Solution and solid-state NMR studies of amphotericin B

Anderson, Thomas

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https://hdl.handle.net/2142/95659 Copy

Description

Title

Solution and solid-state NMR studies of amphotericin B

Author(s)

Anderson, Thomas

Issue Date

2013-04-16

Director of Research (if dissertation) or Advisor (if thesis)

Burke, Martin D.

Doctoral Committee Chair(s)

Burke, Martin D.

Committee Member(s)

• Rienstra, Chad M.
• Hergenrother, Paul J.
• Katzenellenbogen, John A.

Department of Study

Chemistry

Discipline

Chemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• ergosterol
• biomembranes
• solid-state NMR
• solution NMR
• NMR
• nuclear magnetic resonance
• amphotericin

Abstract

Since its discovery in 1955, amphotericin B (AmB) has been a vital clinical agent. It remains the drug of last resort for systemic fungal infections despite its significant toxicity. Despite over 50 years of clinical use, very few cases of AmB resistance have been reported. This lack of resistance has been attributed to its unique mechanism of action. AmB is hypothesized to bind to yeast membranes and self-assemble into membrane-spanning ion channels leading to cell death. AmB thus represents a prototype of a small molecule with capacity to perform protein-like function. However, despite extensive scientific inquiry, this capacity remains poorly understood. An atomistic understanding of this mechanism stands to enable efforts to harness this untapped potential and/or improve the therapeutic index of AmB. The leading model for AmB antifungal activity is self assembly of the natural product into discrete, membrane-embedded barrel-stave pores which disrupt cellular ion gradients and cause cell death. A ring of salt bridges and/or hydrogen bonds at the channel periphery are proposed to stabilize the channel assembly. These polar interactions are proposed to form between the C41 carboxylate and C3' amine of adjacent AmB molecules within the channel architecture. This dissertation describes experiments carried out to directly test these two major hypotheses of AmB antifungal activity. We have developed a functional group deletion strategy to directly test the role of the C41 carboxylate and C3' amine. Derivatives were prepared lacking either one or both of these functional groups and solution NMR conformational analysis was employed to determine the ground state conformation of AmB and our derivatives. The functional consequences of these deletions were then assessed in antifungal assays. Our results indicate that in stark contrast to the salt bridge hypothesis, oxidation at C41 is not required for antifungal activity. To test the long-standing hypothesis that AmB is primarily embedded as discrete ion channels in phospholipid bilayers, we have performed an extensive series of solid-state NMR experiments. These data sets enabled us to assign the 13C signals of AmB and to assess geometric and topological aspects of the structural models. The work described in this dissertation highlights the power of both solution and solid-state NMR for studying the function of small molecules. Moreover, these NMR experiments led to an updated model for the antifungal activity of AmB.

Graduation Semester

2013-05

Type of Resource

text

Permalink

• http://hdl.handle.net/123456789/1816
• http://hdl.handle.net/2142/95659

Copyright and License Information

Copyright 2012 Thomas Anderson

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