Degrading Drugs for Problematic Proteins

In Bio Eats World’s Journal Club episodes, we discuss groundbreaking research articles, why they matter, what new opportunities they present, and how to take these findings from paper to practice. In this episode, Stanford Professor Carolyn Bertozzi and host Lauren Richardson discuss the article “Lysosome-targeting chimaeras for degradation of extracellular proteins” by Steven M. Banik, Kayvon Pedram, Simon Wisnovsky, Green Ahn, Nicholas M. Riley & Carolyn R. Bertozzi, published in Nature 584, 291–297 (2020).

Many diseases are caused by proteins that have gone haywire in some fashion. There could be too much of the protein, it could be mutated, or it could be present in the wrong place or time. So how do you get rid of these problematic proteins? Dr. Bertozzi and  her lab developed a class of drugs — or modality — that in essence, tosses the disease-related proteins into the cellular trash can. While there are other drugs that work through targeted protein degradation, the drugs created by the Bertozzi team (called LYTACs) are able to attack a set of critical proteins, some of which have never been touched by any kind of drug before. Our conversation covers how they engineered these new drugs, their benefits, and how they can be further optimized and specialized in the future.

Show Notes

• How conventional drugs work, and how PROTAC targets proteins differently [1:46]
• Discussion of how PROTAC can only reach proteins within the cell [6:00], how LYTAC targets external proteins [9:56], and how LYTAC pinpoints specific proteins for degradation [12:54]
• Implications for future use of LYTAC [16:37], and what work is needed to turn it into a therapeutic treatment [20:29]

Transcript

Hanne: Hi, I’m Hanne, and welcome to Bio Eats World.

Lauren: I’m Lauren, and this is the first episode of “Journal Club.”

Hanne: So, tell me. What’s “Journal Club” all about?

Lauren: So, “Journal Club” is on Thursdays, and this is where we take a recent scientific article and discuss it, either with the authors of the paper, or with our own internal experts here at a16z. And we highlight what the paper shows, what new opportunities it presents, and how to take those research findings from paper to practice.

Hanne: Okay. So, tell me — what’s this first paper all about?

Lauren: The first paper is titled “Lysosome-targeting chimaeras for degradation of extracellular proteins,” and it was published in Nature.

Hanne: That’s a lot of words. What do they actually mean?

Lauren: The basic idea is that diseases are often caused by proteins that have gone haywire in some way. So, there’s either too much of them or they’re present in the wrong place or at the wrong time. And the idea here is to create a new kind of drug to degrade those proteins. So, if there’s too much of the protein, you’re reducing the levels. If it’s in the wrong place or the wrong time, you’re removing it from that area. And that’s a really exciting new type of drug molecule.

Hanne: Okay, cool. So, what’s important about this paper? How is this moving us forward?

Lauren: This paper is really exciting because it’s targeting a whole new class of proteins, some of which have never been able to be touched by any other kind of drug.

Hanne: And who is the guest joining you for today?

Lauren: Right. Today, we have the senior author on the paper, Carolyn Bertozzi, who is an amazing scientist. She’s a professor at Stanford and her work focuses on creating new methods to perform controlled chemical reactions within living systems. So, we’re going to lead off with Carolyn describing how conventional drugs work.

PROTAC vs. conventional approaches

Carolyn: So, most conventional medicines act by binding to a target — a pathogenic driver. That’s a protein in your body that’s contributing to a disease. And they act by what’s called occupancy-driven pharmacology. They bind to that target and block its function. So, ibuprofen binds to an enzyme and blocks its activity, which then blocks an inflammatory pathway.

Lauren: So, the normal typical drugs are working by binding proteins and blocking their activities. But in the last 10 years, we’ve seen some really exciting alternatives to drugs that rely on this specific model, with the most well-known and the most well-developed being what’s called a PROTAC, or a proteolysis-targeting chimaera. So, how do these new drugs differ from what we just described — the standard typical drugs?

Carolyn: So, that concept came out of academic labs in the early 2000s, and the two people who published the defining papers in this area were Craig Crews from Yale and Ray Deshaies, who at the time was at Caltech — now he leads research at Amgen. And they had this idea that another way to shut down a pathogenic protein would be to target it for degradation. And around that time, there had been some breakthroughs in our understanding of how nature normally degrades proteins, because she has to be able to do that — new proteins get made, old ones get degraded. And a central mechanism for degradation of proteins inside the cell is that they get marked with ubiquitin chains, and that’s a signal for the proteasome — which is like the meat grinder inside of the cell — to chop up these proteins and destroy them. And there are enzymes that put these ubiquitin chains onto proteins that are destined for degradation.

And so, what Crews and Deshaies realized is that you could build a molecule that artificially bridges the gap between a target protein and this ubiquitin machinery. And with that molecule, you could basically get a protein ubiquitinated intentionally, and therefore degraded. So, that was their conceptual idea.

Lauren: So, in the course of the normal function of the cell, you have proteins being produced, but you also have proteins being degraded. And so, one of the main mechanisms for degrading protein is by the ubiquitin-proteasome system. And that’s where the cell says, “Degrade this protein by adding ubiquitin molecules onto it,” and that pulls it to the proteasome where it gets chopped up. But what a PROTAC does is, it’s a molecule that can bind to target protein — so the one that you want to degrade — and brings the enzyme to it that adds the ubiquitin tag — adds the flag — and then that brings it to the proteasome to be degraded.

Carolyn: Right. And the reason that was so transformative is that not all proteins are easy to block, actually, with drugs. There are lots of proteins that are not enzymes, and they don’t even have a pocket, really, where you could put a drug and it would block the function. So, the cool thing about these PROTACs is that they don’t have to bind in a place that would block its activity, but instead bridges the gap to an enzyme that puts the ubiquitin on and drives the degradation. So, the promise, really, is that the PROTAC — or the targeted degradation approach — expands the druggable proteome because now more proteins can be drugged, because you have this other way of doing it — through degradation and not just blocking.

Lauren: So, you’re using the endogenous mechanism that the cell already has for flagging proteins that you want to be degraded, and using it now to target a much wider range of proteins than you could if you were only able to target those that have, you know, a really nice pocket that could be targeted with an activity inhibitor.

Carolyn: Right.

The benefits of LYTAC

Lauren: So, what are some of the limitations of these approaches?

Carolyn: Well, the targeted degradation field began with the PROTACs, but it has expanded over the last 20 years to include other types of protein degraders — but all of these processes function on proteins that are inside the cell — in the cytosol or in the nucleus. And meanwhile, there’s this whole other world of proteins which are outside the cell. So, these are proteins that are displayed on the cell surface — the membrane-associated proteins — many of which, the majority of the molecule is outside, presented on the surface, where it’s not accessible to the proteasome. And as well, there are many proteins that are just completely secreted by the cell and just released into the extracellular space, and those extracellular proteins are about 40% of the human proteome. So, that’s a pretty big chunk of the pie that is not available to the PROTAC strategy.

And many of these proteins — these extracellular and cell surface proteins — are important targets for drug development. And, you know, my lab had been working on a variety of different cell surface molecules and secreted molecules that contribute to things like cancer immune evasion, for example. And many of the molecules we wanted to drug were really not druggable using the conventional blockers. And that’s where the lysosome-targeting chimaera, LYTAC, research started.

Lauren: I see. So, the PROTACs that you described are a really exciting new modality, but they are limited in that they can only target the proteins that are within the cell. And there’s this huge world of proteins that just are not available to be targeted in that way. And they aren’t ones that rely on occupancy of, like, a particular binding site — they can’t be targeted by those types of drugs either. So, they’re really kind of an unmet need for drugs to target them.

Carolyn: I would go even further and say sometimes even targets that can be drugged with a blocker, you can get a more potent effect with a degrader at lower doses, right? So, even secreted and cell surface molecules that have been successfully drugged with monoclonal antibodies, you might actually do better if you convert over to a degradation strategy.

Lauren: Why do you think that is? Why do you think they’re better than activity modulators, or is that not known?

Carolyn: Well, I think with occupancy-driven pharmacology, you can’t ever get, like, 100% of the target protein blocked. There’s always an equilibrium, and you have to constantly pump the system with enough drug to keep the occupancy as saturated as possible. By contrast, the degrader can bind to a target and get rid of it, and then bind to another target, and get rid of it, and bind to another one, and get rid of it. So, you’re just reducing the level of the target protein, but because there’s the potential for one drug molecule to mediate the degradation of multiple targets, you could get a deeper inhibitory effect in principle. And that has now been borne out, even in some early-stage human clinical studies, with PROTACs. The same could very well be true with LYTACs. Of course, it’s a much earlier technology, so we don’t know that definitively, but there’s — I think — a rationale for thinking that way.

Lauren: Yeah. That’s kind of like pharmacodynamics 101, that you have a reversible inhibitor, and you’re going to have this equilibrium, but these degrading molecules, you know — they don’t get degraded when they tag the protein for degradation. They have a benefit of — one degrader molecule can target a huge number of target molecules. So, that’s really interesting, that even in, like, a head-to-head comparison on a known druggable target, that you can possibly get a better effect by degrading as opposed to inhibiting.

How LYTAC targets proteins

So, now that we have the background on why we need this new type of drug, why you decided to go after extracellular and membrane-associated proteins — let’s get into the details of how you develop these molecules. And, as we mentioned, the PROTACs co-opt this endogenous pathway, the ubiquitin-proteasome pathway — but they can’t reach these proteins outside the cell. So, what cellular pathway did you co-opt to degrade those proteins?

Carolyn: So, again, nature degrades these extracellular and circulating molecules, and she does this through what’s called the endosome-lysosome pathway. So, cells will basically internalize and engulf molecules from the extracellular space into endosomal vesicles that go through a maturation process to become the lysosomes. And the lysosomes — people from their cell biology classes might recall — that’s the organelle within the cell that has a lot of degradative enzymes. So, lysosomes can degrade proteins, polysaccharides, lipids. There’s a lot of hydrolases within the lysosome.

And so, we conceived of an idea where we would develop bifunctional molecules, where one part binds the protein that you want to degrade, and the other part binds a lysosomal trafficking receptor system. So, that’s the key — is that lysosomal trafficking system. And it turns out that in human biology, there are about a dozen known receptors whose job it is to grab stuff — either from the membrane or from the extracellular space — and pull it into this endosome-lysosome pathway for degradation. And so, what we have done is hijacked those pathways by basically building molecules that interact with those receptors, and then attaching them to a molecule that binds a target of interest. So, that’s the structure of the LYTAC — a binder on one side for the target, a binder on the other side for a lysosomal trafficking receptor.

Lauren: Right. So, nature has already come up with a way to degrade the proteins that are membrane-associated and extracellular, and you just developed a mechanism that allowed you to say which protein you want to degrade and then extracting it from the extracellular space and degrading it inside the cell.

Carolyn: Yeah. And one of the best known lysosomal trafficking receptors is the so-called mannose 6-phosphate receptor. And mannose 6-phosphate is a sugar epitope that is found on lysosomal enzymes, and that allows them to be trafficked to the lysosome by this receptor — the mannose 6-phosphate receptor.

Lauren: So, you have this sugar molecule that if you attach it to a protein, that’s going to take it into the lysosome. So, how did you engineer the specificity to target the protein that you wanted to the lysosome?

Carolyn: So, you need a binding molecule that is very specific, and ideally also very high affinity, against your target of interest. And, you know, in our early proof of concept studies, we chose targets to degrade for which there already were high affinity, high specificity antibodies available — several of which are already approved human medicines. So, for example, we’re interested in the epidermal growth factor receptor as a target for degradation. This is an important cancer target. EGFR — it’s overexpressed or mutated in many cancer types, where it’s driving the proliferation of cells. And we made a LYTAC out of a human drug called cetuximab — it’s an antibody against EGFR that is used, you know, in the oncology setting. So, that process of taking an antibody against a target and just decorating the antibody with the mannose 6-phosphate groups — that converts it to a LYTAC.

Lauren: Great. So, antibodies are molecules that our immune system produces, and they are incredibly well-tuned to bind one specific protein — and there are many drugs that are actually antibodies — but their main function is to just block that protein. And what you did was you took that therapeutically active antibody and added the glycan molecules that you needed to turn it into a LYTAC. So, now not only is it blocking the protein, but it’s shuttling it into the lysosome to be degraded. It almost gives it, like, an extra function— like, making it even more effective at disrupting their target’s function.

Carolyn: That’s right. Also, the more we learn about biology, the more we are appreciating its complexity. And I think we also are now understanding that most proteins have functions that are not just binary, you know — like an enzyme is either on or off. Most proteins have multiple dimensions to their function. They interact with other proteins. So, when you block a protein through an antibody, or through a small molecule inhibitor, there are probably other interactions of that protein that you’re not affecting, which still contribute to the biology. And when you degrade the protein entirely, you take away all those dimensions of its function. And so, it’s not just that a degrader can be more potent than the inhibitor in an axis of biology — I think the degrader can have more axes of an effect.

Lauren: Do you think that that could lead to possibly off-target effects of disrupting, kind of, a bigger network than you anticipated?

Carolyn: That’s a good question, and I guess it depends on where you draw the line between on-target and off-target. Because, take a protein like EGFR. The biology of that receptor is driven by its interactions with other components of the signaling pathway. You know, EGFR binds its ligand — the epidermal growth factor — and the consequence of that is, that triggers a signaling cascade. And so, if you inhibit the activity of EGFR by just blocking, you don’t affect any of the downstream signaling biochemistry. 

However, if you drive the degradation through the LYTAC approach, and some components of that signaling machinery come down with it, that is actually a direct hit — I would say that’s on target, right? Because you’re hitting not just EGFR, you’re hitting the complex that drives its biology, right? So, again, the biology is never transacted by a protein in isolation — it’s by that protein and the network of its interactors. So, I would argue that if you can degrade some of its interactors, it’s a more profound influence that’s on-target.

Possible future applications

Lauren: So, now that we’ve talked about, you know, the details of your study, how you develop these bifunctional ligands that can bind to a specific target protein and shuttle it into the lysosome for degradation — let’s zoom out and put this research into the broader perspective. What are some of the new opportunities that this work provides?

Carolyn: We’re now exploring therapeutic applications of the LYTAC technology, and we’re interested in extracellular targets that have been very — either difficult, or really just impossible to drug, and there really is no option right now for patients for certain disease settings. So, for example, we’re very interested in diseases that involve aggregation of proteins in the extracellular environment. Proteins that in their misfolded or unfolded forms lead to toxic aggregates that can cause tissue damage. And so, these are diseases that are often called amyloid diseases. The ones that are most familiar to people would be neurodegenerative conditions like Alzheimer’s disease, Parkinson’s disease — it’s been very difficult to figure out, you know, how do you get rid of these protein aggregates that are pathogenic in the extracellular space? They’re not really amenable to inhibition — the process by which they form is often not well-understood. You really just want to get rid of them, right? You want to degrade them. And I think the LYTAC approach is perfectly situated to take on peripheral amyloid diseases. 

For example, there’s a condition called light chain amyloidosis. Antibodies have a heavy chain and a light chain. So, in patients with this condition, there’s too much light chain all by itself, and it’s not stable, and it’s forming amyloid aggregates — which deposit in organs throughout the body and they’re toxic. The standard of treatment for these patients is very poor. So, we think the LYTAC approach could be interesting in that setting.

Lauren: That’s a perfect example because those light chains don’t have an enzymatic function. They don’t have a nice pocket that you would be able to stick a drug in. So, the ability to pull those out of the extracellular space and degrade them with a LYTAC sounds like a perfect match between disease physiology and drug modality.

Carolyn: Yeah. So, that’s an example of a, sort of, secreted pathogenic molecule or system of molecules. There are other membrane-associated targets that we think the LYTAC is well-suited toward. And one class of molecules that my lab is really interested in are called mucins. These are transmembrane glycoproteins that are huge, and they’re kind of the giant redwood trees of the cell surface, so to speak. And they’re known to be associated with cancers. And cancers that overexpress these mucin molecules — they tend to be very aggressive and very difficult to treat. And we’ve done a lot of work to understand, like, what’s the function of these mucins that’s oncogenic. And the bad news, from the perspective of drug discovery, is that a lot of the biology of these mucins is a physical biology. So, they’re pathogenic because of their stiffness, and their rigidity, and their physical effects on the cell surface — not because they interact with a receptor, for example, which maybe you could block, right? 

And so, what do you do when the function of the molecule is a physical one, and not a biochemical one? And I think this is where you just want to get rid of them. I think you just want to degrade them. And fibrosis, right — that’s a disease setting where there’s pathogenic accumulation of collagen scarring. And you know, that’s hard to think about — how to drug that, you know, at least at the end point of the disease, where you have this material that you really just want to degrade. And so, again, I think a LYTAC strategy would be interesting to test in that setting.

Lauren: A lot of really important applications for this. So, what are some of the elements of the LYTAC design that still need to be optimized to turn them into therapeutics?

Carolyn: So, this was the “version 1.0” of the LYTAC technology, and the work that’s now going on is basically the second- and third-generation improvements. And those improvements have taken several forms. So, first of all, we’re interested in improving the structures. So, the second-generation LYTACs have a new chemistry, so that the conjugations are site-specific — that we can engineer the part of the antibody that actually gets coupled to the mannose 6-phosphate groups. And with our new chemistries, we can make different geometries of LYTACs and find what is the best geometry for a given target — and it probably will be target-dependent.

So, we’re kind of now writing the rulebooks, and in the publication, the LYTACs we made are built from these known antibodies. We are now developing LYTACs from other kinds of binders — including small molecules that might otherwise have been blockers — we’re now converting them to degraders through the LYTAC approach. Another dimension that we’re expanding upon is the lysosomal trafficking receptor that we hijack. So, the mannose 6-phosphate receptor was a great starting point — it’s expressed in virtually all cell types. But there are other systems that are more specific for different cell types or different tissues. So, our next LYTAC family are targeting a receptor called the asialoglycoprotein receptor, which is a liver-specific lysosomal trafficking receptor. And we have a preprint that we posted on ChemRxiv on this new generation of LYTACs.

Lauren: Yeah. Liver-specific makes a lot of sense because we were talking about fibrosis — liver fibrosis is a huge problem, and that’s caused by too much collagen in that area that you want to break down. But you don’t want to break down collagen everywhere in the body, you know — that’s really a critical molecule. You could get wrinkles, God forbid! <laughter> It’s really important in your skin, it’s really important in your joints. So to have that specificity of where you want to target the degradation is really important, and an additional, like, strength to this approach.

Carolyn: Yeah. And I think that then hints to a broader universe of LYTACs that target different receptors that are tissue-specific in different settings. That’s the tip of, hopefully, a big iceberg of interesting new degraders.

Lauren: And more to come. So, we’ll end with — what is the key take-home message from this article and from our discussion today?

Carolyn: I think the most important point is that this exciting, still relatively young field of targeted protein degradation has just been set free from the confines of the cell. So, extracellular proteins should now be added to the list of potential targets for a degradation strategy, and we hope with the LYTAC technology that we can bring added benefit to patients.

Lauren: Well, thank you so much for joining me today on “Journal Club.” I really enjoyed our discussion, and I’m so excited to see what comes out of this research.

Carolyn: Thank you.

Lauren: And that’s a wrap for the first episode of “Journal Club.” If you enjoyed this episode, please subscribe, rate, and review wherever you listen to podcasts. And to learn more about how biology is technology, subscribe to our newsletter at a16z.com/newsletters.