Self‐Assembled Amphotericin B Is Probably Surrounded by Ergosterol: Bimolecular Interactions as Evidenced by Solid‐State NMR and CD Spectra

Abstract

Amphotericin B (AmB) is thought to exert its pharmacological effects by forming a barrel-stave assembly with ergosterol in fungal membranes. To examine the interaction between AmB and ergosterol (Erg) or cholesterol (Cho), (13)C- and (19)F-labelled covalent conjugates were prepared as reported previously (N. Matsumori et al. Chem. Biol. 2004, 11, 673-679). The CD spectra of the conjugates in a membrane-bound form suggested that the distance between the heptaene moieties of the ergosterol conjugates AmB-C(2)-(6-F)Erg 2 and AmB-C(2)-Erg 3 is similar to that of AmB in ergosterol-containing membranes, but significantly larger than that of AmB in nonsterol or cholesterol-containing membranes. These observations suggest that, as is the case with ergosterol-containing membranes, the conjugated sterol moiety prevents the close contact between the heptaene moieties within the membrane that would reduce channel conductivity of the AmB assemblies. To further investigate this bimolecular interaction, we recorded the solid-state NMR spectra of conjugates 2 and AmB-C(2)-(6-F)Cho 4, which are composed of uniformly (13)C-labelled AmB and 6-fluorinated ergosterol or cholesterol; the conjugates were expected to facilitate the estimation of distances between the fluorine and carbon atoms. By using rotor-synchronous double resonance (rotational echo double resonance of X cluster; RDX) experiments, we deduced the distance between the fluorine atom and its nearest carbon atom in the heptaene moiety of 2 to be less than 8.6 A. This indicates that the B ring of ergosterol comes close to the AmB polyene moiety. A conformational search of the AmB-ergosterol conjugate by using distance constraints derived from the RDX results suggested that ergosterol molecules possibly surround the AmB assembly, which is in contrast with the conventional image in which ergosterol is inserted into AmB molecules.

Structural Features of Dinoflagellate Toxins Underlying Biological Activity as Viewed by NMR

“…In addition to X-ray co-crystallography approaches, solid-state NMR and solution-state NMR with bicelle/micelle systems seem promising, and thus strategies are extensively explored in biophysical and biochemical research groups including the authors'. [117][118][119][120][121][122] …”

Molecular organization of antifungal antibiotic amphotericin B in lipid monolayers studied by means of Fluorescence Lifetime Imaging Microscopy

“…According to a popular conviction AmB present in biomembranes associates into molecular aggregates in the form of transmembrane pores that affect physiological ion transport [4][5][6][7][8]. The concept that such structures are formed more efficiently in the presence of ergosterol, the sterol present in the membranes of fungi, than in the presence of cholesterol is a key paradigm of selectivity of AmB.…”

Synthesis-enabled functional group deletions reveal key underpinnings of amphotericin B ion channel and antifungal activities

“…Third, both the C41 carboxylate and mycosamine appendages are hypothesized to promote the direct binding of membrane-embedded sterols ( Fig. 1E) (11)(12)(13)(30)(31)(32)(33)(34)(35)(36)(37)). An often invoked alternative model states that indirect effects on global membrane properties, rather than any direct binding interactions, are responsible for the impacts of membrane sterols on the biological activity of AmB (21)(22)(23)(24)(25)(26)(27)(28)(29).…”

“…For example, many computational (11-13, 29, 36, 37) and spectroscopic (9,10,15,34,39,40) studies have been reported, but the membrane-localized, dynamic, and multimolecular nature of this small-molecule-based channel assemblage challenges the current limitations of these techniques. In addition, many studies have been performed with AmB derivatives having covalent modifications of the C41 carboxylic acid and/ or C(3′) amine (14,19,(31)(32)(33)(34)(35)(44)(45)(46). However, relative to many proteins and large peptides, the self-assembly of small molecules can be exquisitely sensitive to steric effects that result from even very minor covalent modifications (47,48).…”

“…Specifically, the C41 carboxylate and/or mycosamine appendages of AmB are proposed to form key polar interactions with the 3-β-hydroxyl group of a sterol. Combined with favorable hydrophobic interactions between the polyene and sterol cores, these polar interactions are predicted to stabilize a direct AmB/sterol complex (30)(31)(32)(33)(34)(35)(36)(37). Studies designed to probe this putative interaction in solution or in liposomes have traditionally relied primarily on UV/Vis and/or CD spectroscopy (10).…”