Nearly seventy years after the discovery of Amphotericin B, scientists are still learning from this enigmatic drug. UW–Madison scientists, in collaboration with scientists at the University of Illinois at Urbana-Champaign and the National Institutes of Health (NIH), applied innovative nuclear magnetic resonance techniques to reveal the structure of Amphotericin B as it’s about to bind to ergosterol. Their results challenge a long-standing paradigm for the drug's mechanism of action and suggest new avenues for drug development.
Reliable yet potent
Amphotericin B (AmB) is powerful and reliable. Used to treat many different types of infections, it saves lives by obliterating serious fungal infections that can't be diagnosed quickly.
One of the reasons AmB is so effective is also, paradoxically, why it can be toxic. It can bind to ergosterol, a major component of fungal membranes, as well as cholesterol, a component of human cell membranes. When AmB binds to ergosterol, it stops fungal infections in their tracks. When it binds to cholesterol, though, AmB can target cholesterol-containing membranes, wreaking havoc on those cells. AmB is so potent that while doctors have adapted to minimize toxicities associated with the drug, up to 80% of patients receiving AmB may still experience a wide range of side effects.
A team led by Chad Rienstra, a professor of biochemistry and an investigator with the Morgridge Institute for Research, recently elucidated the structure of Amphotericin B as it prepares to bind to a sterol like ergosterol or cholesterol. This work comes out of a longstanding collaboration with Martin Burke and Taras Pogorelov, professors at the University of Illinois at Urbana-Champaign. The scientists’ results, which were published in Nature Structural & Molecular Biology, call into question AmB’s mechanism of action.
Amphotericin B "sponge". The sponge contains
two types of monomers (examples in orange and blue).
Image courtesy of Taras Pogorelov,
University of Illinois at Urbana-Champaign.
“Our findings show that Amphotericin doesn’t exist as a single molecule that binds to a single spot in a fungal cell membrane [as previously thought] — it works in a collective manner, forming ‘sponges’ of many Amphotericin molecules that work together like a team to absorb sterol,” Rienstra says.
Results also provide evidence about why fungi find AmB so hard to evade. If a fungus wants to bypass Amphotericin B, it needs to evolve to work with a sterol other than ergosterol. And that requires time the fungus just doesn’t have when AmB is around.
Innovations in NMR
Rienstra’s team needed to make major modifications to their nuclear magnetic resonance (NMR) methods to build the structure of Amphotericin B.
AmB poses a challenge for standard NMR spectroscopy. When an AmB sample is placed in a magnetic field and excited into nuclear magnetic resonance by radio waves, the signals emitted by the AmB nuclei are messier than scientists would like — spectra of AmB, in its sponge-like lattice, have multiple sets of overlapping peaks that are difficult to tease apart.
To make it easier to identify each peak, the scientists altered their sample preparation techniques and NMR sequences. They conducted additional NMR experiments at the National Magnetic Resonance Facility at Madison (NMRFAM) to figure out which sets of peaks were connected. And through their collaboration with the NIH, they also modified the computational models that calculate structures of molecules from spectroscopic data.
Says Rienstra, “We wanted to know how the AmB sponge fits together to accommodate ergosterol. Just like sponges that absorb water, if it’s dried out and crusty, it doesn’t move well and won’t do a very good job of absorbing sterols. Once it’s a little soft, it does a better job of absorbing because then it’s flexible.”
Ultimately, the scientists discovered that their data were consistent with multiple forms of AmB sponges, suggesting that AmB has some sort of inherent flexibility that may be essential for binding sterols. For example, pockets in AmB sponges are irregular, but molecules appear to shift to make room for ergosterol.
With this new knowledge about AmB’s binding structure and eager to develop less toxic versions of AmB, Rienstra’s lab and NMRFAM are developing technologies to help them get there. Rienstra says that higher field magnets and new types of NMR probes that can look at samples that behave like AmB but which can only be procured in smaller amounts will be essential to this work. “This project is a really nice synergy between technology development and application, because as we develop higher field instruments that are more sensitive, we can use smaller quantities of samples,” he says. “That will open up opportunities for studying many other categories, or the sort of ‘extended family,’ of Amphotericin.”
This story was written by Catherine Steffel, Ph.D. Please direct media inquiries and questions about this work to email@example.com.