Direct Interaction between Amphotericin B and Ergosterol in Lipid Bilayers As Revealed by ²H NMR Spectroscopy

Abstract

Although amphotericin B (AmB) is thought to exert its antifungal activity by forming transmembrane ion-permeable self-assemblies together with ergosterol, no previous study has directly proven AmB-ergosterol interaction. To establish the interaction, we measured (2)H NMR using deuterium-labeled sterols and AmB. The (2)H NMR spectra of deuterated ergosterol in palmitoyloleoylphosphatidylcholine (POPC) bilayers showed that fast axial diffusion of erogosterol was almost completely inhibited by the coexistence of AmB. Conversely, cholesterol mobility in POPC membrane was essentially unchanged with or without AmB. These results unequivocally demonstrate that ergosterol has significant interaction with AmB in POPC bilayers. In addition, we examined the mobility of AmB using deuterium-labeled AmB, and found that, although AmB is almost immobilized in sterol-free and cholesterol-containing POPC membranes, a certain ratio of AmB molecules acquires mobility in the presence of ergosterol. The similar mobility of AmB and ergosterol in POPC bilayers confirmed the idea of the direct intermolecular interaction between ergosterol and AmB.

Influence of Structural Isomerism and Fluorine Atom Substitution on the Self-Association of Naphthoic Acid

“…17 Multicomponent systems are also important, probing the role of functional groups in aggregation [18][19][20] and drug delivery. [21][22][23][24] A number of physical techniques can provide information on aggregation processes such as optical absorption, 3 x-ray scattering 3,13,25,26 and nuclear magnetic resonance (NMR) spectroscopy. 10,12,[26][27][28] In general, absorption and scattering based experiments provide information on the bulk properties of a sample, e.g.…”

Discovery of an ergosterol-signaling factor that regulates Trypanosoma brucei growth

“…At this stage, cells satisfy their sterol requirements through dietary supplementation of cholesterol via lipoproteins in full-growth medium (FGM) present in the blood meal ( 10,11 ). Consequently, the existence of a sterol uptake process which permits the formation of BSF membranes containing exclusively cholesterol could provide resistance to a range of antifungal drugs, e.g., amphotericin B, that function typically in membranes formed by ergosterol ( 14,15 ). However, all the genes for ergosterol biosynthesis have been found recently to be expressed in BSF ( 16,17 ) and inhibitors of the post-squalene portion of ergosterol biosynthesis can inhibit growth of BSFs ( 4,(18)(19)(20); therefore, the ability for BSFs to grow as an ergosteroldepleted protozoan with dietary cholesterol as an ergosterol surrogate remains enigmatic.…”

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

“…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).…”

“…It is challenging, however, to perform this same experiment in a yeast cell membrane to determine if this type of direct binding interaction is functionally relevant in vivo. More recently, solid-state NMR studies employing deuterated derivatives of AmB and cholesterol (39, 40) suggested little or no binding between these two molecules because their mobilities were dissimilar (40). Analogous experiments with AmB and ergosterol were interpreted as being alternatively consistent with similar mobilities and thus supportive of a direct binding interaction between these two molecules (40).…”

“…More recently, solid-state NMR studies employing deuterated derivatives of AmB and cholesterol (39, 40) suggested little or no binding between these two molecules because their mobilities were dissimilar (40). Analogous experiments with AmB and ergosterol were interpreted as being alternatively consistent with similar mobilities and thus supportive of a direct binding interaction between these two molecules (40). However, in other solid-state NMR studies, through-space dephasing expected for a direct binding interaction was not observed between nonconjugated 13 C-labeled AmB and 6-fluoroergosterol (34).…”