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

Abstract

Amphotericin B is the archetype for small molecules that form transmembrane ion channels. However, despite extensive study for more than five decades, even the most basic features of this channel structure and its contributions to the antifungal activities of this natural product have remained unclear. We herein report that a powerful series of functional group-deficient probes have revealed many key underpinnings of the ion channel and antifungal activities of amphotericin B. Specifically, in stark contrast to two leading models, polar interactions between mycosamine and carboxylic acid appendages on neighboring amphotericin B molecules are not required for ion channel formation, nor are these functional groups required for binding to phospholipid bilayers. Alternatively, consistent with a previously unconfirmed third hypothesis, the mycosamine sugar is strictly required for promoting a direct binding interaction between amphotericin B and ergosterol. The same is true for cholesterol. Synthetically deleting this appendage also completely abolishes ion channel and antifungal activities. All of these results are consistent with the conclusion that a mycosamine-mediated direct binding interaction between amphotericin B and ergosterol is required for both forming ion channels and killing yeast cells. The enhanced understanding of amphotericin B function derived from these synthesis-enabled studies has helped set the stage for the more effective harnessing of the remarkable ion channel-forming capacity of this prototypical small molecule natural product.

Amphotericin primarily kills yeast by simply binding ergosterol

“…However, we found that AmdeB (Fig. 1A), which contains the complete heptaene motif found in AmB and binds very readily to lipid bilayers, is devoid of antifungal activity (2). Thus, an alternative and previously unidentified major mechanism of action for AmB seemed likely.…”

“…The structures of the AmB/ergosterol complex and the multimeric AmB-based ion channel are unknown, thus making the rational design of such a derivative challenging. However, given its recently established critical role in sterol binding (2), it was clear that the mycosamine appendage should be retained. Alternatively, in both of the leading models for the structure of the AmB ion channel (Fig.…”

“…An advanced understanding of how this natural product interacts with living systems thus stands to enable efforts to develop small molecules that serve as surrogates for deficient or dysfunctional protein ion channels that underlie currently incurable human diseases (2)(3)(4)(5)(6)(7). In addition, in stark contrast to most antimicrobial agents (8,9), resistance to AmB has remained exceptionally rare despite extensive worldwide utilization to treat life-threatening systemic fungal infections for more than half a century (10).…”

“…We recently determined that AmB directly binds membrane-embedded ergosterol, the main sterol found in yeast cells, in a manner that requires the mycosamine appendage at C19 (2). Deletion of this appendage yields a derivative, amphoteronolide B (AmdeB) (Fig.…”

“…Deletion of this appendage yields a derivative, amphoteronolide B (AmdeB) (Fig. 1A), which cannot bind ergosterol, is unable to form ion channels, and has no antifungal activity (2,3). These findings caused us to consider two possible conclusions regarding the mechanism(s) by which AmB kills yeast: (i) ergosterol binding-dependent channel formation is required for antifungal activity, or (ii) ergosterol binding alone is a critical mechanism of antifungal activity, and channel formation represents a second complementary mode of action (Fig.…”