Development of a KRAS-Targeting PROTAC

Monday, 16th August 2021

In this question-and-answer session, Michael Bond (Yale University) talks to our Senior Product Specialist, Alex Moloney PhD, about the development of LC 2, a KRAS G12C-targeting PROTAC^® which is now available from Tocris.

Q - Can you give us an introduction to yourself, your lab, and your research interests?

My name is Michael Bond, I’m a graduate student in Craig Crews’ lab at Yale University and we focus on pushing the field of Proteolysis Targeting Chimeras (PROTAC). My thesis is focused on making PROTACs more tumor specific, both from the proteins we can degrade, like KRAS G12C for which I was involved in developing LC 2, the first-in-class endogenous KRAS G12C degrader. I’m also researching ways to hijack new E3 ligases to make PROTACs more tumor specific.

Q - Can you tell us about the motives for targeting KRAS G12C with a PROTAC?

The main motive was two-fold:

To see if RAS proteins could be degraded using the PROTAC technology. In a previous study by the lab of Nathaniel Gray¹, they looked to degrade KRAS G12C using pomalidomide-based degraders. They were unsuccessful in degrading endogenous KRAS G12C, however they did show they could degrade a fusion protein. This left an open question of whether, in fact, KRAS itself could be ubiquitinated and degraded, and I think our study does a good job of answering that question.

The second motive was based on the data coming out around KRAS G12C inhibitors that had started to hit the clinic; just recently we’ve seen the news of AMG-510 being approved by the FDA². There have been observations that a resistance to these KRAS G12C inhibitors can occur and after about 24 h there’s a rebound in MAPK signaling. Some preliminary work in the field suggests this is due to the activation of other pathways, so we were curious to see if TPD could alleviate some of the resistance that is seen with some of the inhibitors.

Q - So, you think that PROTACs may be a great response to targeting resistance mechanisms?

There is the possibility! The thing about inhibition vs degradation is that degradation removes all the roles of the protein, both its enzymatic functions and its scaffolding functions, so there is always a chance that just inhibition of RAS proteins could allow them to still act as scaffolds to allow flux through other growth pathways. One would hope by employing degradation that you would prevent the possible workarounds by the cell.

Q - You mentioned some previous work of others that you’ve built on. How did you go about picking your warhead ligands for LC 2?

Since the efforts to degrade endogenous KRAS G12C with pomalidomide didn’t seem to work, we wanted to see if VHL could form a better ternary complex that would lead to degradation. I think that is an important point for the whole PROTAC field in general, just because one E3 ligase is not able to induce the degradation of a protein, it does not mean that that protein is not amenable to degradation by PROTACs or other targeted protein degradation technologies. Really multiple E3 ligases should be tested.

The reason we went with the Mirati compound (MRTX 849) was also to move away from the previously mentioned publication¹ that had used a tool compound out of Wellspring called ARS-1620. We selected MRTX 849 over AMG 510 because of the potentially more reactive fluoroacrylamide electrophile that could enable better modification of LC 2 once we had added the VHL ligand.

Q - As with the other KRAS G12C ligands, LC 2 binds irreversibly. Should this be a consideration when designing PROTACs? And can you speak more generally about catalytic vs non-catalytic degraders?

I definitely think this is a big consideration and I would say it’s a weakness of LC 2 being non-catalytic, although, we do get rapid degradation of KRAS G12C with 2.5 µM LC 2 and we continue to see robust degradation for over 72 hours. Because of the irreversible binding that doesn’t enable a catalytic mechanism and as such might limit the antiproliferative effect that you might think you would get by degrading KRAS, simply because we’re not turning over the protein as quickly as a catalytic PROTAC might.

Q - More broadly, thinking about small molecule approaches to targeting a specific protein, what are your thoughts on inhibition vs degradation as a strategy? When should we set out to design a degrader over a more traditional small molecule inhibitor?

I definitely think this is a case-by-case basis for what you’re trying to do. Whether in academia as tool compound or as a preclinical/clinical compound you want to treat a disease. I really think where PROTACs and other degradation modalities are going to shine are where there are scaffolding roles of proteins that traditional inhibitors are not able to disrupt. Some recent publications that have come out of our lab, and show this very nicely, are the FAK kinase and FLT-3 PROTACs^3,4. We see some major advantages of the degrader over the inhibitor. But that might not be the case for everything. There may be some instances where you don’t want to get rid of a protein because its scaffolding role may be important for cell health. One example being the BMS TYK2 inhibitor which is in the clinic⁵. Systemic degradation of TYK2 may not be good for innate immunity whereas an inhibitor may be better for patients.

Q - An area of significant interest in TPD is identifying novel ligands that target tissue-specific E3 ligases. Could you tell us about the opportunities that a broader toolbox of E3 ligase ligands present?

This is the part of the PROTAC field that I am most excited about - how we expand the E3 ligase toolbox. The LC 2 story really highlights the need for more E3 ligases to recruit. Just because one E3 ligase is not able to form the proper ternary complex for ubiquitination, it does not mean that all E3 ligases will be the same. The work coming out of the Dan Nomura and Ben Cravat lab’s is very exciting with their electrophilic compounds that bind to new E3 ligases. Our lab has also shown many different types of E3 ligases, not just the traditional cullin-ring E3 ligases, are amenable to targeted protein degradation. The real benefit to targeting new E3 ligases is to get tissue and disease specific degradation. I think this is the most exciting part. You can imagine, as more degraders enter the clinic that a way to alleviate some of the off-target toxicity is to home in on a specific tissue or disease state in which you want to induce specific degradation. A nice example of this is with the BCL-XL PROTAC (DT 2216)⁶ that really shows the power of getting differential degradation in your target tissue.

Q - In your opinion what are the biggest challenges facing the field of TPD that need to be overcome?

The biggest thing for me is the ligand discovery for new E3 ligases and to a lesser extent ligands for other proteins of interest that we might want to degrade. But I think ligand discovery is certainly the bottleneck. Unlike with the protein of interest, where we need a ligand to bind anywhere, having a molecule that’s going to do a good job of recruiting an E3 ligase requires binding to the same site as the native substrate. So I think we need a better understanding of the biology, and the degron sequences for E3 ligases. I imagine once we get a better hold on this, ligand discovery will catch up pretty quickly.

Thanks Mike great to talk and best of luck in your research.