Targeted Protein Degradation: The Next Frontier in Drug Discovery
What is the protein degradation?
The cellular degradation of proteins represents an essential component of protein turnover because it functions as a quality control system for protein folding while enabling fast cellular response to changing signals and controlling amino acid availability. The ubiquitin-proteasome system (UPS) serves as the main route for degrading the majority of proteins. The UPS can process the entire proteome but it executes this function through precise, orchestrated sequential steps. Proteins destined for degradation undergo covalent post-translational modification by attaching ubiquitin in a process called ubiquitylation. Three enzymes work together in a cascade to execute this process. The E1 ubiquitin-activating enzyme uses ATP to produce an activated ubiquitin-adenylate intermediate which subsequently forms a thioester bond with a catalytic cysteine residue located in the E1 enzyme's active site. The ubiquitin molecule moves from the E1 enzyme to the E2 ubiquitin-conjugating enzyme's catalytic cysteine during a transthiolation reaction. The E3 ubiquitin ligase allows ubiquitin to attach to the target protein by creating an isopeptide bond between ubiquitin's carboxyl terminus and a lysine residue on the substrate. The enzymatic cycle repeats itself to generate polyubiquitin chains which mark the substrate for proteasomal degradation. The enzymatic process depends on E3 ligases to define substrate specificity. E3 ligases represent about 600 predicted members that function as adaptors which identify target proteins through protein-protein interactions before aligning them with the ubiquitylation machinery to enable ubiquitin attachment.
Introduction to Targeted Protein Degradation
Targeted protein degradation (TPD) uses small molecules to selectively cause the breakdown of designated proteins. The initial concept of TPD relied on directing target proteins to ubiquitin ligases but now includes multiple cellular degradation processes. Within ubiquitin-mediated degradation systems small molecules serve as degraders which fall into two primary categories: molecular glue degraders and PROTACs (proteolysis-targeting chimeras). Molecular glue degraders and PROTACs share the common goal of directing target proteins to ubiquitin ligases for degradation yet function through different mechanisms. Molecular glue degraders are single-binding molecules which attach to either the E3 ubiquitin ligase or the target protein but maintain low affinity for the alternate binding partner to create an interaction platform between the E3 ubiquitin ligase and the target protein along with the glue itself. In contrast, PROTACs consist of two separate binding domains (often called warheads) connected by a linker: One protein-binding domain interacts with the E3 ligase and the other interacts with the target protein which positions them adjacent to each other. When an E3 ligase attaches to the target protein in both cases it triggers ubiquitylation followed by proteasomal degradation. The therapeutic strategy of TPD employing molecular glue degraders alongside PROTACs has become a significant tool for research and treatment development. Therapeutic protein degradation as a treatment strategy became feasible through the clinical applications of thalidomide, lenalidomide, and pomalidomide which were later identified as molecular glues. The combination of progress in PROTAC development has led to the introduction of many new degraders into clinical trials. The small-molecule-induced proximity strategy continues to develop rapidly yet its core principles have historically been applied both in natural systems and synthetic methods for many years. These findings deliver important mechanistic understanding that aids in creating new synthetic molecules.
How do the PROTACs and Molecular Glues Work?
(1) PROTACs
PROTACs function as heterobifunctional molecules that interact at once with E3 ubiquitin ligases and target proteins to facilitate the E3 ligase complex-mediated ubiquitination of lysine residues located on the target protein. After a protein becomes polyubiquitinated the 19S regulatory cap of the proteasome identifies it for degradation into amino acids and small peptides. In that period researchers had access to a small molecule MetAP2 ligand but no E3 ligase ligands existed which required researchers to use a 10-amino-acid phosphopeptide that binds β-TRCP instead. These positive findings established the necessity for the development of additional drug - like E3 ligase ligands that would be appropriate for PROTAC applications. Research findings demonstrated that HIFα needs a particular proline hydroxylation at position 564 to bind with VHL. The SCF-β-TRCP model guided the integration of short hydroxyproline peptides into peptidic PROTACs which recruited the VHL E3 ligase and degraded FKBP12 and AR. The peptidic properties of the HIFα component restricted their use in living organisms even though hydroxyproline cores indicated that E3 ligase ligands with drug-like properties were possible. Unfortunately, these PROTACs exhibited only modest potency. PROTACs stand out from monovalent molecular glue degraders because they consist of three distinct units: an E3 ligase component, a target protein binder, and a connecting linker. The modular design of PROTACs enables logical discovery through the union of ligase and target binders but simultaneously introduces drug development challenges. PROTACs usually represent large molecular structures with weights above 700 Daltons which call for comprehensive optimization through empirical methods to obtain pharmacokinetic attributes suitable for clinical use.
Common PROTACs at BOC Sciences
(2) Molecular Glues (MG)
Molecular glues (MGs) first gained scientific interest because they can create or stabilize protein-protein interactions between proteins which do not normally interact. Subsequent studies demonstrated that molecular glues facilitate interactions between E3 ligases and target proteins which initiates protein degradation. MGs represent smaller monomeric molecules that typically meet Lipinski's Rule of Five in contrast to PROTACs which are larger heterobifunctional compounds. The compliance with Lipinski's Rule Five shows that MGs demonstrate better drug properties including improved oral bioavailability and positive pharmacokinetics. The identification of MGs poses a substantial challenge because it requires detection of natural or coincidental interaction points between proteins. Currently three amide-based MGs have received approval for oral use in multiple myeloma (MM) and myelodysplastic syndromes (MDS) treatments. The mounting interest in this field has led to substantial investments while multiple MGs move forward into clinical trials.
(2.1) Non-degradative MGs
The earliest and most significant examples of macrocyclic glycosides (MGs) were microbial macrolides including FK506, rapamycin and cyclosporin A. A Swiss biologist first identified cyclosporin's immunosuppressive properties when it was discovered in 1971. FK506 and rapamycin together display cyclosporin A-like activities through their large shared cyclic polyketide structure. In the years that followed scientists worked rapidly to understand their mechanisms of action. Early 1990s research identified cyclophilin and FKBP12 as the two respective receptors that bind to cyclosporin A and FK506 to create complexes which inhibit calcineurin and deliver immunosuppressive action. Calcineurin exclusively binds to protein complexes but fails to bind to free cyclophilin or FKBP. MGs function as an adhesive substance which enables protein-protein interactions between proteins that typically lack mutual affinities. In 1992 scientists created the term 'molecular glue' to explain its functional action. Scientific studies in 1994 discovered the interaction between FKBP12-rapamycin and the protein kinase mTOR. Mutations found in mTOR and FKBP12 demonstrate rapamycin resistance which supports rapamycin's characterization as a molecular glue.
(2.2) Degradative MGs
Thalidomide together with lenalidomide and pomalidomide represent important degradative MGs that have therapeutic applications. Thalidomide entered the market as a sedative until research identified its teratogenic effects which caused manufacturers to withdraw it from sale. Thalidomide's renewed therapeutic application for leprosy treatment led to the creation of more powerful derivatives including lenalidomide and pomalidomide which demonstrated effective anti-inflammatory and anti-angiogenic properties. Recent findings led to expanded usage of IMiDs in treating various blood cancers including multiple myeloma, MDS, CLL and B-cell lymphoma. Research in 2010 revealed that thalidomide functions by targeting E3 ubiquitin ligase CRBN which clarified the molecular mechanism of its effects. The discovery demonstrated lenalidomide functions as a MG which allows CRBN to degrade specific proteins such as lymphoid transcription factors IKZF1 and IKZF alongside CK1α in 5q deletion myelodysplastic syndrome (MDS). Understanding how IMiDs function has generated major interest in the research and development of new MGs. The emergence of arylsulfonamide-based drugs like indisulam and E7820 broadened the range of available MGs. The discovery in 2017 revealed that indisulam enhances the recruitment of RNA-binding motif protein 39 (RBM39) to the CUL4-DCAF15 E3 ligase complex for its degradation. Research studies demonstrated that BI-3802 functions as a MG since it induces BCL6 oligomerization and enhances its binding to SIAH1 which is an E3 ligase. The interaction between BCL6 and its E3 ligase leads to enhanced ubiquitination and degradation of BCL6 which demonstrates the therapeutic potential of MGs.
Fig. 1 Mechanisms of action of PROTAC, molecular glues, and hydrophobic tagging.1,2
Common Molecular Glues at BOC Sciences
Clinical Applications of Targeted Protein Degradation
TPD represents a revolutionary approach in both drug discovery and clinical practice which provides new solutions for overcoming the limitations associated with traditional small-molecule inhibitors (SMIs). The TPD strategy utilizes intrinsic protein degradation pathways like the ubiquitin-proteasome system and lysosomes to selectively eliminate disease-causing proteins. This method reduces off-target effects while addressing drug resistance and enables targeting of proteins that were previously thought to be 'undruggable'. The PROTAC platform leads as the most advanced TPD technology currently in clinical development. These molecules cause the target protein to get close to an E3 ubiquitin ligase which leads to its ubiquitination and subsequent degradation by the proteasome. ARV-110 and ARV-471 represent the current PROTAC candidates in clinical trials as they target the androgen receptor for castration-resistant prostate cancer and the estrogen receptor for ER+/HER2- breast cancer respectively, and both compounds progressed to Phase 2/3 trials. Vepdegestrant stands as the first PROTAC drug anticipated to receive approval for breast cancer therapy. Molecular glues promote or reinforce protein-protein interactions which result in the breakdown of target proteins. The drugs Lenalidomide and Pomalidomide demonstrate effective treatment options for MM and MDS. Researchers are developing novel molecular glues such as CC-92480 to tackle resistance issues found in current therapeutic approaches. Research has shown that both thalidomide derivatives and indisulam function as molecular glue degraders which underscores the need for discovery efforts targeting new molecular glues to broaden the range of possible targets. The discovery of molecular glue degraders began with identifying small molecules that strengthen the interaction between the oncogenic transcription factor β-catenin and its natural E3 ligase SCFβ-TrCP. Cancers show frequent stabilization and dysregulation of β-catenin which functions as a protein in the Wnt signaling pathway. Glycogen synthase kinase 3 (GSK3) together with casein kinase 1 (CK1) phosphorylates β-catenin to produce a phospho-degron that β-TrCP from the SCFβ-TrCP CRL26 recognizes. The researchers sought to discover small molecules that bypass the phosphorylation requirement and through screening and optimization identified NRX-1933 as a molecular glue that stabilizes the phospho-β-catenin–β-TrCP interaction. The chemical optimization process produced NRX-252114 which demonstrated enhanced recruitment and ubiquitylation of a particular phosphorylated β-catenin variant.
Advances in TPD Technologies
(1) Diverse Degradation Mechanisms
Proteins with improper folding inside cells reveal altered hydrophobic amino acid residues or patches on their outer surface. The heat shock protein 70 (HSP70) chaperone retains the ability to detect hydrophobic areas on partially denatured proteins due to its highly conserved and widespread nature. The co-chaperone E3 ligase C-terminus of Hsp70 interacting protein known as CHIP is recruited to trigger the ubiquitination process of misfolded proteins. Misfolded proteins that present exposed hydrophobic regions get processed by cellular quality control mechanisms and are then destroyed by the proteasome system. Hydrophobic tagging (HyT) technology uses a bifunctional molecule to trigger proteasomal degradation of protein of interesting (POI) by replicating the structure of misfolded proteins. HyT represents a molecular structure where hydrophobic moieties like adamantyl or Boc3Arg attach to a selective ligand targeting a specific POI through a short linker. The molecule Boc3Arg targets the 20S proteasome to degrade POIs through an ATP and ubiquitin-independent pathway. This method cannot be applied broadly as researchers have discovered it interferes with Mammalian Target of Rapamycin Complex 1 signaling.
(2) Combination Therapies
Protocols that test different E3 ligases besides CRBN or VHL could improve specificity while minimizing unintended effects. Selective degradation becomes feasible through the use of newly developed ligands that target E3 ligases which show tissue-specific overexpression. Research is underway to test TPD agents alongside immuno-oncology treatments, targeted SMIs, and traditional chemotherapeutics. This treatment strategy seeks both to generate synergistic outcomes and elevate the effectiveness of therapies.
Challenges of Targeted Protein Degradation
1. Off-Target Effects and Toxicity
TPD molecules carry the risk of breaking down proteins that they were not designed to target which creates unintended side effects and toxicity. To minimize harmful effects scientists need to maintain molecular stability while delivering them specifically to targeted tissues.
2. Limitations of molecular structure
TPD molecules including PROTACs experience difficulties due to their substantial molecular weights and structural complexities which result in poor water solubility along with high first-pass metabolism and problems in cellular permeability and oral bioavailability. The design parameters of linkers in TPD molecules including their length and rigidity along with stereoselectivity play essential roles in improving both affinity and pharmacokinetic attributes. The enhancement of these molecular features proves to be a significant obstacle.
Future Outlook
1. Technological Advancements
Research and development of TPD molecules now benefit from computational technologies such as 3D modeling combined with deep learning and neural networks to speed up their design optimization process. These advanced technologies enable precise prediction of drug reactions while refining molecular designs at a faster rate. Scientists are experimenting with nanoparticles and different delivery systems in order to improve the stability and bioavailability of TPD molecules while achieving targeted delivery. The delivery of PROTACs has shown improved performance through the use of gold nanoparticles and lipid-based nano self-regulating platforms.
2. Develop combination therapy
Scientists are working to discover new E3 ligases to increase the range of targeted degradation applications. This method targets overcoming resistance barriers while enhancing both specificity and effectiveness of TPD treatment. Therapeutic protein degraders (TPD) agents undergo testing alongside immuno-oncology treatments as well as targeted small-molecule inhibitors and conventional chemotherapeutic drugs. The method intends to create combined effects that strengthen the effectiveness of treatment.
References:
- Image retrieved from Figure 1 "Protein degradation via the ubiquitin-proteasome system (UPS)," Kim Y., et al., used under [CC BY 4.0]. The original image was not modified.
- Kim, Yosup, et al. "Targeted protein degradation: principles and applications of the proteasome." Cells 12.14 (2023): 1846.
- Schapira M., et al., Targeted protein degradation: expanding the toolbox, Nature reviews Drug discovery, 2019, 18(12): 949-963.
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- Zhong G., et al., Targeted protein degradation: advances in drug discovery and clinical practice, Signal Transduction and Targeted Therapy, 2024, 9(1): 308.
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