March 30, 2023 PAO-03-23-CL-07
Over the past two decades, targeted protein degradation (TPD) has emerged as a therapeutic modality with the potential to tackle disease-causing proteins that have historically been highly challenging to target with conventional small molecules. TPD utilizes the waste-disposal system of the cell to selectively eliminate disease-causing proteins. Advancements in instrumentation and software have led the pharmaceutical industry to look more closely at TPD for new drug discovery possibilities because of its ability to eliminate — rather than inhibit — disease-causing proteins. Interest in the field was bolstered 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 capabilities of TPD are centered around 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.2 Technological advances in proteomics now show capabilities to screen extensive small molecule libraries for degraders of pathogenic proteins previously considered undruggable.
Unlike traditional small molecule drugs that only inhibit their targets, degradation-inducing molecules deplete their targets and do not require active binding sites to exert their effect. 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 and is thus inaccessible to small molecule inhibitors. The novel therapeutic mechanism makes protein degraders promising therapeutic agents for indications of high medical need unmet 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 recognizes the resulting poly-ubiquitin chains and degrades the neosubstrates into amino acids that are recycled for novel protein synthesis. Unlike conventional small molecule drugs, degrader molecules don’t just inhibit their targets. Instead, they act as catalytic agents that eliminate disease-causing proteins and their associated functions.4
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.5 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 favorable 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. 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 (Figure 1). Finally, new TPD approaches go beyond the proteasome, exploiting autophagic or lysosomal degradation pathways to eliminate extracellular targets and protein aggregates.
Figure 1. Proteomics addresses the relevant questions in degrader drug discovery by identifying candidate degrader molecules that engage (new) E3 ligases or act through different TPD mechanisms; systematically exploring the true target scope of degrader compound libraries in intact cells; mechanistically validating candidates as bona fide TPD targets in the proteome-wide context; and comprehensively monitoring degrader compound selectivity in endogenous cellular environments.
Discovery efforts in targeted protein degradation start either from degrader targets or from degrader chemistries. Both strategies benefit from high-throughput deep proteomic screening, which unbiasedly analyzes the interaction between small molecules 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 crucial information for optimization efforts. In general, proteome-wide selectivity analysis should be performed for any degrader molecule under development for a known target.
In the second case, drug discovery originates from small molecules presumed or designed to be degraders. Testing such compounds against entire proteomes enables systematic identification of their targets. For example, molecular glue compounds, like those that bind to the E3 ligase cereblon, have been clinically validated to eliminate disease-causing proteins. With low molecular weight, favorable drug-like properties, and the ability to promote target recognition in the absence of cavities or binding pockets, molecular glues can address proteins considered undruggable by conventional small molecule drugs. Their discovery, however, has so far relied on serendipitous observations that have led to only a few dozen potential neosubstrates. Thus, the true target scope of molecular glue degraders is largely unknown, despite their enormous therapeutic potential. Only a systematic, proteomics-based discovery approach that connects degrader compounds with targets in an unbiased manner can exploit the full targeting potential of molecular glues (Figure 2).
Figure 2. The agnostic proteomics platform screens and analyzes all types of degrader molecules in high throughput (e.g., PROTACs, molecular glues, monovalent degraders, DUB inhibitors), mechanistically validates potential targets, and monitors proteome-wide selectivity during compound optimization.
Putative degrader targets found in the proteomics screens can readily be validated using a unique proteomics-based validation pipeline (NEOsphere Biotechnologies, Planegg, Germany). Besides identifying neosubstrates at unprecedented scale, the resulting data can be used for degrader library optimization and expansion, as well as for computational approaches, such as AI-based predictive modeling of novel degrader drugs. The high throughput and fast turnaround capabilities of this platform also support proteome-wide selectivity profiling in drug optimization cycles.
This proteomics-based platform comprises four major components (Figure 3):
Figure 3. A unique proteomics-based proteomics platform systematically connects degrader compounds with their targets to unlock and explore the undruggable proteome.
To demonstrate the capabilities of this technology, colon cancer cells were treated for the indicated times with E3 ligase–modulating drugs, and single-shot MS was performed as described. Proteome coverage of up to 11,000 protein groups per sample was achieved with a data completeness of more than 99% and a median protein coefficient of variation (CV) below 10%.
Statistical data analysis revealed statistically significant up- and downregulation events of cellular proteins.
Thalidomide derivatives, such as pomalidomide, mezigdomide, or lenalidomide, are known to induce ubiquitination and proteasomal degradation of zinc-finger proteins by recruiting them to cereblon. As expected, zinc-finger target proteins expressed in the analyzed cell line were downregulated, including ZNF692, ZFP91, and ZNF827. Other neosubstrates were also degraded, such as the lenalidomide-specific target casein kinase 1a (CSNK1A1).
The sulfonamide-type molecular glue indisulam promotes interaction between DCAF15 E3 ligase and the splicing factors RBM39 and RBM23, leading to their ubiquitination and proteasome-mediated degradation. Treatment with the VHL based PROTAC degrader ACBI18 for four hours downregulated its direct cellular targets SMARCA2 and SMARCA4 together with two interacting BAF complex members, while secondary regulation events are detected eight hours later.
Figure 4. The effect of compound treatment on cellular regulation. The volcano plots depict the fold change of proteins in compound treated vs. control cells (x-axis) and the standard as a measure of precision of quantification (y-axis). Statistically significant downregulation events of cellular proteins are shown in red, upregulation events in blue.
Recent technological advances in proteomics promise to eliminate bottlenecks in degrader development by systematically connecting degrader compounds and their targets. The underlying concepts and strategies for undruggable target space exploration include systematic comparisons across diverse cell lines and types to maximize the accessible proteome, selection of most responsive screening models, and scoring and reviewing metrics based on individual treatment and global data assessment to distinguish likely direct degradation from secondary regulation or off-target effects. Optimized screening of both biological and chemical diversity can identify cell type-specific and rare target candidate hits, as well as the sweet spots/regions in chemical space, creating rationales to expand chemistry where more target candidates are likely encountered.
All of these new capabilities of MS-based proteomics can reveal the true target scope of targeted protein degradation and help to advance degrader drugs, to broadly position TPD as an alternative to existing therapeutic modalities.
Prof. Daub is a pioneer and scientific leader in the field of proteomics-based drug discovery with more than 20 years of industry experience in the development and commercialization of state-of-the-art proteomics technologies. He is the scientific founder and CSO of NEOsphere Biotechnologies, a Munich-based company using data-independent high-throughput mass spectrometry (DIA-PASEF) for proteomic screening and mechanistic validation of degrader molecules. Previously, he was SVP Science & Technology at Evotec, where he led global efforts to discover targets for targeted protein degradation. Prior to that, he was a group leader at the Max Planck Institute of Biochemistry and scientific founder of the proteomics company Kinaxo Biotechnologies. Prof. Daub received his PhD in Biochemistry from the Max Planck Institute of Biochemistry and his venia legendi from the Technical University of Munich, where he is also a Professor of Biochemistry.