How to tame a toxic yet life-saving antifungal

Benjamin Thompson

Welcome back to the Nature Podcast. This week reducing the toxic side effects of a vital antifungal drug...

Nick Petrić Howe

...and the mystery of phosphorus at the Milky Way's edge. I'm Nick Petrić Howe...

Benjamin Thompson

…and I'm Benjamin Thompson.

Benjamin Thompson

Each year, it's estimated that well over a million people die as a result of an invasive fungal infection. This figure is comparable with deaths caused by malaria or TB, but historically, fungal infections have had much less funding and research attention than other infectious diseases. As a result, treatment options for severe fungal infections are often limited. One drug is amphotericin B, an antifungal developed in the late 1950s. It's a drug with a lot of positives — it's broad spectrum, meaning it'll kill many fungal species and resistance to it is rare. However, it has some incredibly serious downsides, so it's only really used as a drug of last-resort to treat the most life-threatening infections. This week in Nature, a team of researchers have altered the molecular structure of amphotericin B in an attempt to curb one of its worst side effects. To find out more, I called up Marty Burke from the University of Illinois at Urbana-Champaign, one of the researchers on the paper. Before we talked about how they tweaked the drug, Marty told me about some of the issues with amphotericin B.

Marty Burke

The problem is – it's highly toxic. So when I was doing my own medical school rotations, we used to call it 'ampho-terrible', because its impact on the patients is so severe. There's a short-term toxicity immediately when you give it to a patient, they get kind of almost like a intense flu-like reaction, it's a very unpleasant experience. But more importantly, long term, it's very toxic to the kidneys and this has been the biggest problem. And in many cases that precludes the doctor giving you enough of the medicine to take care of the infection, you're stuck in this situation where you give too much, you can irreversibly impact the kidneys and if you don't give enough, then the patient doesn't get cured. So, kidney toxicity is the big problem and that's the one that we decided to hone in on.

Benjamin Thompson

And I guess, to get a sense of why this toxicity was happening, it almost comes back to the mode of action of this drug then, and actually how it affects fungi themselves.

Marty Burke

Yes, so in previous work, we had discovered that different than everybody thought, the amphotericin kills the fungi by forming a sponge on the surface, and then it rapidly extracts what’s called a sterol molecule or greasy molecule, it's important for the membrane, it extracts that molecule into the sponge, and thereby kills the fungus. And so this sponge-like mechanism, gave us a new understanding of the killing effects, which therefore gave us a new opportunity to think about how we might make the molecule better.

Benjamin Thompson

And so the sterol molecule in the fungal cell membranes is called ergosterol. And there's a similar molecule in the membranes of human cells, and that is cholesterol. And you wanted to see what the relationship was between the two.

Marty Burke

Yes, so in this paper, we were able to show for the first time that same sponge-like mechanism is how it's killing the kidney cells. And so this is the example you just gave cholesterol is the human version of that same sterol and this molecule forms sponges and extracts cholesterol from the human kidney cells and thereby kills them.

Benjamin Thompson

So, you have this antifungal that acts like a sponge, almost sort of tearing cholesterol out of human cell membranes, which is why it's toxic. And that could be the end of the story. But you've tried to avoid that happening in the first instance, by tweaking this molecule, what did you do there?

Marty Burke

So, once we understood how it was killing kidney cells, this put us in a very exciting position to change that so that it still kills the fungal cells but hopefully it doesn't kill the human cells. And what started to look very promising is that we understood – through additional studies – that it binds the fungal sterol very strong, it binds the human sterol, but it's weaker. So we had this kind of thought that what if we could mess the binding up a little bit, so that it loses its ability to bind human sterol but because it binds the fungal sterol so strongly, even if we mess it up a little bit, it'll still bind. So, we did what's called a controlled destabilisation. And by doing that, we were able to show we could no longer detect any binding of human cholesterol. And yet we still saw good binding, less, but good binding to the fungal sterol. That was an important step forward, but it wasn't enough. The controlled destabilisation worked to get rid of the toxicity. But in doing so, we lost potency, meaning it was now less effective against the fungi. So that was a challenge.

Benjamin Thompson

And I get a sense from reading your paper then that this drug didn't work as well, because although it was able to bind to the ergosterol, to the sterol molecule in the fungal membrane, it wasn't able to pull these sterols out quicker than the fungus could make it is that about what was happening?

Marty Burke

That’s exactly right. So when we decrease the binding, we thought, okay, that's the problem, right? That it's now binding too weakly. But actually, it wasn't about the strength, it was about the speed. So after a whole bunch of studies, we found it was extracting ergosterol, but it was doing it slower. We said, 'aha, okay' and then we had discovered a different type of modification on the molecule that caused it to extract faster. So what we did was we put these two changes into the same molecule, one that knocks out the cholesterol binding, and therefore gets rid of the toxicity. A second one that speeds up the ergosterol extraction, and thereby brings back the potency. We made a new molecule, this is the one we call AM-2-19, by the way, named after the student who discovered it, Arun Maji who made that molecule. And that combination of features created a really exciting compund.

Benjamin Thompson

And what can this compound do, then? I mean, I guess you've tested it against a bunch of different pathogenic fungi, I imagine.

Marty Burke

Yes, so we actually tested it against 500 pathogenic fungi in four different labs. And we found that it's very potent, very broad-spectrum. We also were able to show that it evades resistance in the standard assays that you would run in the lab. And so all of those things look like, for all intents and purposes, it keeps all the desired properties of amphotericin, but it's very well tolerated in animals. We were able to put it into mice with very high-doses, and show that their kidneys were spared. And so this now gives that compound a lot of potential to be a replacement for this otherwise very toxic, but clinically-vital drug, we're hopeful that this new compound might actually be able to offer benefit.

Benjamin Thompson

I mean, potential is quite an important word to underline there, I suppose. Because this work is in human cells in a dish in a lab and in rodents as well, there's an awful long way to go, Marty, before this can be considered humans, I'm sure.

Marty Burke

Definitely there's a long road to go. That's very much true. That said, I was able to work with others to co-found a company called Sfunga Therapeutics and full disclosure, I'm a founder, I'm a shareholder, I receive consulting compensation from that company. But we did license this compound to that company and it just began clinical trials. So, this is a process that's continuing and ongoing. And as you're right, it's a long journey ahead, but it is moving forward at this time.

Benjamin Thompson

And what else is missing from this puzzle? What questions are left to understand about this compound and its mode of action?

Marty Burke

The big mystery that's left, that’s unanswered in this paper: why is it faster? So understanding how changing the molecule causes us to change the rate of sterol extraction from the membrane, that's a very interesting question. And it's going to probably require innovation on the experimental front, because we tried all the stuff that we had lying around and it didn't answer the question. And we're gonna have to dive in very deep and try to understand that one next. There's a whole bunch of other things I could give you of interesting unanswered questions, but that's the one I'll highlight.

Benjamin Thompson

And as someone who's worked in a clinic then and seeing the burden of fungal infections, what are you ultimately hoping for this work when it comes out and people take a look at it?

Marty Burke

Fungal infections, unfortunately, are a major unmet medical need and there's a lot of work that has to be done to try to address that. We're hopeful this paper can contribute, both by helping expand the basic science around how some of these important molecules work, which puts the lights on for everyone, right? And we can all come in and try to be creative and innovative and find better ways to treat these diseases. And we're excited that perhaps something out of this paper could be a starting point right for developing new medicines that could be impactful in the clinic.

Benjamin Thompson

That was Marty Burke from the University of Illinois at Urbana-Champaign in the US. To read his paper, look out for a link in the show notes.

Nick Petrić Howe

Coming up, the puzzle of the Milky Way's far out phosphorus. Right now though, it's time for the Research Highlights with Dan Fox.

Dan Fox

Researchers have succeeded in reconstructing the DNA of an ice-age woolly rhino by analysing fossilized hyena droppings. The woolly rhinoceros roamed northern Eurasia until 14,000 years ago, but little is known about the rhino’s European population. One thing that is known is that they were preyed upon by cave hyena. Because of this, paleontologists analysed fossilized remains of the hyena’s faeces recovered from two caves in what is now Germany to see what they could learn about the rhino. From the droppings, the team were able to isolate ancient DNA of a European woolly rhino, and successfully assembled its mitochondrial genome for the first time. They found that differences between the European and Siberian woolly-rhino genomes suggest that the two populations split around 450,000 years ago. The authors say their findings support the idea that fossilized dung is an overlooked source of ancient DNA. Dig through that research in Biology Letters.

Dan Fox

A tiny, multifunctional soft robot could perform intricate surgical procedures inside a beating heart. The motion of a pulsating heart, coupled with the delicate nature of blood vessels makes minimally-invasive cardiac surgery a challenge. To address this, researchers have engineered a millimetre-scale robot that can enter the heart’s chambers along with a stabilizing device that sits in the large vein called the superior vena cava. Once threaded through the vena cava, the stabiliser expands to about 3 centimetres, allowing the tiny robot — made of small balloon like structures — to brace itself and guide robotic surgical tools into the moving heart. The whole apparatus is directed with a Nintendo Wii game controller. The team tested the device using 3D models and pig organs and they hope that future tests on live animals will confirm that the robot performs well and reveal any complications. Take that research to heart by reading it in Science Advances.

Nick Petrić Howe

Life as we know it has a certain set of ingredients. To assemble a living thing, you need a lot of carbon, hydrogen and oxygen, a sprinkling of nitrogen and sulfur and a dash of phosphorus. These elements are found throughout the Milky Way, with the exception of phosphorus. So far, it's only really been seen in the inner part of our Galaxy. Well, that is until now. This week in Nature, there's a report of the detection of phosphorus containing molecules towards the Galaxy’s edge. I caught up with paper author, Lucy Ziurys, to talk about the new discovery and what it may mean for our search for life. She started by explaining why she was interested in phosphorus.

Lucy Ziurys

Well, phosphorus is a really important element in terms of the origin of life. It's one of the so-called six N-CHOPS elements: nitrogen, oxygen, carbon, hydrogen, sulfur and phosphorus that are supposedly necessary for life. And so if you're interested in whether planet is habitable, you want to know if phosphorus is there.

Nick Petrić Howe

And what do we know so far about the sort of distribution of phosphorus in the Galaxy?

Lucy Ziurys

That's the thing we don't know very much, we only see phosphorus in a small segment of our complete Galaxy. And so you know, we don't really know whether it's widely distributed in our Galaxy, we have one data point in our Galactic Centre and that's it. So we know something in the solar neighbourhood around our Sun. But at the far edge of the Galaxy, we have absolutely no information until our discovery.

Nick Petrić Howe

And so you found some traces of phosphorus, you know, quite far out in the Galaxy. Why did you think there might be some there? What led you to sort of look in this region for phosphorus?

Lucy Ziurys

Well, there was a series of clouds at the edge of the Galaxy, we call them Galactic edge clouds. And we actually had detected methanol in a group of them. And before that, another group had found formaldehyde. So, formaldehyde and methanol are both sort of, you know, organic molecules. And so that gave us a clue that, well, if these sort of organic molecules are there, maybe we should look for ones that contain phosphorus.

Nick Petrić Howe

As I understand it, for phosphorus to be formed, like one of the main ideas is it comes from supernovae, and there aren't that many this far out in the Galaxy. So that begs the question, how did that phosphorus get there where you observed it this far out in the outer regions?

Lucy Ziurys

Yes, there are no known supernova remnants beyond about 15,16 kiloparsecs and these clouds are out at 23 kiloparsecs. So, that's quite a big difference. We think, from our observations, that phosphorus has to be produced by lower-mass stars, stars that aren't, you know, 20 to 40 solar masses that go supernova, but stars more on the order of mass within the Sun's range. And there's been some theory that suggests that such stars may actually be producing phosphorus. You know, when they consider all the supernova that have gone off in the Galaxy, and calculate the amount of phosphorus produced by them,they fall short by about a factor of three to what is observed. And so, people have been thinking there must be another source of phosphorus in some other types of lower-mass stars and I think our detections are good evidence for this.

Nick Petrić Howe

Why did you come down on this particular theory and not some other ideas of how phosphorus could be there? Because I understand there could be things called ‘galactic fountains’ that maybe, you know, sort of spray phosphorus quite far. Or, it could have come from somewhere else beyond the Galaxy.

Lucy Ziurys

Yes, we actually looked into those possibilities. At first, we thought it was a galactic fountain, but galactic fountains don't throw their material so far out. Okay, and you know, there's some clouds that are created by galactic fountains, and usually not this far out, and our detections are in a cloud that's more in the plane of the Galaxy. And these galactic fountain clouds were more above and below the plane of the Galaxy. So, those kinds of data point to the fact that this is unlikely to be a galactic fountain. And you know, you could say, well, maybe another galaxy collided with ours, and that's the source of phosphorus. But typically, those galaxies are low in these kinds of elements as well. So you wouldn't expect them to bring phosphorus enrichment to our Galaxy. And so we narrowed out these possibilities and we really do think it has to do with stellar nucleosynthesis. And a new way to form phosphorus that people just haven't considered too much.

Nick Petrić Howe

So, phosphorus is quite hard to detect and as you sort of said, we don't know that much about it. So, how confident would you say you are in these observations that this really is some kind of phosphorus containing molecules that you're seeing out there?

Lucy Ziurys

Extremely confident. When we do our observations, we look for a spectral fingerprints, okay. And if we find those fingerprints, we know that that molecule is there. This is this is the beauty of what we call high-resolution gas-phase spectroscopy. And so we are 100% confident we found phosphorus monoxide. Because we saw a quartet pattern, due to interactions of electron and nuclear spins. The lines are split into precisely four lines with certain frequency separations. It's all measured in the lab, we know those numbers exactly. We went to the cloud and those four lines are exactly there. For the other molecule, we had two separate transitions. And so it's–it's–it's unambiguous.

Nick Petrić Howe

So what do you think then are the implications of this finding?

Lucy Ziurys

Well, I think that number one, it suggests that there's probably another lower-mass source stellar source of phosphorus, such as something called asymptotic giant branch stars. And the other implications is that, you know, we consider some part of the Galaxy to be habitable. In other words, the conditions are such that, you know, life could evolve. And at the outer edge of the Galaxy, we're now finding phosphorus in these edge clouds, we now see all of these six N-CHOPS elements. And so that suggests that, you know, one finds exoplanets at the edge of the Galaxy, they may indeed be habitable.

Nick Petrić Howe

That was Lucy Ziurys, from the University of Arizona in the US. For more on that story, check out the show notes for a link to the paper.

Benjamin Thompson

Finally, on the show, it's time for the Briefing Chat, where we discuss a couple of articles that have been featured in the Nature Briefing. And Nick, I'm gonna go first this week, and I've got a story that I wrote about in Nature, and it's about NASA's OSIRIS-REx mission but in this case, it's got nothing to do with the asteroid sample that it collected.

Nick Petrić Howe

Okay, yeah, because we've talked about OSIRIS-REx a few times recently on the podcast, so nothing to do with the sample return. What is it to do with then?

Benjamin Thompson

Yeah, you're absolutely right. So we talked with reporter Alex Witze a few podcasts ago, she was on the ground, if you remember, actually when the OSIRIS-REx capsule sort of dropped through Earth's atmosphere and landed serenely on the ground with a sample of the asteroid Bennu inside. But other than containing this quite exciting payload, the capsule itself has given researchers the opportunity to learn about what happens when something enters the Earth's atmosphere very, very, very quickly. And in this case, the OSIRIS-REx capsule slammed into the Earth's atmosphere above the west coast of the US at 12 kilometres per second, which is hypersonic, and apparently one of the fastest human-made things ever to do so. And researchers obviously wanted to capture this and learn as much about it as they could.

Nick Petrić Howe

Right, yeah, because I guess this could be similar to something else crashing into Earth's atmosphere very quickly.

Benjamin Thompson

Bingo. So researchers sent up balloons and aeroplanes and set up seismometers, and all sorts of other equipment along the trajectory to collect this data then of what happened when the OSIRIS-REx capsule reentered the atmosphere. As you say, because sometimes things do that, and particularly meteors can hit the atmosphere, but when is really hard to guess, so knowing exactly when this was going to happen, was very, very useful. And the results of this reentry are still being analysed. But when the capsule hit the atmosphere it compressed air in front of it like a piston creating this glowing superheated plasma of ionised gas and sending sort of shockwaves out all over the place. And researchers have managed to sort of pick some things up for definite. And one of those is it made a quite quiet but distinctive double sonic boom–

Nick Petrić Howe

–ooh–

Benjamin Thompson

–and also fired out something called an infrasound signal that's a very-low-frequency vibration. And this may well have bounced off the ground and bounced back upwards as the capsule reentered.

Nick Petrić Howe

Well, I mean, there's a lot of cool terms for anyone who's writing a sci-fi novel about a meteorite impact coming out of this. But what can this sort of tell scientists more broadly about asteroids and meteors coming towards Earth?

Benjamin Thompson

I mean, I think that's the hope because we've seen quite dramatic examples of when asteroids enter the Earth's atmosphere. I remember the dashcam footage from one in 2013, that happened above a city in Russia. And this thing, cut an absolute trail through the sky and then exploded with the force of multiple atomic bombs, really something. But gathering data on that is quite rare. So in this case, knowing it was going to happen is hopefully given more of an insight into what processes occur when an asteroid does cut into the Earth's atmosphere. But it's not just that to be honest with you, it could also help maybe with the design of heat shields to protect reentering craft, because it's really hard to replicate the kind of conditions that these probes face when they enter the atmosphere in a lab. So actually seeing it in situ could help in the future. But also as well, infrasound detection has been used to listen for things like tsunamis and volcano eruptions and so forth. So, being able to really accurately work out what the signals are and where they come from, could potentially help in the future in early warning systems for things like this. And of course, this spacecraft is only the fifth non-lunar mission to return samples to Earth. So it's not something that happens very often. So, I guess we'll find out when researchers have picked through the data, what this reentry can help with.

Nick Petrić Howe

Well, I look forward to seeing what researchers come up with and more interesting facts about 'double sonic booms' and the like–

Benjamin Thompson

–ha ha ha–

Nick Petrić Howe

–but back down here on Earth, I've been reading a story in Nature. And it's about wildcats, specifically wildcats in Scotland, and how genetic research and the sort of genome research could help stop their slow extinction.

Benjamin Thompson

Right, and I've actually seen one of these wildcats in a wildlife park in central mainland Scotland, and it kind of looks like a tabby cat, right. And these things are pretty furious looking. I remember the person looking after it was trying to get it to eat some chickens to weigh it, and it was having none of it, right. It was sort of staying well, well away. And these cats are facing a bunch of different pressures, right?

Nick Petrić Howe

Yeah, so basically one of the biggest threats to these animals. So in this case, they are European wild cats, or the particular kind we're talking about here, they’re also known as Highlander tigers, they are becoming rarer and rarer because of hybridization with domestic cats, so they can interbreed with a domestic cats like you might have as a pet. And that sort of over time is making them less and less wild and more and more just like the domestic cat. So there are some groups that have been trying to prevent this, try and conserve the wildcats. And now new genomes and new genetic research could actually help with that as well.

Benjamin Thompson

Excellent. So, what's the plan then to try and rescue some of these wildcats?

Nick Petrić Howe

So specifically what's happened so far is that in 2019, a group called Saving Wildcats won some funding to do like a captive breeding programme. And so this is a place where they'll breed the cats away from visitors, away from people like you, Ben, who might come and have a look at them, and sort of breed them to try and get a sustainable wildcat population back into Scotland. And so far, they've released 19 wildcats into a set of land that is set aside for this purpose and the goal is release about 60 by 2026. But as I say, there are some sort of genetic research that can kind of help with that. So one of the interesting things that genetic research has revealed is that whilst these cats are under pressure from domestic cat DNA getting into their genomes, this is actually quite a recent thing. So, up until about the 1970s in Scotland, there was very little domestic cat DNA in these wildcats. So, something has changed. And by looking back in the past at different genomes, different wildcat genomes and different domestic cat genomes, researchers can try and unpick what exactly changed here.

Benjamin Thompson

And is the attempt to then to try and sort of undo the changes in the medium- to long-term?

Nick Petrić Howe

That is one of the things that some groups are trying to do. So, one of the things that researchers have discovered by looking at ancient genomes, more modern genomes, and also domestic and wildcat genomes, is that basically, it seems like there was very little mixing at all, even when the animals were living very close together. And this may be because of different behaviours. As you said, these wildcats are quite ferocious, they're quite fierce. They don't actually live very well with humans. So just that may have protected their genome in the past and then more recently, that has changed. We're not exactly sure what has changed in the 70s, and so on. But as you say, one thing that this genetic research can also help with, is to try and kind of undo what's been going on. So, by sequencing the genomes of breeding wildcats, you can work out what parts of their genome are, quote, unquote, 'wild', and what parts are domestic. And through doing that, you can then try and breed them with specific other kinds that have different parts of the genome that are wild, to end up with litters of baby cats that have more and more wild genomes over time.

Benjamin Thompson

So essentially then, you can breed the domestic cat out of the wildcat?

Nick Petrić Howe

That is certainly the hope. But there are some uncertainties with this as well. The big thing is that we don't exactly know what makes a wildcat, a wildcat or just a cat. So these species are quite similar- they're able to interbreed, they look quite similar, as you described, like the main differences, they've got kind of bushy tails, they may be a bit bigger, but they're not hugely different from domestic cats. So we don't exactly know what makes a wildcat a wildcat. And also, it's possible that some of the genes from domestic cats are actually useful for these wildcats to have. So there are many diseases that afflict wildcats that maybe domestic cat genomes have protected them from, we don't really know. So TBD on how well this will work and whether these uncertainties will come into play.

Benjamin Thompson

I mean, while we wait to find out, you say that some captivity-bred wildcats have actually been released into the wild, I guess, what do we know about that?

Nick Petrić Howe

Yeah, so 18 of the 19 released wildcats are still alive, they're occupying their habitat they're hunting so they seem to be quite successful, but it will take some time to really understand like how well they'll do. And in the meantime, to sort of prevent further hybridization, more domestic cat DNA getting into the future wildcats, the team behind the release of sort of implored the nearby residents to spay and neuter their pet cats to avoid further hybridization.

Benjamin Thompson

Well a fascinating story then, it’s one I'll certainly be keeping an eye on because I do like a good story about cats. But in the meantime, let's leave it there for this week's Briefing Chat and listeners for more on both of those stories look out for links in the show notes and a link where you can sign up to the Nature Briefing to have more science news delivered directly to your inbox.

Nick Petrić Howe

That's all for this week. As always, you can keep up with us on X we're @naturepodcast or you can send us an email to podcast@nature.com I'm Nick Petrić Howe...

Benjamin Thompson

...and I'm Benjamin Thompson. See you next time.