Ученая объяснила, как малейшие изменения в структуре пептидов влияют на создание новых лекарств

Dose of Discovery: Anne Conibear on how small changes reshape peptide and protein biology How toxic cone snails and a "combination of synthesis and structure" led one scientist into the world of peptide research. What can cone snail venom teach us about peptides and new medicines? In this episode of Dose of Discovery, Anne Conibear joins Søren and AJ to explain how small chemical changes can affect protein and peptide behavior in the cell. A lockdown project investigating cone snail venom gave Anne the opportunity to explore how these slow-moving predators use potent peptide toxins to paralyze their prey and how small modifications can make those toxins even more effective. She also discusses how scientists build and study proteins in the lab and how understanding post-translational changes could eventually help develop more targeted medicines. Anne reflects on a career that has taken her from South Africa to Australia and Europe and what she’s learned from working in different research cultures. The discussion moves into the different priorities of academia and industry, how teaching has helped shape Anne’s career, and what the future might hold for peptide medicines. You can check out the podcast on Spotify, Apple, Amazon, or by importing our RSS feed into your app of choice. You can also scan the QR code below to pull the podcast up directly on your phone: In this episode - 02:40 – Explainer: What are peptides and GLP-1s? - 07:25 – How Anne got into peptide research - 08:00 – What is NMR? - 12:57 – The chemical changes that alter how proteins behave - 16:23 – In the lab: peptides as a tool and subject of study - 18:43 – Anne’s journey from South Africa to Australia and Europe - 25:38 – Moving between academia and industry - 32:06 – Impacting science through teaching - 33:51 – Wherein lockdown leads to a cone snail venom project - 36:47 – How chemical changes make snail toxins more potent - 40:31 – What does HMGN1 do and why study it? - 44:09 – The effect of post-translational modification on HMGN1 - 49:08 – Wrap-up and key takeaways About the guest Anne Conibear is an assistant professor at the Institute of Applied Synthetic Chemistry at the Technical University of Vienna (TU Wien). Her research combines peptide chemistry and structural biology to understand how proteins are modified after they’re made and how those changes affect their behavior. Her work has touched on everything from improving our understanding of cone snail venom to proteins involved in DNA packing in the cell. Dose of Discovery is cohosted by Søren Hough and Ayokunmi Ajetunmobi (AJ) and produced by Suze Cooper. Read more Making peptides a ‘staple’ of cancer treatment by Søren Hough (Interview with Peter ‘t Hart, Part 1) AI-designed peptides 'blew minds' of researchers, says professor by Søren Hough (Interview with Peter ‘t Hart, Part 2) An academic’s journey into peptide therapeutics by Søren Hough (Interview with Nathalie Grob) The pharma industry finally believes in peptides, says leading professor by Nnenna Ohaka (Interview with David Craik) GLP-1s forced industry’s hand on sustainable peptide synthesis, says expert by Søren Hough (Interview with Fernando Albericio about green synthesis) Episode transcript Søren: Welcome to Dose of Discovery, a dose of breakthrough medicine delivered with your morning coffee. Coffee not included. In today's episode, we'll be speaking with Anne Conibear, assistant professor at the Institute for Applied Synthetic Chemistry at the Technical University of Vienna (TU Wien). So AJ, this is, I think, an important episode. We've been so focused for the most part on nucleic acids, and finally we're getting to talk about peptides, which is obviously another huge area of research and a big area of what we cover at BioXconomy – but not something we've talked about a huge amount on this show. So, yeah. Are you excited? AJ: Very much so. It's hugely topical, not least because of its impact in developing novel obesity drugs through GLP-1s, etc. So, yeah, it's great to finally be talking about peptides with an expert in the field. Søren: Yeah. So let's start by getting a little bit of a lowdown on our guest. AJ: Sure thing. Anne received her bachelor's from Rhodes University in South Africa in 2008 and her master's in chemistry at Rhodes University in 2010. She completed her PhD at the University of Queensland in Australia under David Craik. She was then a fellow at the University of Vienna, where she also worked at Syntab Therapeutics. She was a UQ development fellow at the University of Queensland before joining the faculty of TU Wien in 2022. Søren: Great. And I think one of the things that really jumps out immediately about her background is the continent hopping, which will be interesting to get into, and also the jumps between industry and academia. I think that always speaks to an interesting hybrid. AJ: Yeah, I think it's definitely become more prominent in sciences today to get exposure to industry and academia. It has been interesting that a lot of the folks we have talked to over the course of the podcast, whether they've developed an academic career or ended up in industry, have straddled both sides. And I think it's actually critical, right? Because there's so much learning that you can take from one to the other, and the earlier in your career that you can make use of that, and take advantage of that, in my mind, the more impactful your career is going to be. So, yeah, I'm really looking forward to digging into that with Anne and understanding what she was able to take from each experience. [MUSIC] Søren: Everyone is talking about peptides. You may have heard about GLP-1s, things like Ozempic/Wegovy [semaglutide] and Mounjaro [tirzepatide]. These are peptides, but they're just one kind of peptide. Peptides are essentially, in simple terms, super small proteins. They're little strings of amino acids. So, they're much, much smaller, which allows them to be therapeutically relevant in ways that perhaps full-sized proteins can't. They come in all sorts of shapes and configurations. Sometimes they're little circles with cyclic peptides. Sometimes they're stapled, so they're sort of looped in on themselves. Sometimes they're knotted. There's a lot of different ways that peptides can manifest. When we think about peptides, we often imagine them as a very modern modality, but in reality, peptides have been around for over a hundred years. The first peptide was synthesized in 1901. You compare that to ASOs, which came out in the 1970s, siRNAs in the 1990s, CRISPR obviously in the 2010s; peptides are much, much older and they have huge applications. There are things like GLP-1s, but also well before that, insulin. Lots of people are aware of insulin and insulin costs, and it's really important for people with diabetes, but insulin is also a peptide. And I think perhaps that connection hasn't really been made by the public. The other thing I would just say about peptides is that, though not always, they're often also hormones. We often think about hormone therapy as potentially a different modality, but peptides are often hormones. So things like GLP-1s are hormones, and I think we might have an interesting framing if we start thinking about GLP-1 therapy as a hormone therapy or insulin as a hormone therapy. I think "peptide" can maybe distance us a little bit from that particular modality. The other aspect of Anne's work that I want to preface, because I think it's probably useful for folks to have an understanding of this area of research, is in post-translational modification. This is actually something I investigated in my own PhD. Post-translational modification, or PTM, is essentially when a protein or peptide has been made and it can be modified in some way: either it has been phosphorylated or it has been ubiquitylated, some sort of change has been made to that product, that peptide or protein, and that change can be very, very important in that protein's behavior. Whether or not the protein goes to the nucleus or the cytoplasm, or whether or not that protein goes to sites of DNA damage, or whether or not that protein gets marked to be destroyed or degraded by the cell is all dictated by PTMs. What Anne looks at is PTMs both in the context of proteins but also in peptides. It's just worth keeping that idea in the back of your mind as you listen to this interview, because it should help clarify some of those points. The other aspect of Anne's work that we're probably going to talk about is NMR, which is nuclear magnetic resonance. We don't need to get into the weeds on this, but NMR is a great way of figuring out what the structure of a protein or a peptide is. You can learn a lot about the protein by using NMR. So that is one of the techniques that you will potentially hear about because it's so instrumental in what Anne is doing to, again, look at things like PTMs and how they affect the behavior and potentially the structure of proteins and peptides. We'll be right back with Anne after a word from our sponsors. [MUSIC] Sponsor:Sponsored by Asymchem – a leading global CDMO with deep roots and a global footprint, we deliver seamless end-to-end pharma solutions. From small molecules to diversified modalities and our TIDES capabilities, they're a gamechanger for your complex programs. Trusted by top pharma and biotechs worldwide. With rock solid compliance and a real focus on leveraging the latest tech, we speed up your drug development and cut time to market. Partner with the CDMO that's got your back. Head to asymchem.com. [MUSIC] Søren: We're back with Anne Conibear, assistant professor at the Institute of Applied Synthetic Chemistry at the Technical University of Vienna. Thank you so much for joining us. Anne Conibear: Thanks, Søren. I'm looking forward to having a chat with you. Søren: Fantastic. So, I guess maybe I want to just start with the beginning of your academic journey, and then we can even go back a little bit further if you're so inclined. When you did your PhD, you were working under David Craik. Obviously, David Craik is known as the cyclotide guy, right? That is his big claim to fame. And these are really cool peptides. They're circular and they have some therapeutic potential. Since then, your interests have shifted towards other areas like using peptides as tools and in particular, to using those peptides as tools to understand things like PTMs. Can you talk a little bit about how you came to peptide and protein biology in the first place and then how that eventually led you to focus on PTMs in particular? Anne Conibear: Sure. I didn't actually originally choose the PhD lab or David's lab for peptides. I chose it because of his expertise in NMR spectroscopy, actually. So during my master's program at Rhodes University in South Africa, I was introduced to NMR spectroscopy and was absolutely fascinated by how you could see what molecules look like in 3D. Søren: Do you mind just giving a super quick layman's explanation of NMR? Usually with protein structure, we often talk about things like cryo-EM and crystal structure, and using that approach to determine the structure of a protein, but you have a very NMR focus – is that a size issue? It would be good to understand what NMR is and why you use it. Anne Conibear: Yes, exactly. So as you already mentioned, especially for X-ray crystallography – there has been huge research done on that, and a lot of what these protein prediction prediction programs like AlphaFold are built on, are built on all the knowledge we've gained from X-ray crystallography – so certainly in no way to diminish that, but to get an X-ray crystal structure, you need a crystal. And if you can't get a crystal, you can't get a crystal structure. Especially for these intrinsically disordered proteins, which are very dynamic and are not going to crystallize, they're just not there in the X-ray crystallography data. And even proteins that are well-structured but have a disordered tail or linker, usually that section has no electron density and is practically invisible. So what I see as one of the advantages of NMR is, in combination with other methods like X-ray crystallography and cryo-EM, to then be able to see those dynamic parts of proteins or proteins that are intrinsically disordered. There, the advantage is that usually we're often working in solution. We're looking at a dynamic protein that's in a more native state than being in a crystal. I wouldn't say it's completely physiological. We can also study dynamics. NMR tells us a little bit about populations. And of course, we often see an average, but that can also tell us a little bit about the populations, how fast things are switching between one state and another. That's very important for proteins because most proteins are not static structures, whether it's a pocket that slightly opens up or closes, domains that rearrange around each other, or even a loop that folds back and forth to cover a binding pocket or something. That's where I see one of the big benefits of NMR: being able to “see” those parts of proteins that are invisible to other techniques. So when I was looking for PhDs, I was looking for a lab that had expertise in NMR spectroscopy, and of course David's name came up. Then I saw he also works on peptides, and I'd had interest in biological molecules. I thought, “Oh okay, this could be a good fit.” But I didn't actually originally choose it for the peptide side. Søren: That's really cool. And so, what led you to that from your master's and your bachelor's degree? What is it that attracted you about this field? Anne Conibear: So, when I joined David's lab, I found out a little bit more about peptides and was also fascinated by how you could synthesize them using chemistry that I'd learned during my undergrad course in the lab, but synthesize them as biological molecules and as biologically functional molecules, and then to study their three-dimensional structure. It was that combination of synthesis and structure, and that's stayed with me. Also, the peptide side has stayed with me. Since then I've worked on bigger and bigger peptides, and now into proteins, but still keeping that combination of synthesis and structure and biological function. Søren: One of the things that I am really interested in is what you see the value of studying PTMs is. Why is it that we want to understand how proteins are modified after they've been created? Because it feels quite niche, but in fact, some would argue it is that fourth step of the central dogma that people don't talk about but should, because it's actually really important. Anne Conibear: Yes, and it is exactly that reason. I felt like when I was moving into this field that there was a lot of new information and excitement about genes and mutations and all the information we could get out of our genome, which is of course true and very valid, but there's also so much that happens to proteins after they're transcribed and translated. I found it fascinating how these post-translational modifications can then very finely tune activities of proteins, which means that the cell doesn't have to go all the way back to the beginning and make a new protein if the environment changes or the stimulus changes, but they can modify the proteins that are already there. And also, the many open questions, because they're in some ways much harder to study because that information is not explicitly encoded in the genome. We can use chemical tools and structural tools to start to tease out those fine, subtle regulations of those proteins. Søren: Perhaps it would be helpful to go through some of these PTMs to understand, not in specific phosphorylation or ubiquitylation, but more in terms of what the outcomes are. If you have a protein and you are attaching a ubiquitin group onto it or a phosphoryl group or one of these other elements, or just modifying it with branches, what's the consequence? What actually happens, and what can the effect be, in a more macro sense, on the cell and potentially the organism? Anne Conibear: This depends a lot on the protein and the modification. Perhaps also for a more general audience: the modifications themselves have a huge diversity. We can have anything from very small modifications, whether it's a single methyl group or a phosphate group; to medium-sized modifications, perhaps a lipid chain or a chain of phosphates; or to rather large modifications, such as, as you mentioned, ubiquitin, which itself is a protein. So, the modifications themselves are very diverse and they can change different functions of proteins, whether it's their three-dimensional structure, their electrostatic interactions, the charge they have on their surface, steric interactions with other proteins – perhaps adding on a bulky group that prevents an interaction with another protein – or, often, where they go in a cell. For example, when proteins are modified by a lipid chain, that often directs them to the membrane of a cell. Or, for example, at different stages of the cell cycle, whether they move into the nucleus or out of the nucleus. Or for cell signaling, whether they transfer a signal from the surface of a cell to some process within the cell. Søren: Do you think about these long-term applications of what you're doing, for example your work on HMGN1, which I'm sure we can talk about in a bit? What impact that might have in a clinical setting or in a translational lab, for example? Anne Conibear: Yes, and of course that is always our longer-term goal. We work rather on the basic end of the science side of it, but one of the drivers of that is to understand better how differently modified proteins function in the cell, but also, sometimes, how they might react to a drug molecule. Often, drugs are designed against a protein target, and often that might be a recombinantly expressed protein from bacteria, which might not have all the native modifications, and therefore it's only designed against one variant of the protein, whereas perhaps in the cell there might be ten, 20, or 100 variants of that, and we don't know the effect. We seldom test the effect of drugs on modified proteins, so I think there's a lot of space there for understanding better how the therapeutics we have affect modified proteins, or vice versa. And also, in the future, if we understand how modified proteins work, perhaps we can better design therapeutics targeted toward a modified variant of a protein, which might actually be a stronger disease driver, for example, than the unmodified form, and perhaps even get better selectivity and specificity than we can with the unmodified proteins. Søren: That's really interesting. Before I hand over to AJ, I think it might be helpful to get an idea of what your lab actually does in a general sense. On one hand, you're a peptide lab where you use peptides to build proteins, and maybe if you could talk a little bit about why you do that. And then on the other hand, you also look at peptides themselves because peptides are also post-translationally modified as well, of course, and so you've also looked at that. And so: peptides as an object of discovery and study and then also as a tool. Anne Conibear: Yes, and there, the definition between a peptide and a protein is a rather fuzzy definition because, at least in my view, a peptide is a small protein, and a protein is a large peptide. But basically, we use the tools of chemistry to be able to make peptides. And because the chemistry we use to make peptides – this is using solid-phase peptide synthesis – has some limitations in terms of length, because we're doing many, many, many reactions over and over on the solid phase, side products and deletion products accumulate as the peptide gets longer. It's often more efficient to synthesize a segment of a protein, and there we can really introduce very specific modifications, whether natural or unnatural. So there we can use the tools of chemistry to bring very specific modifications. And then, because we often can't synthesize the whole protein if it's larger, then either we join bits of segments of the protein together using ligation reactions to build up the whole protein, or we perhaps often express the rest of the protein in bacteria, because they're quite good at making bigger proteins but not introducing modifications, and then join the two together. So we focus the synthesis effort on the part that's modified and then build up the rest of the protein around that. Søren: So you would build a truncation and then a peptide is the last bit of it, or vice versa where it's the beginning of the protein or some other element? Anne Conibear: Exactly. And the beginning or the end is usually easier. We can also do bits in the middle, but then you have to have at least three segments and then two ligations. So that is certainly something we've done, also with HMGN1, but the more segments and the more ligations, the harder it gets. AJ: Anne, I'm interested in taking you back through your career and understanding your motivations behind different career moves. By my count, you've carried out research in three different continents from Africa to Australia to Europe, various fellowships, and of course now a faculty position. Can you tell us a little bit about what motivated each of these moves, and then tell us what it's like carrying out research in different parts of the world. What have you taken from different parts of the world to drive your research moving forward? Anne Conibear: Sure. As you say, I have certainly taken a lot from it. I didn't originally plan to move around so much, and I didn't start out my university career saying, "I'm going to go there and then Australia and then Europe and then back and then back." To be honest, if I'd known beforehand, I probably wouldn't have gone that route. But it's not something I regret because, as you say, I've taken a lot from it. And I think that's also again down to curiosity. I think because I'm curious about science, about how things work, I'm also curious about new places and getting to know new people, and how different places work, how different languages work. I think that's helped with the moves, because I've gone through each move with the mindset of, "Let's go and discover something, explore a new place." It's not always easy. You have to step out of your comfort zone, adapt to new cultures, and figure out new systems and how things work. But I think I've gained more from that than I ever would have realized. AJ: That's amazing. And specifically, that period of time where you were moving back and forth between Austria and Australia, what was that like for you? Were you motivated by certain expertise that was held at different institutions and trying to draw bridges and connections between them? Anne Conibear: Yes, partly. To be very practical, I was also sometimes forced to move by the realities of the academic system, and I think that having the flexibility to move also enabled me to follow my interests better, because as we know, in the academic system, you're often tied to these short-term contracts and then have to move around after that. Because I was relatively open to moving to new places, I could then broaden where I looked and follow my interests rather than saying, "I have to stay in this place and then just find whatever I can." That's a personal decision or situation that everyone has to figure out for themselves, with its pluses and minuses. So yes, the moves were motivated in some cases by my contracts ending and needing to find something else. But then also: where do I see myself growing? Where can I move and do something that's linked to what I've done before but not carry on doing the same thing? Where can I go and learn new expertise? As I was mentioning, when I finished my master's in South Africa, I was looking for a lab that specialized in NMR spectroscopy for my PhD, and so found David's lab in Australia, and that's where I got introduced to peptides and then it moved on from there. Then I moved to Christian Becker's lab in Vienna and there got introduced to how to work with bigger proteins and recombinantly express sections of proteins. So that has built my research direction in some ways intentionally and some ways by picking up what was there in the environment and then moving forward with it. And I think that's again one of the biggest benefits of moving around: that you pick up these things kind of without looking for them sometimes. AJ: We tend to get a lot of early career academics listening to the podcast. They're looking to engage with our speakers and understand their career arc and career trajectory. Often, one of the things that is most revealing is the emotional roller coaster of building a career in academia with the holy grail of reaching tenure, which is kind of difficult given the number of PhDs that get produced and the relatively limited number of tenure-track positions. Can you tell us a little bit about your own experience with that and also what it was like eventually achieving a tenured position at the Technical University of Vienna? Anne Conibear: Yes, sure. And I should also be careful to say my tenure is under evaluation at the moment, so it's not a done deal yet. But yes, when I started out in research during my master's and PhD, I also wasn't absolutely fixed on going the academic path. I was open to it, but I think it was very useful that during the PhD, through various mentors, through my supervisor, and also peer groups at the university, it was quite well-communicated to us what it takes. There were also several postdocs in the group who were going through that really difficult stage of trying to move from being a postdoc to being independent, and seeing that as a PhD student, I think in hindsight, was very valuable because I was perhaps a little less naïve than some others in seeing how hard it was for them. So, yeah, always keeping in mind probably a more realistic view that yes, I'm keen to go for it, I'll give it my best shot, but in some cases it does come down to being in the right place at the right time and a bit of luck, but also being intentional. And I think a very important part is being aware of how the system works. Of course, people come with a huge range of experience depending on what background they have, where they've studied, and how actively they've been mentored and what exposure they've had. But learning some of those things through mentors, peer groups, and collaborators, I think, has been very helpful. And I must say, I underestimated in the early steps how important mentorship and community are: that actually, in the end, it's the people. You have to do good science – there's no getting around that – but just doing good science is not enough. AJ: Yeah, for sure. It's probably useful to touch on the fact that you didn't just have exposure to academia, but you also had exposure to industry, right? Which has its own ecosystem and systems and community. In 2019, I think you took a fellowship where you worked between the University of Vienna and Syntab Therapeutics. Can you tell us a little bit about what it was like living between these two worlds, which absolutely rely on each other, but can seem an ocean apart? What was that like? Anne Conibear: So, one of the things that attracted me to that postdoc was, as I was finishing my PhD, also having heard how tough it can be to stay in academia, I was actually moving more in the direction of, "I think I'll move into industry. I think that's where I can see myself." To build a bit of that bridge, I was looking for a postdoc where I could start to put my foot in that industry ballpark because before then I had zero experience with industry. It still ended up being much more on the academic side. I was based in the university. It was a project that was linked with a spin-off company that had come out of my supervisor's previous lab. So it gave me some sense of industry in a very small, academic kind of sense. I also did a short internship at Roche Pharmaceuticals during my PhD and saw a little bit from my PhD supervisor when he had a collaboration with Pfizer. That showed me how this academic-industry collaboration worked, and how that's often portrayed as being the ideal. It’s great, but some of the really down-to-earth differences are where those two cultures clash. So it was helpful for me to realize how different the priorities are there and what the drivers are. Not to say that one is better than the other, but for me, I actually realized during that that if I followed the industry route, what I would miss the most would be the teaching aspect. As far as I could see at the time, academia was one of the few ways that you can really still combine research and teaching on a day-to-day level. There are not that many other careers where you can do that. So that was one of the deciding factors for me. I thought, "I want to stay in research, but if I was doing only research, I'd actually miss the teaching." AJ: I'd love to dig into what aspects of teaching you most enjoy and why that's such a passion for you. But let’s talk about this culture clash between academia and industry because it would be interesting to understand that from your perspective a little bit further. What were you actually doing? What was your involvement between these two worlds, and what was the focus of Syntab Therapeutics? How did you engage with their priorities? Anne Conibear: So, we were making these synthetic immune system engagers. They had one peptide that was binding to cancer cells and one that was supposed to then activate the immune system. I was exploring the chemistry of how to join these together and what ratios to join them. Do we have two molecules of the binder and two molecules of the stimulator, or one and two? Again, very much on the chemistry basic research side. And then the collaboration was that they were then testing these molecules, and that I saw as an advantage because they had the assays set up for testing that we didn't have in the university lab. And I could keep a bit of the freedom because, also as a postdoc, I was under pressure to publish. I made sure that there were still some chemistry synthesis aspects of it that the company was not all that interested in for their purposes, and so I was then free to try and push some publications out on that. That is one of the big differences I noticed: especially at the early stage of the [academic] career, where we have this big pressure to publish because that's what a lot of things are decided on, against what's the priority of the company, what information we can reveal and disseminate. And also having the freedom to follow those interesting things that perhaps are, again, not the priority of the company but are just scientifically interesting, whereas in an industry setting, you probably wouldn't have the freedom to do that. AJ: I suppose it's much more about hitting the next milestone or hitting the next inflection point as opposed to being able to pursue curiosity-driven research, which is a theme we often touch on. Anne Conibear: But that's something I think we can, and I certainly should, learn more from the industry perspective: defining these milestones and making decisions, because I know for myself in academia we often tend to put off decisions and just follow what's interesting. Often we get to a point where in hindsight we should have made a hard decision earlier as to whether to cut something or pursue it. So that's certainly something I can and should still learn. Søren: Yeah, that sunk cost fallacy is definitely a thing. It's a thing in industry as well, but I feel like it can definitely affect academia. AJ: 100%. I just want to tease out that bit you mentioned around teaching and how that's such a fundamentally important aspect of what you do. It's not just the ability to carry out research, but to be able to communicate that research to students and others. Can you expand on what makes teaching such an important core part of your day-to-day? Anne Conibear: Sure. I think, in some ways, myself and my science benefit a lot from teaching, because to teach something you have to really understand it well. I find it very stimulating to hear the questions of students, and especially in discussions with my research students, realizing how differently they think from me and from each other, how differently they approach problems, certainly gives me different perspectives. As scientists, I think many of us have this desire to have some kind of impact, that our science drives something forward. And of course, I also aim for that in my science, but I see a great opportunity for doing that also through my teaching and in mentoring other young scientists, because there, I think, the impact can be far longer term, perhaps far more impactful, both on a one-to-one personal level but also broader on a scientific level. So I also see it as an opportunity to have impact in two directions. Søren: I think that the exchange between professors and students, teachers and students, graduate students and undergrads, is so valuable. Anne Conibear: Yes, and not only the questions, also the way of representing data and sometimes making figures. I'm quite a visual person, so I really like figures and representing data. Sometimes when a student comes to me and says, "Oh, these are the data I got, I plotted it like this," I'm like, "Okay, I wouldn't have plotted it like that, but if you represent it like that, we see different information that perhaps I wouldn't have seen if I just plotted it my standard way." Or they come up with a draft figure for a manuscript and, "Oh, was that really even the question we were asking? But maybe we should ask that question." So, yes, I also learn a lot from that. Søren: Yeah, it's interesting thinking about how things are displayed, and how that can lead to new questions. So, maybe just to pivot back to your lab and your work: I watched a talk that you gave last year, back in 2025, and I was intrigued because I think it kind of speaks to the two aspects of what your lab does. One was HMGN1, which we've mentioned, and I'd like to hear a little bit about that story, but maybe let's start with the first story, which is about the snail that produces a toxin that's actually a peptide that has a modification on it. What led you to study this and where did the research ultimately take you? Anne Conibear: So the question about what led me to study that is, again, not a super scientific reason. I'd gone back to Australia after my postdoc in Vienna and was starting up my small independent research. Of course, having worked in David Craik's lab, I had exposure to conotoxins, although I hadn't worked on them myself during my PhD. The situation was that COVID came, there was a big lockdown, and I was in the lab over a summer responsible for four undergrad students. I had to come up with a project for them that we could do in shifts if necessary, or that each one could do a little bit but not everyone had to be in the lab at the same time together – that was fairly feasible, but that eventually we could put all the bits of data together to build a story. So it was a very practical reason. And then I thought, "Okay, what's a short, simple peptide? Where's a modification? Each student can make one variant, then I can show them how to do synthesis all together, and each one can make a variant when they come into the lab." So that was really the reason, the honest reason, why we did this project – of course also because there were modifications and it was interesting. Conotoxins are great, small, structured peptides that are usually synthetically accessible, interesting to look at with NMR spectroscopy to study their structures. Also, in hindsight [it] turned out to be quite a good model peptide for looking at how these modifications change structure and function, because they're small and we can relatively quickly generate a number of variants, which for a bigger protein would take a lot more effort and be a lot harder to study structurally. Søren: So just to be clear, so these are poisonous snails – maybe I should say venomous snails – so they'll sting you if you step on them? Is that the idea? Just out of curiosity, how does this toxin manifest in nature? Anne Conibear: In nature, they're obviously not out to catch humans. Their natural prey is worms and other snails, sometimes fish, for some of the species. They make this cocktail of toxins in their gut. They have what’s like a hypodermic needle that they shoot into their prey. It very quickly delivers this cocktail of very often very potent toxin peptides that usually have very fast effects on the nervous system and then paralyze their prey. Because as snails, they don't move very fast, and often their prey moves faster. They have to usually paralyze it very quickly so that they can then eat it. Of course, if you then happen to annoy one or step on it, you might also get a dose of these toxins. But they're not usually out to hunt humans. Søren: You found that there was a modification on one of the peptides that these snails produce? What was the impact of that modification? Anne Conibear: Actually, almost all of the conotoxin peptides are modified in some way, and some modifications are more common than others. In the first case, we looked at tyrosine sulfation, which is a much less common modification. At least it's less reported. We think it may actually be there a lot more than we think because it often falls off or is not visible in the mass spec analysis mode that's often used to identify these toxins. So I didn't identify it at first; it was reported in a database and I thought, "Let's synthesize it and see, why is it there? What does it do?" So in this case, we synthesized the sulfated variant and the unsulfated variant, also a variant with a C-terminal amidation and one without, also to see which of these modifications individually or in combination affects the structure and biological function at the N-acetylcholine receptors. Basically, both modifications increase the activity at the N-acetylcholine receptor. We found that the C-terminal amidation has a bigger structural effect than the tyrosine sulfation, which we had sort of predicted that because the sulfation is a bit sticking out on a side chain, whereas the C-terminal amidation curves around and helps to stabilize a helical turn. So it was interesting to see these subtle differences. It wasn't the overall structure, but more the structural stability, and we think that might help to pre-arrange the molecule to fit better into the binding pocket of the receptor. But the sulfation might then add the electronegative charge – because the sulfate group is negatively charged – to promote interactions with the receptor there. We found that both modifications together then further increased the activity. Søren: That's really interesting, too, because we think of the central dogma as “we're making these proteins” and that's kind of it. But in this case, this is what's making that a potentially more potent toxin. And just to clarify, you're talking about receptors in whatever organism has been stung by this snail? Anne Conibear: Yes. So, we tested it on human N-acetylcholine receptors, because it is also active on human N-acetylcholine receptors, but we also tested it on a mollusk analog of the receptor because that's probably what it was designed against. As I said, they're not usually out to hunt humans. But many of these molecules are potent on human receptors, as well. It was interesting to see that the gene for this protein would be exactly the same, but every modification requires some investment of energy and resources to put it there. So my argument was: if these cone snails are investing effort in modifying these proteins and diversifying these toxins, there must be a reason for that. Søren: That makes a lot of sense. So that's one side of the peptide research that you're doing. On the other side, the analogy that I like to use is that you're using peptides almost like LEGO or building blocks to assemble and put together larger proteins or create protein variants. As you mentioned, making a protein with bacteria makes the basic protein, but it doesn't have any of these modifications on it, whereas making it through synthesis, you can actually put these modifications on. So could you talk a little bit about your HMGN1 project: what does HMGN1 do and what were you trying to understand about how post-translational modification affects its function? Why focus on HMGN1? Anne Conibear: One of the reasons we focused on it is because it's intrinsically disordered, which means that it doesn't have a very defined structure. There are many intrinsically disordered proteins in our genome, in our proteome, and many disordered parts of structured proteins as well. These are the parts of proteins that are often most highly modified, as well, so that was the connection there to the modification interest. We assume that's because they're flexible; they're more accessible to the enzymes that modify them, but therefore they're also often the regulatory regions of proteins because they are modified. Because they're flexible, they often have multiple interactions. It's not a simple "one protein interacts with another" or "a protein binds to a specific ligand." They tend to be much more fuzzy interactions and fuzzy conformational ensembles rather than a specific structure. These kinds of conformational ensembles can be well-studied by NMR spectroscopy, because we're looking at them in solution. We're not looking at a solid crystal. They're not the kind of proteins that will crystallize or that you can study using cryo-EM, for example. So one of my motivations for studying HMGN1 was because it was well-suited to structural conformational studies by nuclear magnetic resonance, by NMR spectroscopy. The other reason is, obviously, because it is post-translationally modified, and it was a reasonably accessible size and length to be able to synthesize. Something we often quietly forget on the synthesis side is that even if you can synthesize a protein, at some point, you have to refold it into its native structure, which can often be one of the hardest steps. The advantage of an intrinsically disordered protein is you get to skip that step. So that was also one of the practical reasons for that. But on a biological side, HMGN1, to explain, is a chromatin-associated protein. It binds to both sides of the nucleosome. Each nucleosome is made up of histone proteins wrapped around with DNA, and that helps allow DNA to pack into the cell into a very compact space. But then, when cells divide or when proteins get transcribed or genes get transcribed, the DNA has to unwrap to give access to the DNA, and then wrap up again. So these proteins that control the folding and unfolding of DNA for transcription and for cell division are very important for controlling replication and transcription. Søren: I guess my brain always goes to DNA repair and DNA replication because that's my background, but more broadly, I think it goes to clinical applications and thinking about these things. Was that part of the idea of looking at something like HMGN1 in those contexts? I think broadly with post-translational modifications, there are these downstream consequences to, for example, not packing your DNA properly in the cell, or in some way interfering with things like replication. Obviously, your focus was on the structure side of things – but did you also want to know, in an applied sense, where that understanding could go? It would also be good to know what you found with HMGN1, and what was the effect of PTMs on HMGN1's function? Anne Conibear: So far we've looked at post-translational modifications in the N-terminal domain, which is in the nuclear localization sequence, and in the nucleosome-binding domain. And there, we found that the two phosphorylation modifications in the nucleosome-binding domain seem to abolish the binding to nucleosomes. It's thought that these residues interact with the acidic patch of the nucleosome. So this acidic patch is quite negatively charged, and two phosphorylation modifications introduce negative charge on the binder, and so negative and negative probably repel each other. We would, as a next step, be interested to see if those modifications in the nuclear localization signal then affect where the protein goes in the cell cycle, because it's important that this protein moves into the nucleus and out of the nucleus at the right point of replication so that the DNA gets packed in the right way into cells. At the moment, I'm not seeing HMGN1 being a specific target for therapeutic development – I think that would be very difficult because it's so abundant and because it's intrinsically disordered – but more to understand in which processes it is important, and which modifications are important for these processes. Also, because it is thought to interfere with the modification of histones because it's very closely associated with histones. Probably the same enzymes that are modifying the histones, which can be very important epigenetic markers, are also modifying or competing with HMGN1, and that process is very poorly understood so far. AJ: So Anne, a final question to wrap up. Peptides have obviously exploded into the general public's consciousness with GLP-1s, I think most famously. On the discovery side, we've touched on approaches like cyclotides, but also, you know, stapled peptides, AI-driven design, and other initiatives in peptide synthesis. We've seen a green revolution in solid and hybrid synthesis. We had a previous episode with the CDMO of Asymchem talking about some of these remarkable new initiatives for peptide synthesis. As somebody who is at the coalface of all of this and driving this research forward, what has you most excited about the future of peptide research? Anne Conibear: I think, as you say, the future is exciting, and I think it will be really interesting to see how the peptide community grows, because until fairly recently, it has been a small and quite tight and rather friendly community. I hope it stays that way. But now that it's becoming so much bigger. We're learning about synthesizing peptides on scales that we never even dreamed of before. I think that will drive big changes on the synthesis side. The green aspects of peptide synthesis is a huge area for development. I'm curious to see whether, as the peptide field grows bigger and more and more people from other fields are now doing peptides, as well, it then divides into different applications, like the biological drug applications, or materials, or synthesis, whereas previously it was all together because it was a reasonably small community. That could have pros and cons. I think it will in any case be exciting. I think we have a lot to learn about synthesis on a large scale, but I think it will also drive more innovations in learning how to develop peptides as drugs. For example, now we're seeing many different formulations and modifications of peptides to then get them to where they need to go, have the stability and retention that they need to be drugs. All of those will require the chemistry to do that and also an understanding of what effect these modifications have on the peptides. AJ: Really exciting, and I think you're demonstrating across multiple avenues the ways that peptides can be used in ways that even I – having spoken to all these peptide researchers – I hadn't really thought about as a tool itself to then further understand proteins and how they're working. So it's really cool to hear about your research. Thank you so much for taking the time out to speak with us today. Anne Conibear: Thanks very much. Thanks for the invitation. [MUSIC] Søren: Well, that was fun. It's always a great opportunity to have a chance to speak with researchers early in their career. Just getting that sense of what it's like to start to find your niche in the industry and in academia, and figure out where your contributions are going to come from, is really difficult. So it was cool to get Anne's perspective on coming out of David Craik's laboratory, which is very focused on cyclotides, into quite a different area and to see how she's created a whole new avenue for discovery and cool ways to use peptides in multiple directions. I really appreciated that. AJ: You know, as I was listening to Anne, I thought back to our interview with Erik, actually. And I got a similar kind of vibe in terms of where peptide research is today and where, you know, CRISPR research was in circa 2008 before everything exploded. For Anne to be at the coalface of using peptide research as a tool and using it to understand how peptides work and potentially how they impact therapeutic applications must be super exciting for her. I can only imagine as we track the next ten, 15, 20 years, the size and scope of the impact her research is going to have in the field. I really saw that analogy and I'm really excited to see where it goes. Søren: For sure. I think also on that subject of early career, and I felt this personally, she talked about what it was like to try and work during COVID and lockdown and how it led to this kind of cool story about working with snails and looking at how they produce peptides as a self-defense mechanism. AJ: I thought that was so cool. Søren: It was very cool. Yeah. My PhD was interrupted by COVID, and that was really difficult. It was really disruptive. AJ: For sure. Søren: I couldn't use the equipment I needed. I ended up getting an extension on my PhD by a couple of months just to make up the time. For Anne, I’m sure it was difficult, but she had to look at what she could do, what she could provide as a research project that would be doable in this climate, and it led to this kind of innovative, quite fun story about snail toxins. AJ: The other bit that I thought was really interesting was how much focus Anne puts on visualizing proteins. She talked about NMR spectroscopy quite a bit and studying peptide and protein structure. I think one of the misconceptions the general public has about biology and life science is that everything is about data. Everything is about statistics. Everything is about numbers. Look, you and I have both done PhDs. There's a lot of data, there's a lot of stats, there's a lot of numbers; there's no getting away from that. But you know, one of the more unheralded parts of research in this field is being able to visualize things, right? And using amazing technologies to bring things to life, if you pardon the pun. It's fantastic to see Anne so excited about that aspect of her research. It was certainly one of the more exciting aspects of my research, you know, studying the nervous system and being able to visualize neurons firing and see them light up and see them communicate in real time. It was spectacular, to be honest. So, yeah, I loved hearing that from her. Søren: Yeah. I mean, on the other hand, I would say one of the stereotypes about biologists is that we're biologists because we don't like math, or else we would be physicists! [MUSIC] So we’ll leave it there. If you want more information on peptides, definitely check out BioXconomy. We have an article about peptides being used to treat spinal cord injury, and we'll link that in the show notes along with a series of interviews. We've talked about David Craik over the course of this episode. David Craik, major, major figure in peptides; we interviewed him not long ago as well as a number of other really cool peptide researchers: Peter ‘t Hart, Nathalie Grob, Fernando Albericio, about things like green chemistry and using AI to design peptides. The stories are really, really cool. So we'll add all those into the show notes so people can check those out. With that, I want to say thank you for listening to Dose of Discovery. If you enjoyed the episode, follow the show wherever you're listening so you don't miss the next one. And the next one is going to be very exciting, so I do hope folks will check in! Don't forget to check out BioXconomy for more of our coverage over at bioxconomy.com. Dose of Discovery is an Informa podcast and is produced by Suze Cooper. See you next time. 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