Professor Henrik Daub, Founder and CSO, NEOsphere Biotechnologies looks at the advancements taking place in proteomics and how targeted protein degradation represents a promising approach for developing new treatments for life-threatening diseases.
Advancements in proteomics using mass spectrometry are helping researchers to identify novel, previously undruggable targets and develop degrader drugs to treat life-threatening diseases. Targeted protein degradation (TPD) is one of the most promising approaches for developing alternative treatments for life-threatening diseases. New technological advances in mass spectrometry (MS) based proteomics show great promise in this field by systematically connecting degrader compounds and their targets, which were previously undruggable. TPD taps into a cell’s natural disposal systems to selectively eliminate disease-causing proteins and is gaining interest for new drug discovery possibilities because of this ability to eliminate, rather than inhibit, disease-causing proteins especially those that have previously been challenging to target. Interest in the field was heightened recently when preliminary clinical data for bavdegalutamide (ARV-110), a proteolysis-targeting chimera that flags the androgen receptor for degradation, indicated that the drug is safe and shows some efficacy in men with metastatic castration-resistant prostate cancer.1
The power of TPD stems from small molecules commonly referred to as degraders. However, although protein degraders could become blockbuster drugs in many therapeutic areas, their discovery has been largely by chance to date.2 This is about to change as technological advances in proteomics instrumentation and software now enables researchers to screen extensive small-molecule libraries for degraders of pathogenic proteins that were previously considered ‘undruggable’.
Degradation-inducing molecules explained
While traditional small-molecule drugs only inhibit their targets, degradation-inducing molecules eliminate them and do not require active binding sites. That means entities like molecular glues, heterobifunctional degraders known as proteolysis targeting chimeras (PROTACs), monovalent degraders, and deubiquitinase (DUB) inhibitors can potentially target the large part of the human proteome that lacks active binding sites making it inaccessible to small-molecule inhibitors. The novel therapeutic mechanism makes protein degraders promising therapeutic agents for key indications that are underserviced by conventional medicines.3
Many degrader drugs redirect E3 ubiquitin ligases to non-native target proteins called neosubstrates. After forming a ternary complex with the target and the degrader, the E3 ligase modifies these neosubstrates by attaching multiple ubiquitin molecules. The proteasome recognises the resulting poly-ubiquitin chains and degrades the neosubstrates into amino acids that are recycled for novel protein synthesis.
Established degrader drugs that reprogram E3 ligases come mainly in two varieties: heterobifunctional molecules also known as PROTACs (PROteolysis TArgeting Chimeras) or molecular glue compounds.iv PROTACs consist of a recruitment ligand for the E3 ligase and a targeting ligand (“warhead”) that binds the target protein connected by a linker. Molecular glue degraders are smaller in size and have more favourable drug-like properties. They reshape the substrate interaction site of E3 ligases to create complementary binding sites for non-physiological targets.
Degraders with alternative mechanisms of action are also being developed. For example:
- Monovalent degraders induce degradation by triggering conformational changes in their target proteins rather than by repurposing an E3 ligase.
- DUB inhibitors effectively block removal of poly-ubiquitin chains from target proteins, causing their proteasomal degradation.
- New TPD approaches go beyond the proteasome, exploiting autophagic or lysosomal degradation pathways to eliminate extracellular targets and protein aggregates.
TPD in drug discovery
Discovery efforts in TPD start either from degrader targets or from degrader chemistries. Both strategies benefit from high-throughput deep proteomic screening, which analyses, without bias, the interaction between small molecule and the proteome to guide drug development. The first case applies mostly to heterobifunctional molecules like PROTACs, which are rationally designed to possess binding moieties for harmful proteins. Monitoring their selectivity against all cellular proteins provides researchers with crucial information.
In the second case, drug discovery originates from small molecules presumed or designed to be degraders. Testing these compounds against entire proteomes enables systematic identification of their targets. Molecular glue compounds, like those that bind to the E3 ligase cereblon for example, have been clinically validated to eliminate disease-causing proteins and can address proteins considered undruggable by conventional small molecule drugs. Their discovery has so far only led to a few dozen potential neosubstrates, meaning the true target scope of molecular glue degraders is unknown, despite their enormous therapeutic potential. A systematic, proteomics-based discovery approach to connecting degrader compounds with targets in an unbiased manner is required to exploit the full targeting potential of molecular glues.
Enabling effective processing and analysis of large-scale proteomics data
Putative degrader targets can be identified in proteomic screens and readily be validated using a unique proteomics-based validation pipeline. Besides identifying neosubstrates at unprecedented scale, the resulting data can be used for degrader library optimisation and expansion, as well as for computational approaches such as AI-based predictive modelling of novel degrader drugs. The high-throughput and fast turnaround capabilities of this platform also support proteome-wide selectivity profiling in drug optimisation cycles. It uses data independent acquisition (DIA) in combination with trapped ion mobility spectrometry (TIMS) (timsTOF Pro, Bruker, Billerica, MA). (Figure 1).
Global ubiquination analysis
MS-based proteomics has enabled the rise of ubiquitomics, which refers to the set of proteins that are modified by ubiquitin and the associated ubiquitin chain topologies found under these conditions. Advancements in technology has enabled the high throughput detection of proteins that are modified with ubiquitin, referred to here as ubiquitin site profiling, which has greatly increased understanding of the ubiquitome. One such tool is a scalable workflow for deep and precise in vivo ubiquitinome profiling. Hereby, an improved sample preparation protocol is coupled with DIA-MS and NN-based data processing specifically optimised for ubiquitinomics to routinely and reliably identify and quantify up to 50,000 ubiquitination sites in a single run. Ubiquitinomics thus allows the rapid validation of cellular downregulations being due to E3 ligase-neosubstrate relationships, by analysing degrader drugs in endogenous cellular systems without the need for pharmacological intervention or genetic modification (Figure 2).
Almost all neosubstrates found to be degraded upon five hours treatment of HEK293 cells with the cereblon modulator avadomide (left graph) show induced ubiquitination sites after 30 min of treatment (depicted on the right).
Recent technological advances in proteomics have the power to revolutionise degrader development by systematically connecting degrader compounds and their targets. By enabling systematic comparisons across diverse cell lines and types to maximise the accessible proteome, selecting the most responsive screening models, and scoring and reviewing metrics based on individual treatment and global data assessment, researchers have more power in their hands to make successful treatments based on TPD. MS-based proteomics can help reveal the true target scope of targeted protein degradation, untapping its potential as the next big thing in medicine.
- Békés, M., Langley, D.R. & Crews, C.M. PROTAC targeted protein degraders: the past is prologue. Nat Rev Drug Discov 21, 181–200 (2022).
- Wu, T., Yoon, H., Xiong, Y., Dixon-Clarke, S. E., Nowak, R. P., & Fischer, E. S. (2020). Targeted protein degradation as a powerful research tool in basic biology and drug target discovery. Nature structural & molecular biology, 27(7), 605–614.
- Liena, Q., Han, D., Junfeng, W. Key Considerations in Targeted Protein Degradation Drug Discovery and Development. Frontiers in Chemistry. 10(2022).
- Zuzanna Kozicka and Nicolas Holger Thomä Haven’t got a glue Protein surface variation for the design of molecular glue degraders. Cell Chemical Biology 2021 Jul 15; 28(7):1032-1047.