The antifungal Amphotericin B (AmB) is an old and effective drug—it saved many COVID-19 patients whose compromised immune systems failed to stop secondary fungal infections. But it sometimes causes life-threatening kidney damage. Now, after more than a decade of sleuthing into this toxicity, researchers have not only found an explanation, but used it to devise a powerful antifungal alternative without any obvious side effects in mice and human cells. And the strategy that led to the discovery of the compound, described today in Nature, may offer a route for detoxifying other antimicrobial drugs.
“This is really inspiring work,” says Leah Cowen, a mycologist at the University of Toronto. “They leveraged molecular insights into how the drug works to dial up the properties they wanted and dial down properties they didn’t want.”
Worldwide, fungal diseases kill some 1.5 million people annually, about the same as tuberculosis or malaria. But in contrast with antibiotics, where dozens of classes of effective drugs are available, there are only three classes of antifungals, and each faces problems of toxicity, growing resistance, or limited effectiveness. “We are very much in need of new antifungals that are safe and effective,” Cowen says.
AmB, produced by a Streptomyces bacterium, was first isolated in 1955 from soils near the Orinoco River in Venezuela. And though chemists learned to synthesize it, the complex molecule was painstaking to make from scratch. In 2012, researchers led by Martin Burke, a chemist at the University of Illinois Urbana-Champaign (UIUC), reported they had developed a way to quickly produce closely related analogs of AmB from modular building blocks. And tests on one such analog revealed that AmB kills fungi by stripping them of ergosterol, a key structural support in their cell membranes. Human cells don’t use ergosterol. But cholesterol, a closely related sterol, performs much the same function in human cells. And Burke and his colleagues found that AmB likely causes renal damage by stripping cholesterol out of the membranes of kidney cells and weakening them.
Burke’s team churned out numerous new variants using various techniques, each with a slight tweak to its chemical structure, to see whether any had reduced toxicity. One was initially promising enough to license to a biotech company, but ultimately proved not safe enough in animal studies.
Burke and his colleagues went back to the drawing board. They developed ideas on where tweaks to the molecule might further reduce its toxicity by studying high-resolution images of AmB binding to both ergosterol and cholesterol recently provided by Chad Rienstra’s group at the University of Wisconsin-Madison. “The atomic resolution models were really the key to zoom in and identify these very subtle differences,” says Corinne Soutar, a UIUC graduate student and the paper’s co–first author. They then created another derivative, in which they swapped the positions of a single hydrogen atom and a hydroxyl group, and found that this blocked the compound’s ability to bind to cholesterol, making it even less toxic. However, it stripped out the membrane ergosterol too slowly. Fungi like yeast “could make new ergosterol faster than we could remove it,” Burke says. “We pushed the pendulum too far, We got rid of the toxicity but lost potency.”
Additional insights from the earlier imaging studies offered a solution. Burke’s team tweaked a carboxylic acid group on the core of the molecule, speeding up how quickly the antifungal removed ergosterols. Today in Nature, Burke and his colleagues report that in cell culture the compound, dubbed Am-2-19, is at least as effective as AmB, if not more so, in killing more than 500 different fungal species. Studies on mice showed that Am-2-19 thwarted three common, hard-to-treat fungal infections with no signs of toxicity, even at high doses. And tests on human blood and kidney cells produced no warning signals. Am-2-19 has been licensed to Sfunga Therapeutics, which has launched a phase 1 human safety trial.
Beyond the hope that Am-2-19 will make a safer form of AmB, Burke says the same strategy of teasing out the key molecular interactions could help detoxify other antifungals that are in the same class as AmB and are already on the market. Several such compounds work through the same ergosterol-stripping mechanism and are similarly toxic. Burke says: “The lights have been turned on.”
