lipids. Higher affinity of NYS has been observed for ergosterol vs. cholesterol, however, the strongest attractions were found for its mixtures with DPPC. The obtained results have been verified with biological studies reported previously for both antibiotics (NYS and AmB). A thorough analysis of the Langmuir experiment results performed for both polyenes enabled us to conclude that the presence of DPPC can be considered as a key factor affecting their antifungal activity as well as their toxicity towards host cells.     

Polyene antibiotics in the membrane environment.

The cytotoxic activity of the polyene antibiotics mainly depends on the appearance of the drug species which arises from drug-sterol complexation. The unsaturation and intact macrolide ring of the polyenes are the requirements for the biological activity. All the polyene antibiotics can form the complex with the sterol having 3 beta-OH group, and planar ring and a hydrophobic side chain. Aromatic polyene antibiotics with positively charged head group have been considered as most potential antifungal agents.     

Interactions between membrane sterols and phospholipids in model mammalian and fungi cellular membranes — A Langmuir monolayer study

This paper is aimed at investigating sterol/phospholipid interactions in the exact proportion that occurs in fungi/mammalian cells. We have performed a thorough analysis of surface pressure (π)–area (A) isotherms with the Langmuir monolayer technique, complemented with Brewster angle microscopy (BAM) images. The following mixtures were analysed: cholesterol (Chol)–dipalmitoyl phosphatidylcholine (DPPC), Chol–dioleoyl phosphatidylcholine (DOPC), ergosterol (Erg)–DPPC, and Erg–DOPC. For each system, two different concentrations of the sterols were used, 13 and 30%, corresponding to the range of concentration found in various natural membranes.The obtained results show the existence of attractive interactions between phospholipids and cholesterol. Mixtures with ergosterol behave quite differently, i.e. either the interactions are repulsive (mixtures with DPPC) or the system is ideal (mixtures with DOPC). The obtained results have implications in the polyene antibiotics mode of action, i.e. the polyenes may interact easier with ergosterol, present in fungi cells, as compared to cholesterol — the main sterol of the mammalian cellular membranes.     

Solution NMR structures of the polyene macrolide antibiotic filipin III

The solution structure of filipin III, an antifungal polyene macrolide biosynthesized by Streptomyces filipinensis and widely used for the detection and the quantitation of cholesterol in biomembranes, has been calculated with a set of geometrical restraints derived from 1H NMR in DMSO-d(6) at 25 degrees C. Filipin III appears as a rod-shaped molecule of 18 A length. Its amphiphilic structure is made of an all-syn 1,3-polyol motif, stabilized by intramolecular hydrogen bonds on one side, and a conjugated pentaene moiety on the other side of the molecule. The overall shape is comparable to cholesterol, and the molecular structure of filipin III affords a first molecular basis to the comprehensive understanding of the interactions possible in the filipin III-cholesterol complex which is still unknown at the atomic resolution.     

Investigation of polyene macrolide antibiotic-induced permeability changes in vesicles by 31P nuclear magnetic resonance

The permeability of egg yolk lecithin (EYL) vesicles to Pr3+ has been measured by 31P nuclear magnetic resonance (nmr) spectroscopy. Measurable Pr3+ leakage into the internal aqueous compartment of EYL vesicles at ambient (21 degrees C) temperature required the presence of small (7--10 mol%) amounts of dicetyl phosphate (DCP). The permeability of DCP-containing vesicles is decreased by incorporation of sterol (cholesterol greater than ergosterol approximately 5.6-dihydroergosterol greater than zymosterol) into the lipid bilayer. Addition of the polyene macrolide antibiotic, nystatin, to DCP-containing EYL vesicles with and without sterol resulted in increased Pr3+ permeability at the three temperatures studied (21--37.5 degrees C). Permeability changes observed upon addition of nystatin to sterol-impregnated, DCP-containing vesicles varied with sterol structure: ergosterol approximately 5,6-dihydroergosterol greater than cholesterol approximately zymosterol. These results are compared with other polyene macrolide induced permeability changes on model and natural membrane systems. Permeability changes induced by nystatin in sterol-free EYL vesicles were generally greater than for comparable sterol-containing vesicles. This is attributed to a nonspecific interaction of the antibiotic with the latter vesicles.     

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.     

Rates of amphotericin B and filipin association with sterols. A study of changes in sterol structure and phospholipid composition of vesicles

The influence of structural modifications in sterols and phospholipids on the rate of polyene antibiotic-sterol interaction was studied. For filipin and amphotericin B association with sterols in vesicles, a preferential interaction was found with sterols whose side chain length is close to that of cholesterol. Introduction of trans double bonds into the sterol side chain did not alter the rate of interaction in vesicles. The delta 7-bond of the sterol appears to be of critical importance in amphotericin B-sterol interaction, whereas the delta 5-bond is not essential. These observations are relevant to the well-known effects of amphotericin B on cell membranes containing ergosterol compared with those containing cholesterol. The dependence of the rates of sterol-polyene antibiotic interaction on the phospholipid composition of the vesicles indicates that phospholipid vesicles may be an inadequate model for reaching a comprehensive understanding of the effects exerted on biological membranes by these agents.     

Phosphatidylcholine liposomes containing cholesterol analogues with side chains of various lengths

The effect of the length of the side chain of sterols on their interaction with phosphatidylcholine was studied by measuring the permeability properties of liposomes constituted with sterol analogues with side chains of various lengths. The sensitivities of liposomes constituted with these sterol analogues toward digitonin and polyene antibiotics were also examined.The effects of sterols on phase transition of phosphatidylcholine were examined by measuring their effects on permeability increase due to perturbation of phase equilibrium and by differential scanning calorimetry. An analogue with a short side chain, isopropyl (C-22), had a very similar effect to cholesterol in suppressing the permeability increase, suggesting that the full length of the side chain is not necessary for this effect.The permeability of egg yolk phosphatidylcholine at 42°C was suppressed as much by the analogue C-22 as by cholesterol. Androstene-3-β-ol, an analogue without a side chain, however, had little suppressive effect. Thus it is concluded that the condensing effect of sterol requires a side chain, but not the full length of side chain.Liposomes constituted with analogues having a side chain with more than 5 carbon atoms showed maximum reactivity with a polyene antibiotic, amphotericin B, whereas those constituted with analogues having a side chain with less than 4 carbon atoms showed weaker reactivity. These findings indicate that a side chain with more than 5 carbon atoms is essential for the maximum interaction of liposomes with amphotericin B. Unlike amphotericin B, filipin reacted almost equally well with liposomes containing C-22 and with those containing cholesterol. Thus the chain length of the side chain of sterol is less important for interaction of liposomes with filipin than for their interaction with amphotericin B.Liposomes containing analogues having a side chain with more than 6 carbon atoms showed maximum reactivity with digitonin. Thus for the maximum interaction of liposomes with digitonin, the side chain of sterol should be longer than 6 carbon atoms.     

Dissociation kinetics and equilibrium binding properties of polyene antibiotic complexes with phosphatidylcholine/sterol vesicles

The interactions of sonicated vesicles with the polyene antibiotics amphotericin B, candicidin, mediocidin , and a water-soluble, guanidine derivative of amphotericin B were examined by UV-visible spectroscopy at concentrations below which the polyenes become self-associated. The association constants, Kapp, and the numbers of binding sites per sterol or phospholipid molecule (n) were determined at 30 degrees C and pH 7.4. A single class of binding sites was found, with no evidence of cooperativity. For the binding of mediocidin , amphotericin B, and the guanidine derivative with phosphatidylcholine (PC), PC/cholesterol, and PC/ergosterol vesicles, Kapp was in the range of (1.0-3.0) X 10(6) M-1; Kapp was higher for candicidin-vesicle interaction, reaching 9.0 X 10(6) M-1 with PC/ergosterol vesicles. Binding of the guanidine derivative of amphotericin B to PC vesicles lacking sterol was extensive (n = 0.46); since the other polyenes, which have low aqueous solubilities, had n less than 0.05, positive charges in the mycosamine moiety appear to enhance the extent of polyene antibiotic interaction with the glycerophospholipid head group. Higher values of n (and, therefore, of nKapp ) were found with sterol-containing than with sterol-free vesicles, suggestive of penetration of the polyenes toward the interior of the bilayer when sterol is present. For binding to PC/sterol vesicles, nKapp followed the order of candicidin greater than guanidine derivative of amphotericin B greater than amphotericin B much greater than mediocidin . The values of n and nKapp were appreciably higher for amphotericin B-ergosterol than for amphotericin B-cholesterol interaction in vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Potential-dependent formation of single conducting ion channels in lipid bilayers induced by the polyene antibiotic levorin A2

The aromatic polyene antibiotic levorin A₂ forms ion channels permeable to monovalent cations, in lipid membranes containing cholesterol or ergosterol. Channel conductivity is in the range 0.3–0.5 pS. The channel has two main states: conducting (open) and nonconducting (closed). The potential-dependent formation of levorin A₂ channels is observed in lipid membranes. The system responsible for the ion-channel selectivity is localized on the hydrophilic side of the lactone ring of the polyene molecule.     

Structural analysis of P450 AmphL from Streptomyces nodosus provides insights into substrate selectivity of polyene macrolide antibiotic biosynthetic P450s

AmphL is a cytochrome P450 enzyme that catalyzes the C8 oxidation of 8-deoxyamphotericin B to the polyene macrolide antibiotic, amphotericin B. To understand this substrate selectivity, we solved the crystal structure of AmphL to a resolution of 2.0 Å in complex with amphotericin B and performed molecular dynamics (MD) simulations. A detailed comparison with the closely related P450, PimD, which catalyzes the epoxidation of 4,5-desepoxypimaricin to the macrolide antibiotic, pimaricin, reveals key catalytic structural features responsible for stereo- and regio-selective oxidation. Both P450s have a similar access channel that runs parallel to the active site I helix over the surface of the heme. Molecular dynamics simulations of substrate binding reveal PimD can “pull” substrates further into the P450 access channel owing to additional electrostatic interactions between the protein and the carboxyl group attached to the hemiketal ring of 4,5-desepoxypimaricin. This substrate interaction is absent in AmphL although the additional substrate -OH groups in 8-deoxyamphotericin B help to correctly position the substrate for C8 oxidation. Simulations of the oxy-complex indicates that these -OH groups may also participate in a proton relay network required for O₂ activation as has been suggested for two other macrolide P450s, PimD and P450eryF. These findings provide experimentally testable models that can potentially contribute to a new generation of novel macrolide antibiotics with enhanced antifungal and/or antiprotozoal efficacy.     

Regio- and Stereospecificity of Filipin Hydroxylation Sites Revealed by Crystal Structures of Cytochrome P450 105P1 and 105D6 from Streptomyces avermitilis

The polyene macrolide antibiotic filipin is widely used as a probe for cholesterol and a diagnostic tool for type C Niemann-Pick disease. Two position-specific P450 enzymes are involved in the post-polyketide modification of filipin during its biosynthesis, thereby providing molecular diversity to the “filipin complex.” CYP105P1 and CYP105D6 from Streptomyces avermitilis, despite their high sequence similarities, catalyze filipin hydroxylation at different positions, C26 and C1′, respectively. Here, we determined the crystal structure of the CYP105P1-filipin I complex. The distal pocket of CYP105P1 has the second largest size among P450 hydroxylases that act on macrolide substrates. Compared with previously determined substrate-free structures, the FG helices showed significant closing motion on substrate binding. The long BC loop region adopts a unique extended conformation without a B′ helix. The binding site is essentially hydrophobic, but numerous water molecules are involved in recognizing the polyol side of the substrate. Therefore, the distal pocket of CYP105P1 provides a specific environment for the large filipin substrate to bind with its pro-S side of position C26 directed toward the heme iron. The ligand-free CYP105D6 structure was also determined. A small sub-pocket accommodating the long alkyl side chain of filipin I was observed in the CYP105P1 structure but was absent in the CYP105D6 structure, indicating that filipin cannot bind to CYP105D6 with a similar orientation due to steric hindrance. This observation can explain the strict regiospecificity of these enzymes.     

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.     

Potential-dependent formation of single conducting ion channels in lipid bilayers induced by the polyene antibiotic levorin A

The aromatic polyene antibiotic levorin A2 forms ion channels permeable to monovalent cations, in lipid membranes containing cholesterol or ergosterol. Channel conductivity is in the range 0.3-0.5 pS. The channel has two main states: conducting (open) and nonconducting (closed). The potential-dependent formation of levorin A2 channels is observed in lipid membranes. The system responsible for the ion-channel selectively is localized on the hydrophilic side of the lactone ring of the polyene molecule.     

Sterol and fatty acid composition of polyene macrolide antibiotic resistant Torulopsis glabrata.

Successive reculturing of Torulopsis glabrata on media containing increasing concentration of the polyene macrolide antibiotics nystalin or lucensomycin resulted in the segregation of cultures resistant to these antibiotics. Isolates resistant to lucensomycin showed good resistance to nystatin, and vice versa. Analysis of the sterols and fatty acids of sensitive and polyene resistant T. glabrata revealed that compositional changes occurred in both classes of lipids upon acquistion of resistance. The sterol composition of nystatin and lucensomycin resistant cultures possessed reduced amounts of, or no ergosterol (the major sterol of the sensitive parent culture), and increased amounts of sterols which were biogenetically more primitive than ergosterol. Resistant cultures in which ergosterol was absent possessed a fatty acid composition that did not differ significantly from the parent sensitive culture grown under identical conditions. Resistant cultures containing significantly reduced amounts of ergosterol were found to possess altered fatty acid compositions. Generally it was observed that these latter cultures possessed fatty acids containing shorter and more saturated chains. These results are considered to indicate that alteration in both lipid and sterol composition is involved in determination of culture resistance to polyene macrolides.     

Ergosterol in POPC membranes: physical properties and comparison with structurally similar sterols

The physical properties of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/ergosterol bilayers in the liquid-crystalline phase were determined using deuterium nuclear magnetic resonance (²H NMR) and vesicle extrusion. For the ²H NMR experiments, the sn-1 chain of POPC was perdeuterated, and spectra were taken as a function of ergosterol concentration and temperature. Analysis of the liquid-crystalline spectra provides clear evidence that two types of liquid-crystalline domains, neither of which is a liquid-ordered phase, having distinct average chain conformations coexist in 80:20 and 75:25 POPC/ergosterol membranes over a wide temperature range (from −2 to at least 31°C). Adding ergosterol to a concentration of 25 mol % increases POPC-d₃₁ chain ordering as measured by the NMR spectral first moment M₁ and also increases the membrane lysis tension, obtained from vesicle extrusion. Further addition of ergosterol had no effect on either chain order or lysis tension. This behavior is in marked contrast to the effect of cholesterol on POPC membranes: POPC/cholesterol membranes have a linear dependence of chain order on sterol concentration to at least 40 mol %. To investigate further we compared the dependence on sterol structure and concentration of the NMR spectra and lysis tension for several POPC/sterol membranes at 25°C. For all POPC/sterol membranes investigated in this study, we observed a universal linear relation between lysis tension and M₁. This suggests that changes in acyl chain ordering directly affect the tensile properties of the membrane.     

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.     

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.     

Solution NMR structure of five representative glycosylated polyene macrolide antibiotics with a sterol-dependent antifungal activity.

Glycosylated polyene macrolide antibiotics, as nystatins and amphotericins, are amphiphilic structures known to exert antifungal activity by disrupting the fungal cell membrane, leading to leakage of cellular materials, and cell death. This membrane disruption is strongly influenced by the presence and the exact nature of the membrane sterols. The solution structures of five representative glycosylated members, three tetraenes (pimaricin, nystatin A1 and rimocidin) and two heptaenes (candidin and vacidin A) have been calculated using geometric restraints derived from 1H-NMR data and random searches of their conformational space. Despite a different apparent structural order, the NMR solutions structure indicate that the hydroxyl groups all clustered on one side of the rod-shaped structures, and the glycosyl moieties are structurally conserved both in their conformation and their apparent order. The molecular structures afford an understanding of their selective interaction with the membrane sterols and the design of new polyene macrolides with improved activities.     

Composition and characteristics ofCandida albicans cells differing in resistance to polyene antibiotics

Development of resistance to polyene antibiotics in a highly resistantCandida albicans strain was shown to be accompanied by the complete loss of the ability to synthesize ergosterol and the substitution of other sterol components as well as by higher amounts of free fatty acids. No significant differences in lipid and protein composition have been noted between slightly resistant cultures ofC. albicans and initially susceptible ones. Sterols of resistant cultures (added in the solution and incorporated in the composition of native membranes and liposomes) have the same affinity for polyene antibiotics as do sterols of a sensitive strain. It was found that the resistance of the slightly resistantC. albicans strain did not depend on the cell wall. The ability of some detergents to reduce resistance to polyene antibiotics was shown.