Molecular organization, localization and orientation of antifungal antibiotic amphotericin B in a single lipid bilayer

Abstract

Amphotericin B is a popular antifungal antibiotic, a gold standard in treatment of systemic mycotic infections, due to its high effectiveness. On the other hand, applicability of the drug is limited by its considerable toxicity to patients. Biomembranes are a primary target of physiological activity of amphotericin B and both the pharmacologically desired and toxic side effects of the drug relay on its molecular organization in the lipid phase. In the present work, molecular organization, localization and orientation of amphotericin B, in a single lipid bilayer system, was analysed simultaneously, thanks to application of a confocal fluorescence lifetime imaging microscopy of giant unilamellar vesicles. The results show that the presence of sterols, in the lipid phase, promotes formation of supramolecular structures of amphotericin B and their penetration into the membrane hydrophobic core. The fact that such an effect is substantially less pronounced in the case of cholesterol than ergosterol, the sterol of fungal membranes, provides molecular insight into the selectivity of the drug.

Lipid Systems for the Delivery of Amphotericin B in Antifungal Therapy

“…Despite several decades of clinical use, AmB mechanism of action at the molecular level remains elusive and several models have been proposed based on extensive experimental research and theoretical studies [45][46][47][48]54,55,[58][59][60][61][62][63][64][65][66]. AmB has been shown to bind sterol-containing membranes of eukaryotic cells and to insert into the lipid bilayer forming pore-like supramolecular structures that can act as transmembrane ion channels, leading to increased membrane permeability, K + leakage, and disruption of ion transport [44,45,48,53].…”

“…AmB has been shown to bind sterol-containing membranes of eukaryotic cells and to insert into the lipid bilayer forming pore-like supramolecular structures that can act as transmembrane ion channels, leading to increased membrane permeability, K + leakage, and disruption of ion transport [44,45,48,53]. Recent studies also suggest that different oligomerization of AmB in lipid bilayers modulated by membrane sterols contribute to the higher toxicity of the drug to fungal cells, since it has been found that ergosterol promotes association of AmB dimers into tetramers responsible for membrane permeabilization while cholesterol hinders AmB aggregation in the lipid matrix [44,46,48,55,62]. Disruption of ergosterol biosynthesis is responsible for resistance to Due to its amphipathic nature, AmB is able to self-associate in aqueous solution forming water soluble dimers and oligomers that can further associate to form insoluble polyaggregates, which act as a monomer reservoir [25,[43][44][45][46].…”

ABCA1 transporter reduces amphotericin B cytotoxicity in mammalian cells

“…2b (A1G cells) are preferentially formed in the environment of cholesterol and possibly contain the sterol as an element of such structures. It has been demonstrated that presence of either ergosterol or cholesterol in the lipid membranes was necessary for incorporation of AmB into the hydrophobic core of the membranes [43]. In the case of the MMG cells exposed to AmB, an increase of the fluorescence anisotropy can be observed (Fig.…”

“…A relatively intense fluorescence signal in the cell surface region, characterized by a long fluorescence lifetime (> 6 ns), coded with red color (Fig. 2b, upper panels), can be assigned to antiparallel dimeric structures of AmB [43,44]. The same structures are characterized by relatively low fluorescence anisotropy values (see the bottom panels in Fig.…”

“…3b, bottom panel). This particular band can be assigned to higher dimension molecular structures of AmB, including tetramers, represented by short fluorescence lifetimes and able to penetrate sterol-rich lipid membranes [43]. Such structures are considered as responsible primarily for disturbance of the ionic equilibrium of cell membranes.…”