Amphotericin also interacts with membranes, which are notoriously difficult to study. Many labs, including Burke's, have tried to figure out the three-dimensional structure of amphotericin by crystallizing it, a standard technique. So far, all attempts have failed.
So the team turned to one of the most powerful tools for studying non-crystalline samples: solid-state nuclear magnetic resonance (NMR) spectroscopy, which measures how atomic nuclei respond to changes in a magnetic field.
"NMR is a great technique for detecting signals and interpreting the structural properties of molecules in solution," said Rienstra, who led the NMR work with graduate students Mary Clay and Tom Anderson. But few labs use it to track interactions between small molecules in membranes, he said. "We developed several new NMR experiments to study amphotericin in the presence of membranes."
Previous studies had found evidence that amphotericin opens up ion channels in membranes, perhaps making them more leaky to charged atoms that could disrupt a cell. Most scientists assumed that this was the drug's main mode of action.
But the evidence also suggested that amphotericin interacted with sterols, such as cholesterol in animal cells and ergosterol in yeast. Rienstra and Burke focused on amphotericin's influence on sterols, hypothesizing that this might be a key to its toxicity.
The initial NMR data supported this idea, indicating that very little of the drug less than 5 percent actually formed channels in membranes. Using NMR and other experimental tools, the Rienstra/Burke team found that most of the amphotericin aggregates on the exterior of membranes, extracting sterols out of membranes like a sponge. Cell death follows soon after.
"For over 50 years, the mechanis
|Contact: Diana Yates|
University of Illinois at Urbana-Champaign