A Post-PKS Oxidation of the Amphotericin B Skeleton Predicted to be Critical for Channel Formation Is Not Required for Potent Antifungal Activity

Abstract

The clinically vital antimycotic agent amphotericin B represents the archetypal example of a channelforming small molecule. The leading model for self-assembly of the amphotericin B channel predicts that C(41) carboxylate and the C(3′) ammonium ions form intermolecular salt bridges/hydrogen bonds that are critical for stability. We herein report a flexible degradative synthesis pathway that enables the removal of either or both of these groups from amphotericin B. We further demonstrate with extensive NMR experiments that deleting these groups does not alter the conformation of the polyene macrolide skeleton. As predicted by the leading model, amphotericin B derivatives lacking the mycosamine sugar that contains the C(3′) ammonium ion are completely inactive against Saccharomyces cerevisiae. However, strikingly -and in strong contradiction with the current model -the amphotericin B derivative lacking the C(41) carboxylate is at least equipotent to the natural product. Collectively, these findings demonstrate that the leading model for the mechanism of action of amphotericin B must be significantly revised -either the C(41) carboxylate is not required for channel formation, or channel formation is not required for antifungal activity.The leading model for the antifungal action of amphotericin B (AmB, 1) involves its selfassembly into a membrane-spanning ion channel. 1 This natural product thus represents a potential prototype for small molecules with the capacity to perform ion channel-like functions in living systems. Efforts to harness this potential and/or improve the notoriously poor therapeutic index of this clinically vital antimycotic 2 would benefit from a molecular-level understanding of this channel activity.Although the evidence that AmB can self-assemble in lipid membranes to form discrete ion conducting channels is strong, 1,3 the molecular architecture of this channel assemblage and its role in antifungal activity remain poorly understood. 4 Despite this, the leading "barrelstave" model 5 is an often cited textbook classic (Fig. 1A). 6 Extensive computer modeling studies predict that this complex is stabilized by a ring of salt bridges 7a and/or hydrogen bonds 7b-c at the channel periphery between oppositely-charged C(41)-carboxylate and C(3′)-

Amphotericin primarily kills yeast by simply binding ergosterol

“…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).…”

“…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.…”

The Production of Reactive Oxygen Species Is a Universal Action Mechanism of Amphotericin B against Pathogenic Yeasts and Contributes to the Fungicidal Effect of This Drug

“…The chemistry of AMB has been widely studied, with particular emphasis on how chemical modifications of the molecule affect its biological activity (see seminal reviews in references [28][29][30][31][32]. Some of these molecular changes that result in the inability to form pores still retain antifungal activity (33), suggesting that AMB acts through ergosterol sequestration (34).…”

Cell Wall Changes in Amphotericin B-Resistant Strains from Candida tropicalis and Relationship with the Immune Responses Elicited by the Host

“…However, recent findings suggest that AmB elicits its fungicidal effect through multiple mechanisms. In this sense, AmB induces ergosterol sequestration, which causes alterations in the cell membrane that result in killing of the cells (3,4). In addition, AmB induces a significant accumulation of reactive oxygen species (ROS), which has been correlated with cell damage, apoptosis induction, and death (5)(6)(7)(8)(9).…”