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Title:Synthesis-enabled understanding of the mechanism of action of amphotericin B and the development of increased therapeutic derivatives
Author(s):Endo, Matthew Masashi
Advisor(s):Burke, Martin D
Department / Program:Chemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
amphotericin B
Abstract:The polyene macrolide amphotericin B (AmB) remains a critically vital antifungal as the last line of defense against a wide range of life-threatening fungal pathogen. Despite its clinical usage for over half a century, AmB has evaded the development of clinically relevant microbial resistance. AmB has been shown to form ion channels similar to that of their protein counterparts, which has led to the proposal that AmB kills yeast cells via membrane permeabilization. The capacity for ion channel formation and cytotoxicity of AmB are thought to be dependent upon membranous sterol, but the role of sterols in this mechanism and whether membrane permeabilizaton and biological activity are even linked has remained unclear. Thus, the complete understanding of the mechanism of action of AmB would enable the development of new antifungals with an improved therapeutic index, as well as guide the pursuit of new antimicrobials that evade resistance. To elucidate the operative mechanism, we pursued a systematic functional group deletion strategy where derivatives of AmB are synthesized lacking a single protic functional group to understand its role in AmB’s activity. The C35 hydroxyl group of AmB has been proposed to be critical for ion channel formation and so we accessed the derivative lacking the C35 hydroxyl via an iterative cross-coupling (ICC) strategy. The resulting derivative maintained the capacity to bind membranous ergosterol, but could no longer cause membrane permeabilization. Despite the lack of channel activity, this derivative still demonstrated potent fungicidal activity. Deletion of the mycosamine sugar yielded a derivative that could no longer bind ergosterol and was completely inactive against yeast. Collectively, these results led us to conclude that the primary mechanism by which AmB kills yeast is the binding of the ergosterol and that channel formation is a complementary mechanism that marginally increases AmB's potency. This finding suggests that toxicity to humans is likely due to the binding of the major mammalian sterol: cholesterol. Given the importance of the mycosamine appendage on the binding of sterol, we pursued an atomistic understanding of this interaction. The axial C2' hydroxyl group of AmB has been proposed to be critical in binding both sterols. Surprisingly, derivatives lacking or epimerizing the C2' hydroxyl maintained the capacity to bind ergosterol but could no longer bind cholesterol. Consistent with sterol binding being the operative mechanism for toxicity, both derivatives exhibited potent antifungal activity but no toxicity in human cells and mice. However, synthetic access to both derivatives limited their further pursuit. We hypothesized that the sterol selectivity resulted from a ligand-selective allosteric modification and proposed that a similar effect could be achieved by derivatization of the accessible C41 carboxylate. Similar to the C2' modified analogues, the new AmB ureas also demonstrated a preferential binding for ergosterol over cholesterol. This corresponded with their potent activity against a wide range of fungal pathogens as well as their substantial decrease in toxicity to human cells and mice. Despite their decreased toxicity, the AmB ureas maintained the ability to evade resistance similar to that of the parent compound.
Issue Date:2016-07-21
Rights Information:Copyright 2016 Matthew Endo
Date Available in IDEALS:2016-11-10
Date Deposited:2016-08

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