This paper (open access) is not going to be to everyone’s taste, but the people who are working in its area – and there’s an ever-increasing number of them – will want to read it closely. It’s a close look at the red-hot fields of targeted protein degradation and “molecular glues”, and for those who aren’t into those, the next paragraphs will give you some background. If you’re already into degradation – that didn’t quite come out right – skip ahead if you like:
So the idea behind targeted protein degradation (and now many related ideas besides) is the creation of a “bifunctional” molecule to hijack an existing cellular pathway. For TPD, you’re latching onto the protein ubiquitination system. Ubiquitin is a short protein tag that’s appended to lysine residues of other proteins, yet another post-translational modification, and Ub resides can be “stacked” onto each other into various sorts of oligomeric chains. If the right ones get added in the right quantity, it’s a signal that the protein thus tagged is ready for the cellular scrap heap, like a fluorescent orange waste disposal sticker. These Ub tags are applied by a set of enzymes (there’s a whole collection of them in cells) that are specialized for just that purpose.
Polyubiquitinated proteins are taken up by the proteasome, which as I’m fond of saying is a tubular structure resembling the planet-destroyer thingie in the old Star Trek episode “The Doomsday Machine“. Proteins enter one end of the proteasome and are ripped to shreds by a wall of protease enzymes facing the inside, and recycled amino acid rubble comes out the other end. So what if you could target specific proteins for this fate before their time?
That’s the plan. What you do is find a molecule that binds to some component of that ubiquitinating machinery (usually the part called the E3 ligase). There are many of these known, targeting different ligases, with (weirdly enough) the infamous thalidomide as one of the first to be proven for a particular E3. Then you find a molecule that binds your target protein – it can be an inhibitor, an allosteric site binder, whatever, just so long as it binds well. Now the so-obvious-it’s-dumb part: you take both of those molecules and build a linker between them to turn them into One Big Odd-Looking Molecule. One end of your new beast will grab onto the target protein, and the other end will grab onto the ubiquitination complex, and you will thus bring them into a close proximity that otherwise they would surely not otherwise enjoy.
The crazy thing is, it works: we have many examples now of such bifunctional degraders that manage to get into cells and cause drastic, sudden reductions in the amount of their targeted proteins as they get dragged, no doubt kicking and protesting, off to the proteasome shredder. What’s more, the bifunctional molecule survives the experience and goes back to degrade again, so you get true catalytic cycles out of the things. These have been shown to work in animal models, and several have now been dosed in human clinical trials – it’s a whole new way to attack targets of disease that we’ve never had before. We can block up active sites of proteins, but that leaves many of their other functions with other binding partners undisturbed. We can genetically silence expression of proteins, but that gives the cells time to come up with emergency backup plans in many cases. But reaching in during the middle of the cellular business day and zapping targeted proteins out of existence – that’s something new!
The idea doesn’t stop there. You can potentially bring *any* two proteins together if you have molecules targeting each partner. So you could haul a kinase over and have it phosphorylate the other, or a phosphatase to have it take any nearby phosphates off (this stuff has been demonstrated), and once you start thinking in that direction a whole list of ideas will occur to you. It’s not a small field now, but it’s going to get a *lot* bigger.
Molecular glues are another bizarre thing, with some similarities and some differences. In this case, you’re bringing two proteins together with a single small molecule – and some of them really are small – that binds to both of them simultaneously and may even arrange the two protein surfaces into a more favorable shape. I would not have thought that this was likely, or even possible, if you’d tried to sell me on it twenty years ago. But that’s what thalidomide itself does (when it’s by itself), and it’s also what how the auxin plant hormones work. I would have told you that this would be like trying to stick two cruise ships together by jamming a sailboat in between them, but proteins are a lot more complicated and interesting than your average cruise ship. People are currently searching all over the place for more of these things and trying to understand when they can work and how.
OK, then. By now there are a lot of examples of bifunctionals and several molecular glues, but the field has been, well, extremely empirical. Take those linkers that you have to build into make the bifunctionals. How long should they be, and what should they look like? Alkane chains, polyethylene glycol-like stuff, amides, sulfonamides, triazoles made via click chemistry? There are a lot of ways to do that, and they vary greatly in polarity, molecular weight, conformational flexibility, and more. People have tried all kinds of variations, and what we know for sure is that different linkers vary widely in their effectiveness once you’ve settled into a given system. But there are very few useful systematic rules; often enough you just make things and keep making them until something works, and when it works you’re not quite sure why.
The tricky part is that a ternary complex has to form for all these ideas to work, and that takes you into pretty complex kinetic and thermodynamic territory. As you can picture, there are a lot of possibilities about what binds to what and in what order, and plenty of rate constants that can kick the outcomes all over the place. This new paper tackles these in a comprehensive way. One key, as it details, is to separate the ideas of cooperativity and binary affinity. Many bifunctionals have very high binary affinity (each end binds strongly to its partner) but are nothing special in cooperativity. And many molecular glues are at the other end of the spectrum: the glue molecule has terrible binding (one-on-one) to Protein A, and terrible binding to Protein B, but bring all three of them together and you get a nice tight complex. Counterintuitive, to say the least, but this “induced cooperativity” is what makes the whole thing work, as the protein structures shift around. And it’s what makes screening for these things rather labor-intensive, and it also shows you how my cruise ship analogy breaks down, because they aren’t quite so flexible.
If you overdo the binary affinity part (through very tight interactions or high concentrations), bifunctionals suffer from what’s been called a “hook effect” – the binary interactions get so favorable that the ternary complex doesn’t form properly, which is truly not what you want. Any consideration of thermodynamics will of course break down into entropy and enthalpy, and there’s plenty of that to go around here. The enthalpy terms (energies of the actual binding interactions) can be cancelled out by the entropic penalties of assembling a more ordered ternary complex, particularly one that involves flexible parts that might have suddenly had to get a lot less flexible. And there are all sorts of potential ternary complexes, some of which are more productive than others.
The middle of the paper goes into detail on all this, and the latter part talks about cellular context. Which frankly, is even more of a black box. You get into all sorts of fuzzy discussions about the “tone” of the various systems (how much capacity they have, how much range, how much disruption of them might be needed to achieve the desired effects, and so on). The best way forward here seems to be the development of better high-throughput cellular assays, although no one’s going to turn up their nose at any new fundamental insights, either.
So if you’re a degrader/glue type, have a look. You’ll run into some things you already knew, but you’ll find others that you hadn’t devoted enough thought to, as well. Very few of us have devoted enough!
The post Hard Thinking About Protein Degradation and Bifunctional Molecules first appeared on In the Pipeline.
