Spotlight on: Professor Danny Huang
The discovery in the 1970s that certain proteins are ubiquitinated before degradation was awarded the 2004 Nobel Prize in Chemistry. Ubiquitination has increasingly been recognised as important in various cell functions. Its best-known function is as a mediator of protein degradation: the tagging of a protein with ubiquitin marks it for degradation by the proteasome.
As a brief recap, this degradation pathway requires a consecutive cascade of three enzymes: E1 activating enzymes, E2 conjugating enzymes and E3 ligases, which attach the ubiquitin to the substrate protein to be degraded.
Here Professor Danny Huang tells us how the field of ubiquitin signalling has rapidly evolved over the past twenty years, from hardly knowing which proteins are involved in this huge system, to the present day, when the power of ubiquitin-mediated degradation is making its way into clinical trials. I began by asking Danny:
What got you interested in ubiquitin and its role specifically in cancer?
It all started with my postdoc. I did my PhD in Australia, and when I graduated, the opportunities in Australia were quite limited, so I was looking abroad to see if I could do a postdoc. And of course back in those days, it wasn’t like now where you can just go online to find a job. You’d have to go to conferences and hope that someone was recruiting, or look at the job listings in journals. I came across a booth at one of the meetings in Australia, and it was from the St Jude Children’s Research Hospital in Memphis, Tennessee. I applied and was invited to go over there to meet some of their PIs. One of the projects really caught my eye, which was the ubiquitin signalling project. At that time – back in 2002 – the ubiquitin system pathway was not very well characterised. So what appealed to me at the time was that this was a field that was largely unexplored. And I could see from the literature that the ubiquitin modification was related to a lot of different biological processes. Of course, at the time its implication in cancer was not very prevalent, but it was known that the protein MDM2 was a ubiquitin ligase that targets the important tumour suppressor p53.
Over the last 15–20 years the field has really exploded, and the field has evolved so rapidly in the past several years that we were able to ask the question: could we use this system to degrade any protein we want? This latter is a method called PROTAC, and it’s a prevalent method of targeting any protein. It’s basically a molecule that you design that on one end binds an E3 ubiquitin ligase and on the other end binds your target protein to be degraded. So it brings your target protein – which is not normally a substrate of the E3 ligase – close enough to the E3 that it gets ubiquitinated and subsequently degraded. So now we are seeing an explosion of this new modality in drug development, and the first one is now going through clinical trials.
So we have seen the journey from understanding the basic biology of this pathway and evolving into all sorts of approaches to target the system: targeting specific E3–substrate pairs (e.g. the aforementioned MDM2–p53) for targeted cancer therapy, targeting the E1 enzyme, targeting the proteasome, and targeting the enzyme that removes the ubiquitin mark. What drives me in my research is trying to understand the basic biology of this system and trying to see if there’s a way we can target this system for cancer therapy.
You mentioned that back in 2002 the pathways were not very well characterised and our understanding has advanced enormously since then. What would you say during that time has been the most surprising or the most interesting development?
Well I was trained as a structural biologist. So, our interest is to capture the three dimensional structure of a protein. When I first started back in 2002, we only knew that there was E1, E2 and E3. We didn’t even know what they looked like. But in order to target a protein you need to know its 3D structure; once you have that you can decide where you want to target it to inhibit its function. So, when I started we didn’t know the structure of E1, we knew what E2s looked like, and we knew a handful of E3s (we now know there are about 700). And we didn’t really know how they all worked together. In my postdoc I solved the structure of E1, and that paved the way for understanding how ubiquitin or ubiquitin-like protein is activated by E1 and how that ubiquitin or ubiquitin-like protein then passes over to E2. And that was important because that ultimately led to a company taking our structure and developing a small molecule inhibiting E1’s function. That inhibitor is now in clinical trials and it’s also a commonly used reagent for groups studying the ubiquitin system.
What are some of the questions that you and your group are currently trying to get to the bottom of?
When I left my previous lab, we had very limited knowledge on how E3 ligases work and what substrates they act on. So that was my goal when I started my lab, to understand how they work. There were a lot of structural biologists working on this pathway, but it is very difficult to get a protein’s structure – purifying the protein and getting it to crystallise. Sometimes that process works quite smoothly and you could get the structure in six months, for example, but likewise it could take up to five years. And specifically we wanted to look at E3s that are important for cancer, in other words E3s that are implicated either as tumour suppressors or as oncogenes. Our goal is to understand what they look like and how they work. And a lot of these are mutated in cancer, so we want to know how those mutations affect their function and whether we can target them for potential therapeutics.
|MDM2 (violet), p53 (blue), ubiquitin (yellow). (Shutterstock)|
The key E3s that we have been studying over the last 5–10 years are CBL and the oncogene MDM2 which targets p53. p53 is mutated in around 50% of cancers – in most of the other 50%, you get overexpression of MDM2, resulting in the same outcome: loss of p53. So one idea in the field is to reactivate p53 by inhibiting MDM2. That kind of angle has been developed in the field for 10–15 years, and various drugs are now in clinical trials. Unfortunately, the first glimpse from these trials is that the drugs are quite toxic. You can imagine that if there is too much p53, it would just shut down the cell and induce apoptosis, and that’s exactly what is happening with these drugs. They have severe side effects, as they affect not just the cancer cells but cells everywhere, and so the consequence is that you get severe p53 activation everywhere. This gives the patients adverse side effects. So there’s a need to find a better way, to reduce this toxicity.
So, I collaborated with the Beatson’s former director, Professor Karen Vousden, who is also an MDM2–p53 expert, and together we demonstrated that by using an alternative inhibition strategy, we could dampen this toxicity. If you think about how an E3 functions, you can target it in several different ways. One way is to block the E3 from binding to p53. A second way is to let it bind but to stop its activity, to stop it from ubiquitinating p53. From our structural approach we could understand how its E3 ligase activity functions, which we could then inhibit. So even though MDM2 can bind p53, it cannot ubiquitinate it. We are exploring this approach to see if we can reactivate p53, and our recent data suggests that this is not toxic in mice, and so we think this approach might work.
Another angle is focused on the CBL ubiquitin ligase. It’s a similar story to MDM2, except that CBL is not an oncogene but rather a tumour suppressor. But the problem is that when it is mutated it becomes an oncogene. So again we used our structural approach to understand what it looks like, and we then collaborated with external laboratories to develop a small peptide inhibitor molecule that we could target to a specific site on this protein so we could interrogate whether inhibition of this interaction could impact its oncogenic effect.
|Cartoon showing the components of the PROTAC system. Diagram courtesy of Alice Wicks|
PROTACs is another area that I’m quite keen to explore. Then in terms of PROTACs, this work really depends on collaboration with a chemist, and we have been collaborating with Dr David France at the University of Glasgow. He makes the molecule and we test it in our systems here. You need a small molecule that binds an E3 and then you also have another small molecule that binds your target of interest. Then you take these two small molecules and connect them with a linker (see diagram). So you can imagine then that this E3 and this substrate are going to see each other because you have this PROTAC molecule linking them together. The E3 will then ubiquitinate the target, which will then get degraded. This is the idea of a PROTAC.
All of this sounds like it has a lot of potential. What do you think the future holds for the field?
Any small molecule that has been designed over the last 20 years can be engineered to PROTAC – that includes not only all the approved drugs but also those that didn’t make it to approval. This latter group can be converted to PROTAC. For example, if you have a kinase inhibitor, the inhibitor blocks the kinase activity. But if you PROTAC it, instead of just inhibiting it, you cause this kinase to degrade, so it’s like a knock-out. So what we’ll see is that there will be an explosion of new molecules over the next five years. It’s already moving right now.
So what we need to see is whether this modality of targeting for degradation works better than the traditional inhibition of the protein. The thing with PROTAC is that you don’t need a very high affinity binding to the target. If you look at current drugs, they all bind with single- or double-digit nanomolar affinity as that allows them to bind very tightly, but the idea with PROTAC is that you don’t need that because you’re not trying to inhibit it, you just need to form this complex long enough for the E3 to ubiquitinate the target. So you don’t need high-affinity small molecule – even a single-digit micromolar affinity molecule will work. This is the advantage of the PROTAC modality.
There’s already one PROTAC in clinical trials and I foresee maybe a couple more coming in this year. Then we will see perhaps targets that haven’t been druggable before, and now you have this PROTAC approach you can now drug it. Taking KRAS as an example, there’s no drug that can inhibit it completely. There are a lot of molecules that bind KRAS, just not with high enough affinity to inhibit it. And a lot of those are now being designed into PROTACs. So we might see previously undruggable proteins being able to be drugged for the first time.