The clinically vital, but severely nephrotoxic, antifungal Amphotericin B has a unique mechanism of action;rather than bind to a macromolecular target, it self-assembles into membrane ion channels in yeast membranes. The effectiveness of AmB arises from its affinity for ergosterol in yeast membranes. However, AmB is severely nephrotoxic due to a competing affinity for cholesterol in human cell membranes. Lack of a suitable model membrane for studying AmB has resulted directly in a lack of detailed molecular understanding of its sterol specificity, thus severely limiting the design of more effective and /or less nephrotoxic derivatives. The typical model membrane employed is the liposome, however, AmB forms extremely large """"""""hyper-aggregates"""""""" in liposomes. Interestingly, analysis of membrane protein structure has also been limited by tendency for aggregation and poor model membranes. Recently, nanoscale discoidal lipid bilayers (nanodiscs) have proven to be effective model membranes for studying structure of monomeric membrane proteins. We propose to harness the advantages of the nanodisc to analyze differences in structure and stoichiometry of the AmB/cholesterol and AmB/ergosterol channels. Quantitative analysis and UV and CD spectroscopic analysis will determine the AmB/nanodisc ratio in the presence of each sterol. UV and CD spectra of AmB will serve as a probe of the physical state of the incorporated AmB. Solid state NMR experiments (SSNMR) will be used to determine the channel length preference for cholesterol and ergosterol. ISCSIP rotational echo double resonance SSNMR will identify those AmB carbon atoms interacting with P atoms of the lipid. Finally, SSNMR analysis of AmB in the nanodisc will allow determination of specific atoms involved in AmB/ergosterol and AmB/cholesterol binding, as measured by changes in 13C chemical shift of the AmB carbons upon binding the sterol. Collectively, these studies will illuminate differences in the cholesterol and ergosterol AmB channel complexes which will serve as a starting point for the rational design of more effective, less nephrotoxic AmB derivatives.
The proposed research will study the mechanism of action of the clinically vital, yet toxic, antifungal drug amphotericin B (AmB). Experiments will probe the interaction of amphotericin B with cell membranes and sterols present therein to advance understanding of the fundamental mechanism of AmB effectiveness and toxicity. Results of these experiments will enable preparation of amphotericin B derivatives that are more effective and/or less toxic.
|Anderson, Thomas M; Clay, Mary C; Cioffi, Alexander G et al. (2014) Amphotericin forms an extramembranous and fungicidal sterol sponge. Nat Chem Biol 10:400-6|
|Lee, Suk Joong; Anderson, Thomas M; Burke, Martin D (2010) A simple and general platform for generating stereochemically complex polyene frameworks by iterative cross-coupling. Angew Chem Int Ed Engl 49:8860-3|