Direct and Stereospecific Interaction of Amphidinol 3 with Sterol in Lipid Bilayers

Abstract

Amphidinol 3 (AM3), a polyhydroxy-polyene metabolite from the dinoflagellate Amphidinium klebsii, possesses potent antifungal activity. Although AM3 permeabilizes phospholipid membranes only in the presence of sterol, the detailed molecular basis by which AM3 recognizes sterols in membranes remains unknown. Here, we investigated the molecular interaction between sterols and AM3 in membranes from the viewpoint of stereospecific molecular recognition using ergosterol, cholesterol, and epicholesterol, which is the 3-OH epimer of cholesterol. Dye leakage assays, surface plasmon resonance experiments, (2)H and (31)P NMR measurements, and microscopic observations revealed that AM3 directly interacts with membrane sterols through the strict molecular recognition of the stereochemistry of the sterol 3-OH group. The direct interaction enhances the membrane binding efficiency of AM3, which subsequently permeabilizes membranes without altering membrane integrity.

Stereochemical Studies of the Karlotoxin Class Using NMR Spectroscopy and DP4 Chemical‐Shift Analysis: Insights into their Mechanism of Action

“…For 4,pore formation was only seen when 3b-OH sterols were present in the membrane and not in the presence of 3a-OH sterols such as epicholesterol. [14] HSQC overlay studies were conducted to provide further insight into the sterol-binding interactions of the karlotoxins. By using aseries of increasing ratios of cholesterol added to 2, HSQC NMR experiments were conducted.…”

“…The only physical effects noted were reversible anorexia and a single case of necrotizing pancreatitis. [15] …”

Bioactive Structure of Membrane Lipids and Natural Products Elucidated by a Chemistry‐Based Approach

“…We have reported that AM3 adopts a hairpin‐like conformation with a relatively rigid turn structure near the center including the tetrahydropyran rings . Based on these findings obtained from NMR results, a structural model of AM3 in the membrane was proposed (Figure c) . As described above, the turn region, which is conserved among other amphidinols, recognizes the 3β‐OH group of sterols via hydrogen bonds.…”

“…Measurements of the 1 H NMR T 1M values of AM3 in bicelles containing Mn 2+ , which revealed how far apart the 1 H position was from the membrane surface, showed deeper insertion of the olefinic terminus into the membrane interior, suggesting the predominance of an extended conformation for the hydrophobic polyene portion. We have reported that AM3 adopts a hairpin‐like conformation with a relatively rigid turn structure near the center including the tetrahydropyran rings . Based on these findings obtained from NMR results, a structural model of AM3 in the membrane was proposed (Figure c) .…”

A highly convergent synthesis of the C1–C31 polyol domain of amphidinol 3 featuring a TST-RCM reaction: confirmation of the revised relative stereochemistry

“…For instance, the amphidinols exhibit antifungal, cytotoxic, hemolytic and anti-diatom activity, in which AM3 ( 1 ) exhibits the most potent antifungal activity (MEC = 4–9 μg per disk against Aspergillus niger ), albeit with hemolytic action (EC 50 = 0.009–0.4 μM against human erythrocyte cells). Interestingly, the mechanism of action for this agent has recently been attributed to its ability to form barrel-stave pores, similar to amphotericin B, which is induced by the stereospecific molecular recognition of membrane sterols 2,3. Specifically, the bis -tetrahydropyran core, which is highly conserved in this family, hydrogen bonds with the 3β-OH of ergosterol and cholesterol to permit the permeabilization of the membrane.…”