Structure and function of amphotericin B aggregates

Amphotericin B (AmB) has long served as the last line of defense for treating severe mycoses due to its broad spectrum activity and lack of clinically observed resistance; however, mortality rates from these infections remain high due to AmB’s dose limiting renal toxicity. Since its FDA approval, researchers have strived to understand AmB’s mechanism of antifungal activity to inform the development of a potent, renal sparing derivative. Decades of efforts by the Burke and Rienstra groups to characterize AmB-sterol interactions in liposomal and fungal cell assays have culminated in the sterol sponge model, in which AmB extracts ergosterol from fungal cell membranes, thereby killing yeast, as AmB’s primary mode of action. Recent studies have confirmed that the sterol sponge mechanism is conserved among the large family of glycosylated polyene macrolides and that this mechanism also drives AmB’s renal cell toxicity. The latter observation resulted in the actionable goal of designing AmB sponges that selectively extract ergosterol over cholesterol. Rationally achieving this goal required the development of tools to probe small molecule-small molecule interactions using techniques amenable to the study of a natural product which is difficult to crystallize and forms insoluble aggregates. As described herein, these challenges were overcome through innovations in sample preparation techniques, adaptation and optimization of solid-state NMR (SSNMR) experiments designed to study proteins, as well as adaptation of NMR structure determination software to elucidate first-in-class SSNMR structures of apo and sterol-bound AmB aggregates. Analysis of these structures combined with molecular dynamics simulations provided a rationale for imparting selectivity for Erg over Chol, and this selectivity predicted by atomistic resolution models was shown to translate to renal-sparing properties in vivo. The strategies, tactics and tools that were developed and deployed to achieve this result should enable the structural characterization and/or future design of other small molecules that similarly operate via higher-order aggregation to target specific membrane lipids. Further studies of AmB derivatives and other glycosylated polyene macrolides will advance our understanding of the unique and resistance-refractory mode of action of these natural products and/or further enable their use as prosthetic ion channels.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Chemistry

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2023

Author dc:creator

• Soutar, Corinne P.

Contributors dc:contributor

• Burke, Martin D
• Rienstra, Chad M
• Mitchell, Douglas A
• Pogorelov, Taras V

• Amphotericin B
• Ssnmr
• Microed

Statement dc:rights

• Copyright 2023 Corinne Soutar

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/121206

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F121206