Synthesis and biological evaluation of non-polyene analogs of amphotericin B

Synthesis of the first analog of a polyene macrolide antibiotic containing a rigid, non-polyene backbone has been accomplished. The sterol recognition surface of amphotericin B has been modified in an effort to better understand the role of the polyene backbone. Its antifungal activity is reduced significantly compared with amphotericin B.     

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.     

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.     

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.     

Effects of lipid constituents on membrane-permeabilizing activity of amphidinols

Amphidinols (AMs) are a new class of polyhydroxyl polyene compounds with potent antifungal activity. Membrane-permeabilizing activities of AM2, AM3, and AM6 were examined using fluorescent-dye leakage experiments with various phosphatidylcholines (PCs) and sterols. All the AMs tested showed the potent activity to cholesterol-containing liposomes. In the absence of the sterol, AM2, AM3, and AM6 had no membrane-permeabilizing activities to membranes of saturated PC. In liposomes consisting of unsaturated PC, AM2, which possesses an additional ether ring in a polyhydroxyl chain, showed membrane-permeabilizing activities with a moderate efficacy, while AM3 or AM6 did not. The potentiation by sterols was prominent even at 0.5% (wt/wt) and structure-dependent, which ruled out the possibility that alteration of the membrane physical properties induced by sterol was chiefly responsible for this sterol effect. The finding that their activity was not affected by membrane thickness implies that AMs permeabilized membrane by a different mechanism from that of polyene macrolide antibiotics.     

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.     

Roflamycoin--a new channel-forming antibiotic

Ion permeability of lipid bilayers was studied in the presence of a new antifungal pentaene antibiotic, roflamycoin, the structure of which differs considerably from that of the well-known polyene channel-former amphotericin B. Both of them, however, show the property of increasing the membrane permeability only in the case of sterol-containing membrane when added on both its sides. The conductance is strongly dependent on the concentration of the antibiotic in the solutions and of sterol in the membrane. Unlike the amphotericin B channels, roflamycoin channels are potential-dependent and have short lifetime (approx. 1 s) and high conductance (approx. 100 ps in 1 M KCl), which increases linearly with the salt concentration and is not blocked by the familiar blockers of amphotericin B channels. The two antibiotics seem to have a common mechanism of channel formation, viz. the formation starts from two semi-pores assembled in the opposite monolayers from several molecules of the antibiotic and sterol. However, the inner diameter of the roflamycoin channel is larger because of the different antibiotic-to-sterol ratio in the channel aggregate. It is believed that the difference in the ratio is due to the presence of the methyl group in the polyene chain of roflamycoin, and the considerable difference in lifetimes of the two types of channels depends on the terminal groups of the antibiotics.     

Roflamycoin — a new channel-forming antibiotic

Ion permeability of lipid bilayers was studied in the presence of a new antifungal pentaene antibiotic, roflamycoin, the structure of which differs of which differs considerably from that of the well-known polyene channel-former amphotericin B. Both of them, however, show the property of increasing the membrane permeability only in the case of sterol-containing membrane when added on both its sides. The conductance is strongly dependent on the concentration of the antibiotic in the solutions and of sterol in the membrane. Unlike the amphotericin B channels, roflamycoin channels are potential-dependent and have short lifetime (approx. 1 s) and high conductance (approx. 100 ps in 1 M KCl), which increases linearly with the salt concentration and is not blocked by the familiar blockers of amphotericin B channels. The two antibiotics seem to have a common mechanism of channel formation, viz. the formation starts from two semi-pores assembled in the opposite monolayers from several molecules of the antibiotic and sterol. However, the inner diameter of the roflamycoin channel is larger because of the different antibiotic-to-sterol ratio in the channel aggregate. It is believed that the difference in the ratio is due to the presence of the methyl group in the polyene chain of roflamycoin, and the considerable difference in lifetimes of the two types of channels depends on the terminal groups of the antibiotics.     

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.     

Ultrastructural alterations induced by Amphotericin B in the plasma membrane of Epidermophyton floccossum as revealed by freeze-etch electron microscopy

Freeze-etch electron microscopic observations demonstrated that Amphotericin B, a potential antifungal polyene induced profound ultrastructural alterations in the plasma membrane of cells of a human pathogenic fungus, Epidermophyton floccosum; aggregation of membrane-associated particles (85-Å) and formation of depressions (or craters) on the inner fracture face.Thin-sectioning electron microscopy showed “vesiculation” of plasma membranes of Amphotericin B-treated Epidemophyton cells.It can be suggested from the preliminary results that the fungal cells which contain ergosterol as the major sterol are highly sensitive to Amphotericin B.     

Membrane composition modulates the interaction between a new class of antineoplastic agents deriving from aromatic 2-chloroethylureas and lipid bilayers: a solid-state NMR study

This work presents the investigations of the interactions between nystatin, a polyene antibiotic, and phospholipids with various head groups (phosphatidylcholine and phosphatidylethanolamine) and acyl chains of different length and saturation degree. The experiments were performed with the Langmuir monolayer technique. Among phosphatidylethanolamines, DMPE, DPPE and DSPE were studied, while phosphatidylcholines were represented by DSPC and DOPC. The influence of the antibiotic on the molecular organization of the phospholipid monolayer was analysed with the compression modulus values, while the strength of nystatin/phospholipid interactions and the stability of the mixed monolayers were examined on the basis of the excess free energy of mixing values. The results obtained proved a high affinity of nystatin towards phospholipids. Nystatin was found to interact more strongly with phosphatidylcholines than with phosphatidylethanolamines. The most negative values of the excess free energy of mixing observed for the antibiotic and DOPC mixtures prove that nystatin favors the phospholipid with two unsaturated acyl chains. The results imply that nystatin/phospholipid interactions compete in the natural membrane with nystatin/sterol interactions, thereby affecting the antifungal activity of nystatin and its toxicity towards mammalian cells.     

Absorption and fluorescence spectra of polyene antibiotics in the presence of cholesterol.

The alterations in the absorption and fluorescence spectra observed for the polyene antibiotics filipin and nystatin in the presence of cholesterol are due to an exciton interaction (polyene aggregates) and cannot be attributed to a specific sterol-antibiotic complex. Filipin and nystatin molecules partition into the sterol aggregates, these structures being very efficient to induce exciton interaction; the observed splitting profile indicates that the chromophores are in a stacked arrangement (parallel transition dipoles). For filipin incorporated in lipid bilayers, the sterol is able to induce the same type of aggregate, at variance with nystatin.     

Natamycin sequesters ergosterol and interferes with substrate transport by the lysine transporter Lyp1 from yeast

Natamycin is a polyene macrolide, widely employed to treat fungal keratitis and other yeast infections as well as to protect food products against fungal molds. In contrast to other polyene macrolides, such as nystatin or amphotericin B, natamycin does not form pores in yeast membranes, and its mode of action is not well understood. Here, we have employed a variety of spectroscopic methods, computational modeling, and membrane reconstitution to study the molecular interactions of natamycin underlying its antifungal activity. We find that natamycin forms aggregates in an aqueous solution with strongly altered optical properties compared to monomeric natamycin. Interaction of natamycin with model membranes results in a concentration-dependent fluorescence increase which is more pronounced for ergosterol- compared to cholesterol-containing membranes up to 20 mol% sterol. Evidence for formation of specific ergosterol-natamycin complexes in the bilayer is provided. Using nuclear magnetic resonance (NMR) and electron spin resonance (ESR) spectroscopy, we find that natamycin sequesters sterols, thereby interfering with their well-known ability to order acyl chains in lipid bilayers. This effect is more pronounced for membranes containing the sterol of fungi, ergosterol, compared to those containing mammalian cholesterol. Natamycin interferes with ergosterol-dependent transport of lysine by the yeast transporter Lyp1, which we propose to be due to the sequestering of ergosterol, a mechanism that also affects other plasma membrane proteins. Our results provide a mechanistic explanation for the selective antifungal activity of natamycin, which can set the stage for rational design of novel polyenes in the future.     

Structural analysis and biosynthetic engineering of a solubility-improved and less-hemolytic nystatin-like polyene in Pseudonocardia autotrophica

Polyene antibiotics such as nystatin are a large family of very valuable antifungal polyketide compounds typically produced by soil actinomycetes. Previously, using a polyene cytochrome P450 hydroxylase-specific genome screening strategy, Pseudonocardia autotrophica KCTC9441 was determined to contain an approximately 125.7-kb region of contiguous DNA with a total of 23 open reading frames, which are involved in the biosynthesis and regulation of a structurally unique polyene natural product named NPP. Here, we report the complete structure of NPP, which contains an aglycone identical to nystatin and harbors a unique di-sugar moiety, mycosaminyl-(α1-4)-N-acetyl-glucosamine. A mutant generated by inactivation of a sole glycosyltransferase gene (nppDI) within the npp gene cluster can be complemented in trans either by nppDI-encoded protein or by its nystatin counterpart, NysDI, suggesting that the two sugars might be attached by two different glycosyltransferases. Compared with nystatin (which bears a single sugar moiety), the di-sugar containing NPP exhibits approximately 300-fold higher water solubility and 10-fold reduced hemolytic activity, while retaining about 50% antifungal activity against Candida albicans. These characteristics reveal NPP as a promising candidate for further development into a pharmacokinetically improved, less-cytotoxic polyene antifungal antibiotic.     

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)     

Sterol metabolism during germination of conidia of Aspergillus fumigatus.

The sterol content of germinating conidia of the opportunistic pathogenic fungus Aspergillus fumigatus has been correlated with germination phase and sensitivity to polyene antibiotics. The sterol and sterol ester contents of walls did not change during germination. The sterol ester content of membranes and cell sap remained constant during germination, whereas the sterol content increased during the outgrowth of germ tubes. On the basis of differential extraction studies it was concluded that the loss of resistance to polyenes that occurred in the early stages of swelling of conidia during germination was not due to a movement of sterol or sterol ester out of the wall. Radioactive-labelling experiments demonstrated that, although the amounts of conidial wall sterol and sterol ester did not change during germination, they were metabolically active. Changes in the turnover rate of wall and membrane sterol and sterol ester during germination were investigated and their relationship to a possible mechanism for the change from resistance to sensitivity to polyene antibiotics is discussed.     

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)     

Natamycin blocks fungal growth by binding specifically to ergosterol without permeabilizing the membrane

Natamycin is a polyene antibiotic that is commonly used as an antifungal agent because of its broad spectrum of activity and the lack of development of resistance. Other polyene antibiotics, like nystatin and filipin are known to interact with sterols, with some specificity for ergosterol thereby causing leakage of essential components and cell death. The mode of action of natamycin is unknown and is investigated in this study using different in vitro and in vivo approaches. Isothermal titration calorimetry and direct binding studies revealed that natamycin binds specifically to ergosterol present in model membranes. Yeast sterol biosynthetic mutants revealed the importance of the double bonds in the B-ring of ergosterol for the natamycin-ergosterol interaction and the consecutive block of fungal growth. Surprisingly, in strong contrast to nystatin and filipin, natamycin did not change the permeability of the yeast plasma membrane under conditions that growth was blocked. Also, in ergosterol containing model membranes, natamycin did not cause a change in bilayer permeability. This demonstrates that natamycin acts via a novel mode of action and blocks fungal growth by binding specifically to ergosterol.     

Amphotericin B alters the affinity and functional activity of the oligopeptide chemotactic factor receptor on human polymorphonuclear leukocytes

Leukocyte chemotaxis is initiated by the binding of chemotactic factors to specific, high-affinity receptors. Amphotericin B, a polyene antibiotic that binds to membrane cholesterol, inhibits human neutrophil (PMN) chemotaxis. We examined the effects of this drug on PMN functions mediated by the oligopeptide chemotactic factor receptor. The antibiotic irreversibly inhibited chemotaxis and depressed the binding of the radiolabeled chemoattractant, fMet-Leu-[3H]Phe, to its receptor without affecting the receptor's specificity. The drug lowered the binding affinity of the receptor by up to fivefold and slightly increased its number. Doses of amphotericin B that depressed receptor affinity and inhibited chemotaxis did not diminish lysosomal enzyme secretion or superoxide anion production. Nystatin, a less potent polyene antibiotic, also diminished chemotactic factor binding, but to a lesser degree than amphotericin B did. A chemically unrelated antifungal agent had no effect on either binding or chemotaxis. Thus, pharmacologic manipulation can alter the affinity of the chemotactic factor receptor on human PMN; this alteration is associated with a change in receptor function. The data suggest that receptor affinity regulates or at least reflects its functional state, and that the transduction mechanisms for various biologic responses mediated by the chemoattractant receptor are heterogeneous. By pharmacologic alterations of receptor affinity, one may be able to modulate specific biologic responses elicited by chemoattractant receptor-ligand interactions.     

Cholesterol as a target for toxins

A mechanism is proposed for the way in which cholesterol facilitates channel formation by polyene antibiotics and bacterial protein toxins. Central elements of the model are: (i) interactions between the ring system of the sterol and rigid elements of the polyene or toxin molecule, and (ii) the specific orientation of cholesterol within the membrane.