How targeted protein degradation will live up to the hype 

Protein

Dr Benedict Cross, CTO, PhoreMost, explores the drug discovery potential that targeted protein degradation (TPD) offers.  

The ubiquitin proteasome pathway (UPP) has been a subject of interest for therapeutic intervention since the first proteasome inhibitors to treat multiple myeloma were developed and approved in 2015. The targeted recruitment of this vital proteostasis pathway to deliberately and eliminate specific disease-associated proteins has dramatically amplified drug development activities in this system over recent years1. Targeted protein degradation (TPD) drug discovery has yielded the first bivalent degraders to enter the clinic, and progress is being made to understand and classify a second important branch of drug, the monovalent degraders, or molecular glues. With a vast number of researchers and companies vying for success, will this modality live up to the hype and the substantial investment preference it has garnered?  

Increased understanding of the diversity of the UPP, the rules for molecular glue function, and the integration of computational platform solutions are poised to deliver real therapeutic and product success. Even the usual ebbs and flows of biotech and VC interest are unlikely to dent the ubiquitous promise and translated progress of the degradation modality.  

Targeting proteins with bivalent drugs 

The most frequent and reliable component in the exploitation of proteostasis for drug development has been the class of enzymes called E3 ligases. These components control the final and most malleable step in the UPP, adding a small ubiquitin tag to the substrate protein to trigger its degradation by the proteasome (see Fig 1). The diversity of E3 ligase enzymes is an important biological feature which helps to explain some of the excitement around TPD strategies. There are estimated to be over 700 distinct proteins that can be classified under this single bracket (see: https://www.proteostasisconsortium.com/pn-annotation/), excluding the activating and conjugating E1 and E2 enzymes. This diversity is key to the specificity and plasticity of the cell’s ability to control protein abundance and maintain homeostasis and presents a vast potential of ligandable nodes to recruit for induced degradation.  

Figure 1: The ubiquitin proteasome pathway: Short-lived or misfolded proteins are targeted by the E3 ligases for polyubiquitination, triggering proteolysis and recycling by the proteasome, controlling protein abundance in response to stress, disease or as part of homeostatic regulation. 

TPD drugs work by either directly connecting or spatially associating a Protein of Interest (POI) and an E3 ligase. Bivalent degraders, such as proteolysis-targeting chimeras (PROTACs), can be designed and built using modular adducting of ligands to each of the POI and the E3 ligase via a linker (Fig 2). This rational process is systematic, yielding hundreds of published or disclosed molecules2, but this avalanche of drug variants should not belie the substantial medicinal chemistry efforts required to achieve activity for these unusually large molecules. Moreover, rules for ideal physiochemical properties rarely apply to these drugs, and it is therefore extremely challenging to predict their bioavailability and pharmacokinetic properties, even with improvements in computational modelling3. 

The next wave of bivalent degraders: beyond cereblon 

To overcome this challenge, there is a major effort underway by biotech and pharma to drastically increase the number of E3 ligases or UPP components that are recruitable to achieve degradation. Drugs which have made it to early clinical analysis mostly converge on just one E3 ligase, cereblon, which was initially discovered through the mechanistic analysis of the imide-containing drugs such as thalidomide4 (Ito et al., 2010). These ligands have proven a momentous source of new drugs, but come with known liabilities in tolerance, resistance and durability inherent to cereblon5.  

Enabling other E3 ligases has massive potential to address degradation of targets so-far unassailed by cereblon, and reduce concerns over safety and resistance to this drug mechanism. Identifying alternative E3 ligase mechanisms also holds the vital promise of adding a selective dimension to the modality, where an E3 ligase has expression or activity that is restricted to tumours to produce cancer-specific degradation. To accommodate the substantial complexities of bivalent drug discovery, more chemical diversity is needed to ensure better routes to oral bioavailability of the final drug candidates, which can be rapidly enabled through new ligases beyond cereblon. 

Figure 2: Bivalent and monovalent degraders: Bivalent degraders use target ligands linked to E3 ligase ligands to recruit the protein of interest to the E3 complex. Monovalent degraders bind only one part of the equation and induce novel interactions by surface remodelling. 

When one is more than the sum of the parts: monovalent degraders and molecular glues 

An increasingly attractive alternative to bivalent degradation approaches is to attempt to achieve a similar phenotypic outcome but through simpler, or more traditional, drug-like molecules. Monovalent degraders can in principle circumvent some of the medicinal chemistry challenges presented by bivalent drugs and may in time become the preferential therapeutic degrader molecules.  

Monovalent degraders are lower molecular weight than bivalent degraders and operate by remodelling the surface of the bound protein, adapting the structural and biochemical properties to create a neo-interface, resulting in new or improved binding protein-protein interactions. Molecular glues exhibit a therapeutic effect by increasing the affinity between an effector protein (e.g. an E3 ligase) and a neosubstrate client (e.g. a target POI). Whilst the desired function of these drugs is well understood, the precise principles of mechanism appear partially dynamic in each instance, and any convincing rules for design of new molecular glue degraders have so far eluded discovery, barring extensive functional screening campaigns6, which are not always possible for the most promising targets.  

One major hurdle to the design and discovery of new monovalent degraders is the limited sensitivity of current technologies for identifying native protein-protein interactions that could provide interfaces suitable for new glue design. There are many tools and approaches available which could drive the identification of such interactions and subsequent development of novel molecular glues. A series of betacatenin degraders was recently discovered to stabilise a native E3 ligase interaction7, and this concept would be an ideal framework for future discoveries with more powerful and sensitive proteomics approaches. Cryo-electron microscopy (CryoEM) has been pivotal in providing structural and mechanistic data on cereblon-based molecular glues8, and will further enable our understanding of degraders based on other E3 ligases. Machine-learning and large language model-based protein structural prediction provide a new set of tools which could be used to support molecular glue design, given the appropriate input data.  

Taken alone, none of these tools are sufficient to crack the degrader code; the biology of proteostasis is a drastically more complex puzzle than current computational methods are capable of solving. Combining new biological insights with these detection and design technologies is an ongoing challenge for the biotechnology community. A comprehensive platform that could induce novel protein-protein interactions at scale and in real biological systems would make a great impact in this field, facilitating routine delivery of new monovalent degrader medicines. 

The outlook for degradation 

The TPD concept remained relatively low profile until 2019, when the initiation of the first clinical trials  triggered huge optimism and investment2. Degradation as a modality is the ideal drug modality for the application of the substantial wealth of data from loss-of-function-based discoveries such as those from CRISPR, and scientists are only just beginning to understand the specific benefits of degrading a target compared to inhibiting it. Momentum of discovery has gained pace through the last few years as more companies and research groups have realised the importance and expansive scope of this approach. These pioneers will enable new applications across diverse disease areas, propelling the modality to address the challenges of unmet pathologies with new powerful drugs. 

About the author 

Dr Benedict Cross is the CTO and Head of Platform at PhoreMost, a pre-clinical drug discovery company based in Cambridge, UK. He is a geneticist & biotechnologist and has pioneered the use of computationally engineered mini-proteins to enable new medicine development, included degrader-based drugs. Cross has research expertise in proteostasis, functional genomics and chemical genetic screening and has authored over 30 peer reviewed studies and patents working at the intersection of biology, drug discovery and data science.  

References  

1: Békés, M., Langley, D.R. and Crews, C.M. (2022) ‘PROTAC targeted protein degraders: the past is prologue’, Nature Reviews Drug Discovery, 21(3), pp. 181–200. Available at: https://doi.org/10.1038/s41573-021-00371-6. 

2: Fang, Y. et al. (2023) ‘Targeted protein degrader development for cancer: advances, challenges, and opportunities’, Trends in Pharmacological Sciences, 44(5), pp. 303–317. Available at: https://doi.org/10.1016/j.tips.2023.03.003. 

3: Mostofian, B. et al. (2023) ‘Targeted Protein Degradation: Advances, Challenges, and Prospects for Computational Methods’, Journal of Chemical Information and Modeling, 63(17), pp. 5408–5432. Available at: https://doi.org/10.1021/acs.jcim.3c00603. 

4: Ito, T. et al. (2010) ‘Identification of a Primary Target of Thalidomide Teratogenicity’, Science, 327(5971), pp. 1345–1350. Available at: https://doi.org/10.1126/science.1177319. 

5: Hanzl, A. et al. (2023) ‘Functional E3 ligase hotspots and resistance mechanisms to small-molecule degraders’, Nature Chemical Biology, 19(3), pp. 323–333. Available at: https://doi.org/10.1038/s41589-022-01177-2. 

6: Słabicki, M. et al. (2020) ‘The CDK inhibitor CR8 acts as a molecular glue degrader that depletes cyclin K’, Nature, 585(7824), pp. 293–297. Available at: https://doi.org/10.1038/s41586-020-2374-x. 

7: Simonetta, K.R. et al. (2019) ‘Prospective discovery of small molecule enhancers of an E3 ligase-substrate interaction’, Nature Communications, 10(1), p. 1402. Available at: https://doi.org/10.1038/s41467-019-09358-9. 

8: Watson, E.R. et al. (2022) ‘Molecular glue CELMoD compounds are regulators of cereblon conformation’, Science, 378(6619), pp. 549–553. Available at: https://doi.org/10.1126/science.add7574. 

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