For decades, Amphotericin B (AmB) has served as the last line of defense for treating life-threatening, systemic fungal infections due to its broad-spectrum antifungal activity and evasion of antimicrobial resistance. However, the mortality rate for immunocompromised patients with systemic fungal infections remains high due to AmB’s toxicity. Given the growing population of immunocompromised individuals, it is increasingly important to better understand the structural underpinnings responsible for AmB’s antifungal activity and human cell toxicity. We have recently shown that AmB primarily kills yeast by forming an extramembranous sterol sponge. We have also shown that a synthesized derivative of AmB which retains binding to ergosterol but does not bind cholesterol, retains potent fungicidal activity but shows no toxicity to human cells <i>in vitro</i>. These results suggest that modulating the sterol selectivity of AmB sterol sponges represents a promising approach for retaining potency and reducing toxicity. To illuminate the structural underpinnings that would enable this goal to be rationally pursued, we have generated an atomic-resolution structure of one of these fungicidal AmB sponges using solid-state NMR (SSNMR) together with simulated annealing and DFT calculations to show that AmB forms clathrate-like structures encasing void volumes similar in size to sterol molecules. UV-vis and SSNMR studies performed on AmB-sterol aggregates reveal key structural features that differ from those of the non-sterol bound AmB sponge. Collectively, these results suggest that AmB modifications which impart sterol selectivity may do so by modulating the structures of AmB aggregates in a way that favors binding to ergosterol more readily than cholesterol. These insights are being leveraged to design and develop derivatives that minimize toxicity to human cells while retaining potent fungicidal action.

Insights into the structure-function relationship of amphotericin B-sterol aggregates

ACS Spring 2022

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