Botanical mystery solved: how plants make a crucial malaria drug

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1:53The Cologuard. Nature.

2:01Welcome back to the Nature Podcast. This week, I'm picking some puzzling plant biochemistry. I'm Benjamin Thompson.

2:23Plants of the Cinchona genus are fairly unassuming things. Native to South America, they're sometimes described as large shrubs and sometimes as small trees. But some species make some incredibly useful chemicals. For reasons that aren't entirely understood, these plants produce a diverse bunch of nitrogen-containing compounds, collectively known as cinchona alkaloids. The most well-known of these is quinine. Perhaps you know it as quinine, the earliest known naturally occurring malaria treatment. But there are others too, including molecules that play important roles as catalysts for chemical reactions.

3:08And although these alkaloids can be made in the lab, cinchona plants, also called cinchona plants, remain the biggest source of the molecules. But there's a catch. Nobody quite knows how the plants make them. Figuring out the biochemistry underpinning their production is of great interest to researchers, as it might allow them to adapt it to produce useful new molecules. But, to date, much of the molecular pathway remains a mystery. Things might be changing, however, as this week Nature has a paper that has shown evidence of solving this mystery, revealing the steps in the chemical pathway that the plants use.

3:51One of the authors of this work is Sarah O 'Connor from the Max Planck Institutes for Chemical Ecology in Germany. I called her up and asked her to lay out what previous efforts had uncovered about the pathway prior to this work. So there were a number of chemists in the 1960s who had done a lot of what's called isotopic feeding studies. So they made radioactive simple starting materials and they fed them to chinchona plants and then isolated the radioactive products and then worked out what the structures were. And based on those very tedious, difficult experiments, there were a couple of chemists who worked out what some of the chemical intermediates were in this biosynthetic pathway.

4:39So that's essentially what we had to work with. We had a little bit of a chemical framework for what the steps might be in this biosynthetic pathway. But given how well known the molecule is, it was a little bit surprising how little was known about how it was made. And this seems then like a detective story to work out how you get from the start to the end. And you say not much is known maybe about the middle. How in general do you go about trying to figure that out? I think you have to look very carefully at the extracts of the plant and really try to work out if there are any compounds in there that other people might have missed that might be biosynthetic intermediates along the way to the product that you're interested in.

5:23And that's what happened to us. We managed to stumble across one of these intermediates that nobody had ever seen before. And this really enabled us to be able to look for the genes that might encode these enzymes that might be responsible for synthesizing these new intermediates. So what you're saying, then you found a piece of the puzzle somewhere along the way. And from that, you could work out where the next piece goes. Exactly. Yeah. It was sort of like maybe we found, you know, a corner piece of the jigsaw puzzle. And I guess to make sure that these different intermediates that you found and that you pieced together were part of this, you had to essentially feed them to a plant to see if they were incorporated into the final product.

6:02Is that right? Exactly. So we would find a new intermediate and then we would make an isotopically labeled version of it and feed it back to the plant and make sure that we saw that isotopic label being incorporated into the final downstream quinine product. So essentially, we're trying to get a chemical roadmap. So you sort of get all of your chemical landmarks in place, and then you can go to all the sequence data and try to map the enzymes onto that. Right, because this is a biochemistry paper, and working out, I guess, the enzymes, the protein machines that catalyze one intermediate becoming another is a separate part of your work then and how challenging was that and how did you go about it?

6:43Yeah so a plant might express I don't know 40 ,000 or so genes and in that set of 40 ,000 genes you've got to find the one that encodes this single enzyme responsible for the usually very specific transformation and this is really where we've benefited enormously from all of the advancements in sequencing technology. So essentially what we can do is we can look and see which genes a single cell is expressing. We did most of our work in leaf. So you do get some of these compounds being synthesized in the very young leaves of the chinchona plant. Leaves have epidermal cells, just like people do.

7:22And it turns out these alkaloids are synthesized in epidermal cells. So instead of looking at the genes that were just expressed in young leaves, we could look at the genes that were just expressed in young leaf epidermal cells. And so that already eliminated a whole lot of extraneous genes that weren't involved in this process. So we could look at our sequence data, and we have these amazing computer programs now that will look at the sequence of a gene and make a best guess as to what type of a gene it is and try to work out which one of those genes might be most likely to catalyze some of these reactions.

8:02And when you sort of laid it all out in front of you then, what your evidence suggests is this full pathway. I mean, how did you feel? Because you say people have been trying to crack this knot for quite a long time. This was a hard one. This was a really hard pathway to solve. And, you know, sometimes the first pieces can be very difficult to come by. And then as you start filling in more and more information, you know, the puzzle comes together and it gets a little bit easier to solve. Except it turned out that the last enzyme that we found, which is in the middle of the pathway, it was the toughest one to find.

8:35We knew exactly the reaction that it would catalyze from all of the other experiments that we had done. And because we had found the enzyme before it and the enzyme after it, and the problem was we had no idea what type of protein might perform this reaction. So we really had to do both some very fancy state-of-the-art bioinformatics analysis, and then we combined that with some very old-fashioned, old-school biochemistry, where we ground up the tissues of the Chinchona plant in a mortar and pestle, and extracted out the proteins. And then we compared the proteins in the extract with the genes in this sequencing database and then combine the data sets.

9:20And from that came up with a list of 20 genes that might possibly catalyze this reaction. And one of the genes in those 20 actually did catalyze the reaction. And one of the things you did to add weight to the evidence that you're showing in this paper is you took the whole system and put it into a plant that doesn't do this. Yeah. So this is a plan called Nicosiana benthamiana. This is a model system that's been worked on by many, many people. And we can take our Chinchona alkaloid biosynthetic genes. And what we can do is we can introduce those genes into the leaf of the Nicotiana benthamyana plant.

9:58So now Nicotiana is able to express the proteins that those genes encode. And we can also syringe in the upstream early substrates that those proteins utilize that Nicotiana benthamiana does not make. And after we do that, we wait two days and then we can cut off the leaf of that Nicotiana plant. We can grind it up with a little bit of methanol and then we can see these chinchona alkaloids in the Nicotiana benthamiana methanolic extracts. Obviously, a lot of people have been looking at this pathway for a long time. How confident are you that this really is what's going on? And what do you think other folk will make of it when they see your paper?

10:38I hope they like it. But so, I mean, I think we are very convinced that these enzymes are doing what we think they're doing. And as additional validation, what we also did, not for every gene that we reported in the paper, but for a number of them, we also worked out a way to turn off those genes in chinchona. We could then look to see if the alkaloids in chinchona got messed up, and they did. So that's really nice evidence that these plants are actually using these genes in the pathway. And what questions are left, would you say? What does this work not do? The one step that we were a little bit more cautious about in the manuscript was the very last step.

11:23It converts quinanone into quinine. And this step, we were never able to get to work in Nicotiana benthamiana for reasons that we don't know. and we are on the hunt to see if the Chinchona plant has a more efficient version of this enzyme. And we think that we are going to have the best chance to find this gene in the root tissue rather than the leaf tissue, which is what we use primarily for this study. The other point that has us a little bit stumped is in the Chinchona plant, you find two versions of quinine. You find a version with a double bond and a version with a single bond. and we can only reconstitute the version with the single bond.

12:06So clearly the Chinchona plant has a trick that enables the production of the mixture. And so we have some ideas for how the plant might make that double bond. And when you do get to the bottom of it and answer all these questions, what use do you think this knowledge may have in future? So these are very valuable compounds and they're not just valuable in and of themselves, They're valuable as what we call scaffolds. So in other words, if you can take the scaffold of this molecule, but maybe make little changes to it, you can dramatically alter the pharmacological properties of these compounds.

12:44So we're really interested in the idea of being able to use nature's biosynthetic machinery and to try to trick it into making analogs or derivatives or non-natural versions of these compounds that might have new and improved medicinal properties. And we hinted at it in the paper because we showed that if we took all these genes in Nicoshana benthamiana and we fed it, for example, the natural substrate that had a fluorine on it, you know, this atom that you don't find very often in nature, but nevertheless is often found in drugs because it very often will endow a molecule with good medicinal properties, we could see that these biosynthetic enzymes could actually turn over the substrates with this fluorine molecule incorporated in it.

13:31So it would be a way to get fluorinated quinine derivatives. And lastly, it seems that one question folk have been scratching their heads about is why these plants produce these alkaloid compounds at all. I mean, does your work offer any insight into what they're doing, do you think? This paper does not. And I had not really given much thought to this question earlier in my career. But I am now located at this institute, which has ecology in the name. And many of my colleagues here, they're interested in exactly that question. And so we've been able to do a few studies looking at the toxicity and the impact on insects that might be eating the leaves of the chinchona plant.

14:13So maybe then that they're essentially an insecticide of sorts. That's a working hypothesis, yes. Sarah O 'Connor from the Max Planck Institute for Chemical Ecology there. To read her paper, look out for a link in the show notes. Finally this week, and in a slight change to our regularly scheduled programming, it's time for the research highlights with Dan Fox.

14:39Improvements in the quality of batteries will help electric vehicles to keep up in the tough conditions imposed by global warming. Lithium-ion batteries are very durable in mild climates. But as temperatures rise, durability starts to disappear. They degrade three times faster in hot US climates than in more temperate ones, raising the question of whether global warming will cause problems for electric vehicles. Researchers combined models of electric vehicle and battery degradation with predicted climate data in various cities around the world to calculate how these batteries might fare under a variety of conditions.

15:18They found that batteries produced between 2019 and 2023 would lose an average of just 3 % of life expectancy at the higher temperatures, compared with an average of 8 % for batteries produced from 2010 to 2018. The authors suggest that advances in battery technology can double as an important climate adaptation strategy as well. You can find that paper in Nature Climate Change.

15:48Ancient Peruvians had a particular fancy for parrots, importing live specimens from the other side of the Andes mountain range, hundreds of kilometres away. Members of the pre-Inca Ishma culture, which flourished in what is now Peru between the years 1000 and 1470, used vibrantly coloured parrot feathers to make ornaments and other items. Researchers analysed DNA from feathers discovered in an Ishma tomb. All of the colourful feathers came from four large parrot species native to the Amazon rainforest, including the red and green macaw, which can grow to nearly one metre in length. The team think the birds were transported alive over the Andes from the Amazon, a journey of hundreds of kilometres.

16:33Isotopes in the feathers suggest the parrots were reared on maize and other foods typical of coastal Peru, but the genetic diversity observed in the feather DNA was similar to that of wild populations, indicating that the birds were not bred in captivity. The authors say that such long-range transport and trade of live birds challenges the notion that pre-Incan cultures did not interact much, except during regional conflicts. You don't have to cross a mountain range to reach that research, it's in Nature Communications.

17:08And that's all for this time. We'll be back later in the week with the Briefing Podcast, but until then, you can reach out to us on social media, we're at Nature Podcast, and of course, we're on email too, podcast at nature.com. I'm Benjamin Thompson. Thanks for listening.

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