Beyond Inhibition: Targeted Protein Degradation Unlocks New Avenues for Drug Discovery

Targeted protein degradation (TPD) represents a novel therapeutic strategy for historically challenging disease-causing proteins. The concept of proteolysis-targeting chimera (PROTAC) molecules, leveraging the ubiquitin-proteasome system for targeted degradation, emerged two decades ago. Since then, TPD has transitioned from academic research to a robust industry pursuit, with numerous companies actively developing preclinical and early-stage clinical programs. The successful clinical validation of PROTACs against established cancer targets in 2020 opened doors for targeting previously “undruggable” proteins.

How does cellular protein degradation occur?

Protein homeostasis, or proteostasis, is a complex process that regulates protein concentration, folding, transport, and disposal in cells. The ubiquitin-proteasome system (UPS) is responsible for eliminating short-lived and misfolded proteins, while lysosomes degrade long-lived proteins, aggregates, and organelles. The canonical ubiquitination pathway involves the conjugation of ubiquitin to target proteins through an E1-E2-E3 enzymatic cascade. The UPS involves the attachment of ubiquitin molecules to proteins through a series of enzymatic reactions, with K48 and K63 linkages being the most common. Proteasomes, along with ubiquitin ligases and de-ubiquitinating enzymes, play a crucial role in the UPS-mediated protein degradation.

Lysosomes receive degradation substances through endocytosis, phagocytosis, or autophagy, and are involved in the degradation of cell surface proteins, pathogens, and damaged organelles. Lysosomes are membrane-bound organelles that act as the cell’s recycling center, containing a powerful cocktail of digestive enzymes called hydrolases that can break down all major classes of biomolecules. These enzymes function optimally at an acidic pH, which is maintained within the lysosome lumen by proton pumps in the lysosomal membrane. Lysosomal protein degradation is an essential process for maintaining cellular health and function. It helps to remove damaged or misfolded proteins, recycle cellular components, and provide the cell with amino acids for the synthesis of new proteins. Dysfunction of the lysosomal protein degradation pathway can lead to a number of diseases, including lysosomal storage disorders, neurodegenerative diseases, and cancer.

How can protein degradation pathways uncover new drug targets?

Traditional drug discovery focuses on protein activity regulation, but TPD technologies have emerged as promising approaches. TPD strategies rely on the UPS or lysosome pathways to selectively degrade intracellular or membrane proteins. TPD technologies such as PROTACs, molecular glues, and degradation tags offer promising approaches for studying cellular pathways and developing therapeutic interventions. TPD via lysosomes expands the range of substrates that can be degraded, including membrane proteins and extracellular proteins.

UPS-based targeted protein degradation strategies utilize E3 ligases as targeting proteins for degradation. PROTAC and molecular glue are two major technologies that rely on the UPS for the degradation of proteins of interest (POIs). PROTAC molecules consist of an E3-recruiting ligand, a POI-targeting warhead, and a flexible linker. The addition of PROTAC promotes the formation of the POI-PROTAC-E3 ternary complex, inducing ubiquitination and degradation of the POI. The first PROTAC molecule, Protac-1, was developed to degrade target protein MetAP-2 and subsequent studies demonstrated its effectiveness in degrading estrogen and androgen receptors. Small molecule-based PROTACs are more readily taken up by cells and have potential for drug development. Whereas, molecular glue degraders induce the interaction between a ubiquitin ligase and a POI, leading to POI ubiquitination and degradation.

Figure 2: Schematic of the ubiquitin proteasomal system.

Lysosomes mediate the degradation of proteins and organelles through endocytosis, phagocytosis, and autophagy. LYTAC (lysosome-targeting chimera) is a technique that induces the degradation of extracellular and membrane proteins via the endosome-lysosome pathway. Bispecific Aptamer Chimera utilizes DNA aptamers to target lysosome-targeting receptors and degrade membrane proteins. AbTAC is an antibody-based technology that targets membrane proteins for lysosomal degradation. GlueTAC utilizes nanobodies and covalent interactions to degrade cell-surface proteins. AUTAC (autophagy-targeting chimera) triggers autophagy to degrade cytoplasmic proteins and cellular organelles. ATTEC (autophagosome tethering compound) tethers proteins to autophagosomes for degradation, including mutant huntingtin proteins and lipid droplets. CMA-based degraders utilize chaperone-mediated autophagy to degrade pathogenic or misfolded proteins.

What conditions could TPD therapies be used for?

The explosive growth of TPD in the pharmaceutical industry, highlights its potential in cancer treatment, neurodegenerative diseases, inflammatory diseases, and viral infection.

Multiple TPD molecules, primarily based on PROTAC technology, have demonstrated promising therapeutic effects in cancer. Take the estrogen receptor (ER), a critical protein for breast cancer development. ARV-471, a PROTAC molecule specifically targeting ER, efficiently degrades the receptor and significantly reduces tumor burden in preclinical studies. This paves the way for its current phase II clinical trial, where ARV-471 is tested as a single agent or combined with another cancer drug.

Another exciting example is ARV-110, a PROTAC molecule targeting the androgen receptor (AR) in prostate cancer. This molecule shows particular promise for patients with tumors harboring mutations that render them resistant to existing AR-targeted therapies. These successes highlight TPD’s ability to tackle previously “undruggable” targets, making it a valuable tool in the pharmaceutical industry.
Beyond these, TPD researchers are making strides in targeting other crucial cancer proteins like STAT3, BCL-XL, and IRAK-4.  Molecules like SD-36 effectively degrade STAT3 in leukemia and lymphoma cells, while DT2216 targets BCL-XL in tumor cells with reduced side effects compared to traditional inhibitors.  IRAK-4 degraders are also being developed, with one already in phase I clinical trials for autoimmune diseases.
Figure 3: SD-36 molecular structure. 

The potential of TPD extends beyond cancer. Neurodegenerative diseases like Alzheimer’s and Parkinson’s are often associated with protein misfolding and aggregation.  Researchers are exploring TPD molecules targeting proteins like tau, a key player in Alzheimer’s disease.  Initial attempts using PROTAC technology have shown success in degrading tau and reducing its neurotoxicity.

Inflammatory diseases and viral infections are also potential targets for TPD. BTK, a protein involved in both inflammation and cancer, is being targeted by PROTAC molecules that may degrade both wild-type and mutant forms, overcoming the limitations of current BTK inhibitors.  For viral infections like COVID-19 caused by SARS-CoV-2, TPD offers a novel therapeutic approach. Initial studies utilizing PROTACs to degrade the HCV NS3/4A protease demonstrate the potential of this strategy. Furthermore, researchers are exploring the use of autophagy-lysosome pathway targeting technologies like AUTAC and ATTEC to eliminate key viral proteins.

For research purposes there are several PROTACs available at Biosynth to support research into cancers such as a bcl2 degrader-1, flt-3 degrader 1, and B-raf degrader 1.

PROTAC bcl2 degrader-1

PROTAC B-raf degrader-1

PROTAC flt3 degrader-1

Figure 4: Molecular structures of PROTACs to diverse targets.

How are TPDs developed?

The process for TPD molecule generation depends on the specific degradation pathway.

Target identification: The initial challenge is finding the right target for designing a TPD. For UPS, the scope of peptide discovery platforms for such intracellular targets is to identify small-molecule ligands for intracellular targets. This can be done with high throughput screening to identify a ligand. Alternatively, tools such as CLIPS™ (Chemical Linkage of Peptides onto Scaffolds) offers a uniquely versatile approach for finding ligands for the target. CLIPS^TM is a broadly applicable technology for constraining the 3D conformation of peptides for intracellular and extracellular targets. Unlike other constraining methods, CLIPS™ chemistry can create redox-stable mono-, bi- or tri-cyclic and bibridged formats and is compatible with the presence of side-chain-unprotected amino acids. This can be utilised to generate vast phage display arrays for identifying targets of both the lysosomal and UPS degradation pathways.

For lysosomal degradation strategies, specific cell receptors are used to pull extracellular targets into the lysosome for degradation. There are a couple of technologies in which ligands binding to cell receptors are conjugated with ligands binding to extracellular proteins of interest. In this case, peptides are ideal modality for extracellular targets, this would be well suited to CLIPS™ technology as one of the absolute strengths of this technology is the wide structural diversity of different CLIPS™ scaffolds that can be used, be it hydrophobic or hydrophilic in nature, or with very different length, sizes and bite angles. We currently have a selection of well over 50 different scaffolds. The benefit of CLIPS™ is that every scaffold induces unique conformation to peptides, resulting in a unique binding mode, which means that the binding of peptides attached to it changes when the nature of the scaffold has changed.

Design: This requires a combination of biological and chemical expertise to generate a successful TPD therapy. During biological design understanding of protein-protein interaction interfaces is crucial. For example, you need to identify suitable binding sites on the target protein and the E3 ligase for the PROTAC to effectively bridge them. This is followed by selecting the right protein target for degradation. This involves understanding the protein’s role in disease and its druggability through PROTACs. A lot of these issues for lysosomal projects are less of a consideration as there isn’t a need to cross the cell membrane.

Other strategies like AUTACs may involve designing molecules that bridge the target protein to autophagosomes (cellular waste disposal units) or incorporate specific motifs for recognition by cellular degradation pathways.

From the chemist’s perspective, the ability to design and synthesize small molecule fragments that can bind to the target protein and the E3 ligase is vital. Computational tools and medicinal chemistry knowledge can be an advantage in this area. A chemist will also consider the linker design. The linker region of the PROTAC connects the target protein-binding moiety and the E3 ligase-binding moiety. Designing a linker with the optimal length and flexibility is crucial for facilitating efficient protein degradation.

Pharmacokinetics: Understanding absorption, distribution, metabolism, and excretion (ADME) processes is vital. You need to design a TPD that gets absorbed well, reaches the target tissue, and avoids rapid breakdown by the body.  In addition, knowledge of potential toxicities is important. Lab experiments (in vitro) and animal studies (in vivo) are essential to measure the PK profile of a TPD. This data is used to optimize the dosing regimen, route of administration, and formulation for best therapeutic effect.

Manufacturing:

1. Synthesis: This is where the actual TPD molecule is created. It’s a complex organic synthesis process often relying on multi-step reactions. Chemists typically use specialized equipment and techniques to build the molecule with the desired structure and functionality. PROTACs are bifunctional molecules, meaning they have two distinct functional groups. This complexity can make synthesis and purification more challenging compared to traditional small molecule drugs. This may involve complex techniques for biologics like bispecific antibodies used in some AbTAC approaches.
2. Purification: After synthesis, the reaction mixture will likely contain a variety of components besides the desired PROTAC. Purification techniques like chromatography are employed to isolate the target molecule from unwanted byproducts and reaction leftovers.
3. Quality Control (QC): Once purified, the TPD needs rigorous quality control checks to ensure it meets pre-defined specifications for purity, structure, and potency. Techniques like mass spectrometry, nuclear magnetic resonance (NMR), and functional assays are used for confirmation. Production must be to GMP standard and be accompanied with clear protocols for inspection by regulatory agencies.
4. Scale-Up: Initial synthesis and purification are often done on a small scale for research purposes. If a TPD shows promise as a drug candidate, the manufacturing process needs to be scaled up for larger quantities needed for clinical trials and potentially commercial production. This involves optimizing reaction conditions, developing robust purification protocols, and potentially using specialized equipment for large-scale synthesis. Finding a partner that is able to provide multikilogram GMP production is crucial at this stage.

What are the challenges in TPD drug discovery?

While the field is young, familiarity with current research and development in TPDs can be beneficial. This includes staying updated on clinical trials of TPD therapies. TPD is a promising new approach, but there are still significant hurdles to overcome in its development:

Target Selection: It is believed there are over 600 viable targets but not all proteins are good candidates for TPD. While it holds promise for “undruggable” targets, understanding which ones will respond well and have minimal off-target effects is still an ongoing area of research.  Advice on target selection is key at this stage to assess the feasibility of the design and validate it.

Design: A major challenge is designing specific and effective TPD molecules, particularly small-molecule degraders. These molecules need to precisely target the disease-causing protein while avoiding unintended interactions with others. The current understanding of how these degraders work on a molecular level is still evolving, making it difficult to predict their behavior in complex biological systems.

Limited E3 ligase repertoire: Most PROTAC therapies currently rely on a small number of E3 ligases, the cellular machinery that tags proteins for degradation. This can limit the range of targets reachable and potentially lead to competition between drugs that use the same E3 ligase. Researchers are working on expanding the usable E3 ligase toolbox for more targeted therapies.

Off-target effects: Since TPD relies on hijacking the cell’s natural degradation system, there’s a risk of the drug accidentally tagging unintended proteins for destruction. This could lead to unforeseen side effects. Minimizing off-target effects requires careful design and rigorous testing of TPD molecules. For peptides, this issue is negligible, due to their high specificity. This can be further mitigated by introducing unnatural amino acids, as these prevent immunogenic recognition.

Delivery: Some TPD drugs, especially those with complex structures, like an E3 ligase linked to the target binder, can struggle to enter cells efficiently. This is particularly the case for intracellular targets as peptide drugs have low cell permeability struggling to cross the cell membrane effectively. However, even in cases where both binders are small molecules, the overall construct becomes “big” and has a low permeability.

Pharmacokinetics: Drugs may be cleared from the body too quickly. It is crucial to develop methods to deliver TPD drugs effectively and ensure they reach their target for a sustained period. The inverse must also be considered; lack of clearance can lead to an accumulation of toxic side effects. To be an effective treatment, a TPD drug must be able to alter proteostasis but within very narrow parameters.

Despite these challenges, TPD is a rapidly evolving field with immense potential. Researchers are actively developing new strategies to address these limitations and pave the way for more effective and specific TPD therapies.

How do you choose a partner for your TPD project?

The field of targeted protein degradation is relatively new. As a result, manufacturing processes for TPDs are still evolving, and new technologies and strategies are being developed to improve efficiency and cost-effectiveness.

Many pharmaceutical companies will agree to take on these projects. However, Biosynth has the expertise and infrastructure to handle challenging manufacturing processes like TPD production. Manufacturing protein-targeted degraders is a complex process that requires expertise in organic chemistry, purification techniques, and quality control procedures. Our expertise and manufacture are are able to produce specially designed TPDs with optimum properties for delivery, target selection, pharmacokinetics and reduced off-target effects.

You need a coherent package with experts from across the life sciences to deliver a TPD to market. That is Biosynth. We achieve a synergistic approach to both chemical and biological production. With our end-to-end manufacturing services, we are science-led and customer-focused to solve problems, taking pride in delivering projects that others cannot. Our expertise and capability run across Complex Chemicals, Peptides, and Key Biologics, all from one trusted partner.

From the initial target selection, Biosynth can provide specialist advice to optimise the feasibility of the targeted selected. With our peptide discovery platform, we support you in finding macrocyclic lead peptides binding to your target of choice. Taking into consideration your planned application and route of administration, we will choose the most suitable conditions, phage libraries, and CLIPS™ scaffolds to make your project a success. Our peptide discovery and optimisation team are already successfully working on multiple TPD projects. We validate your hits and leads in vitro, confirm their binding affinities (including Kon and Koff rates as well as Kd values) and provide modified versions for in vivo With our high-throughput peptide library synthesis platform, we identify the core binding motifs of your lead peptides to facilitate your IP filing and patent applications. We have more than 200 non-natural amino acids in stock, which we can incorporate in the peptide libraries to screen and further optimize your leads to have the best of all properties you are looking for. Finally, we can provide your purified lead compound for R&D in different scales and GMP-compliant for your clinical trial.

Figure 5: Workflow for target discovery to optimisation.

About Biosynth

Our complex chemistry specialties include enzyme substrates, carbohydrate and nucleoside chemistry, with manufacturing services from the first idea to the finished product, from route scouting to GMP or ton scale production. For peptides, we also have a full end-to-end offering, from lead discovery and optimization, library production, through to GMP NCE or neoantigen projects.  

Across biologics we have a synergistic offering, with an extensive range of custom bioprocessing enzyme projects for production of key products. We are also able to offer custom antibody projects, and epitope mapping. Through the Biosynth group we also offer development of antibodies, antigens and supply of plasma for in vitro diagnostics.  

The trusted supplier, manufacturer and partner for the pharmaceutical, life science and diagnostic sectors, along with customers across food, agrochemistry and cosmetics, we have facilities across three continents and a rapid global distribution network. Our main chemical research and manufacturing laboratories are in Switzerland, the United Kingdom, Slovakia and China, with peptide production in the USA, the Netherlands and UK. Enzyme projects are based in Austria and biological IVD reagents in Ireland.  Our R&D resources and production facilities are modern and versatile, allowing us to produce chemicals on the milligram to multi-ton scale, and at ISO 9001 and GMP, with peptides at mg to multikilogram scale.