The Role of Protein Degraders in Biopharmaceutical Research
What are Protein Degraders?
Therapeutic molecules known as protein degraders target specific proteins for deletion inside cells through the body's existing protein breakdown processes. The implementation of this approach represents a crucial departure from standard small-molecule inhibitors (SMIs) and antibodies since these traditional methods only block protein function instead of eliminating the protein itself. The drug discovery field has come to view protein degraders as crucial instruments because they enhance development processes. Protein degradation primarily occurs through the Ubiquitin-Proteasome System (UPS). Through covalent attachment of ubiquitin molecules to target proteins the system activates their degradation by the proteasome system. The role of E3 ubiquitin ligases in protein degradation pathways involves substrate recognition followed by the initiation of ubiquitination. PROTACs function as dual-component molecules composed of a target protein ligand which is connected to both a linker and an E3 ligase ligand like CRBN or VHL. Proteasomal degradation of target proteins occurs through ProTAC molecules which enable protein ubiquitination by bringing target proteins into proximity with E3 ligases. Lysosomes serve as essential elements within cellular mechanisms responsible for protein breakdown. LYTACs guide extracellular proteins to enter lysosomes which then degrade these proteins. The molecular structure includes an antibody or ligand that targets proteins and a lysosome-targeting component which assists in internalizing proteins and directing them into lysosomes. Small molecules known as molecular glues operate by causing protein degradation through the manipulation of protein-protein interactions (PPI). These molecules stabilize target protein binding to E3 ligases which initiates ubiquitination followed by protein degradation. Multiple myeloma patients receive treatment through the use of lenalidomide and pomalidomide.
Fig. 1 Cellular protein degradation pathways and chemical-mediated targeted protein degradation methods.1,2
Types of Protein Degraders: PROTACs, Molecular Glues, and More
Degraders based on the ubiquitin-proteasome pathway (UPP)
1. PROTACs
Researchers have extensively investigated PROTAC within targeted protein degradation (TPD) applications. Traditional PROTAC molecules function as heterobifunctional compounds which attach to an E3 ubiquitin ligase and a protein of interesting (POI) through two separate binding domains. The PROTAC molecule consists of three parts: The PROTAC molecule includes an E3 ligase component, a POI ligand section and a linker that joins these two elements. The UPS activates POI ubiquitination and degradation when the ternary complex of POI-PROTAC-E3 ligase forms which subsequently leads to PROTAC release and recycling for further degradation events. Unlike classical inhibitors PROTAC operates through a catalytic mechanism of action (MOA) and relies on an "event-driven" process. As TPD drug research advances quickly, besides conventional PROTACs multiple new UPP-based degradation strategies have experienced significant growth.
Traditional PROTAC
The first small-molecule PROTAC that directed MDM2 E3 ligase toward androgen receptor (AR) protein degradation was reported in 2008 but required micromolar concentrations to achieve protein breakdown. MDM2 functions as the main cellular inhibitor of the p53 tumor suppressor while being crucial in cancer treatment approaches. Most interestingly, Hines et al. Hines et al. developed the first MDM2-based PROTAC A1874 for targeting bromodomain-containing protein 4 (BRD4) which resulted in 98% degradation of the protein of interest while achieving nanomolar efficiency. The small-molecule PROTAC demonstrated dual action by triggering BRD4 degradation while stabilizing p53 and thus blocking both BRD4 and MDM2 more effectively than conventional BRD4 small inhibitors.
In 2012 researchers produced the first small-molecule ligand that targets VHL ligase. Researchers determined the crystal structure of VHL when bound with its most effective inhibitor and showed the inhibitor occupied the hypoxia-inducible factor 1α (HIF-1α) binding site thereby establishing the groundwork for future ligand development. Scientists engineered a VHL-based PROTAC compound in 2015 that facilitated the breakdown of the estrogen receptor α (ERα). The research-produced VHL-PROTACs exhibited precise targeting capabilities and effectively removed ERα proteins from live mice. Researchers developed various small-molecule PROTACs derived from VHL which used lapatinib and gefitinib inhibitors as POI warheads to target receptor tyrosine kinase (RTK) degradation. Scientists developed PROTACs which demonstrated enhanced suppression of cell proliferation and downstream signaling compared to traditional RTK inhibitors while also overcoming resistance problems caused by target protein mutations.
Peptide-based PROTACs
The initial bifunctional PROTAC molecule was developed in 2001 to exploit SCFβ-TRCP E3 ubiquitin ligase via IκBα phosphopeptides for MetAP-2 degradation. The use of IκBα phosphopeptide phosphorylation to recruit E3 ligase demonstrated limited dependency. The new generation of PROTAC molecules which use peptide ligands has been termed bioPROTACs. Schneekloth et al. Schneekloth et al. achieved cell permeability in PROTAC molecules by adding a polylysine-penetrating peptide at the junction and demonstrated their ability to enter cells and degrade target proteins. Researchers in this study employed the briefest peptides derived from HIF1α to engage the VHL ligase which substantially accelerated peptide PROTACs development.
Nucleotide-based PROTACs
Public nucleotide-based PROTAC technology currently employs oligonucleotides to function as POI ligands. DNA repair and replication mechanisms require transcription factors together with RNA-binding proteins as essential protein components for transcription and RNA-dependent processes. Researchers have reported various types of PROTACs including RNA-PROTACs for RNA-binding proteins as well as O'PROTACs that use oligonucleotides and TRAFTACs which focus on transcription factors. The Lin28 protein which functions as a stem cell factor and oncoprotein stands as the primary target of the first RNA-PROTAC because of its potential as a drug target for multiple diseases. RNA-PROTACs bind to the RNA-binding site of RBPs through structurally modified oligoribonucleotides that replicate the natural RNA-binding element sequence of RBP. This PROTAC achieved exceptional degradation of Lin28 while maintaining high selectivity and minimal toxicity.
2. Molecular glues
Molecular glues represent monovalent degraders which possess smaller molecular weight compared to PROTAC molecules while enabling target protein degradation through E3 ligase surface remodeling and protein–protein interaction enhancement between E3 ligases and target proteins. Because molecular glues exhibit physicochemical properties comparable to traditional small-molecule drugs and stay within the 'rule of five' parameters, they demonstrate theoretical advantages over PROTACs thanks to their lower molecular weight and enhanced oral bioavailability and pharmacokinetic/pharmacodynamic profiles. At present, there are mainly two kinds of mature molecular glues: The first mature molecular glues category includes IMiDs such as thalidomide and its derivatives while the second category contains aryl sulfonamide drugs.
SERDs and SARDs
Monomeric degraders targeting ERα, AR, HER2, PI3Kα, BTK and cIAP have recently emerged as research and application areas. Among the monomeric degraders studied for their effects on ERα and AR, publications of monovalent degraders stand out as the most significant and important work. Selective estrogen receptor modulators (SERMs) along with aromatase inhibitors (AIs) were employed in previous treatments of ERα-positive breast cancer to inhibit ERα transcriptional activity and reduce estrogen production. Fulvestrant functions as an ER-targeting selective estrogen receptor degrader through its binding to ER which generates conformational instability that results in ERα degradation and enables overcoming resistance to AIs and SERMs.Researchers have identified a thieno[2,3‑e] indazole derivative that functions as oral selective estrogen receptor degraders (SERDs) to address ER-positive breast cancer and this compound has undergone preclinical trial evaluation.
Common Molecular Glues at BOC Sciences
Degraders based on the lysosome pathway
Two main pathways, the ubiquitin-proteasome system and the lysosomal degradation pathway, control the majority of intracellular protein degradation. The lysosome degradation system consists of two parts known as the autophagy-lysosome system and the endosome-lysosome system which operate on distinct protein depletion principles. The application of lysosome pathway-based degraders has substantially expanded the range of targets that can be degraded. AUTACs remove organelles including mitochondria whereas LYTACs break down membrane proteins and extracellular proteins demonstrating unique research outcomes versus traditional ubiquitin–proteasome pathway TPD agents.
1. AUTACs and ATTECs
Autophagy-targeting chimeras (AUTACs) attach a warhead for POI to a guanine derivative which marks the protein for degradation through autophagy pathways. Scientists designed AUTACs to target both proteins and malfunctioning mitochondria for selective autophagy. The AUTAC which targets mitochondria enhances mitochondrial turnover and prevents apoptosis when there are mitochondrial injuries. The subclass known as autophagosome-tethering compounds (ATTECs) functions within this system by linking a POI warhead directly to a ligand without using a linker which integrates them into monovalent degraders. It is worth noting that ATTEC works like molecular glues: ATTECs enable direct connection of POI warheads to LC3-binding ligands to facilitate autophagic degradation of POIs without the need for linkers. The research in 2019 introduced mHTT (mutant huntingtin)-LC3 linker compounds that reduced mHTT levels in living organisms while revealing that protein degradation could be achieved through autophagosome-tethering compounds thereby creating new opportunities for pharmaceutical development.
2. Lysosome-targeting chimeras (LYTACs)
During 2020, scientists explored a technology known as lysosome-targeting chimeras (LYTACs) which targets proteins located outside cells and attached to cell membranes. LYTACs have two binding domains: The oligoglycopeptide groups and antibodies or small molecules to which target proteins bind are connected through a linker. Researchers have employed LYTACs to specifically break down apolipoprotein E4 (ApoE), EGFR, and programmed death ligand 1 (PD-L1). The development of TPD agents based on lysosomal systems represents the leading advances in drug design and shows great potential for future preclinical and clinical applications.
Applications in Drug Discovery and Therapeutics
1. Dual-target PROTAC
Classical small-molecule PROTACs work by forming complexes with POIs which then target E3 ligases to degrade non-native neosubstrates. The research described homo-PROTACs as a small molecule strategy that induces E3 ubiquitin ligase dimerization leading to its self-destruction. While classical PROTACs use POIs and E3 ligases to degrade target proteins, homo-PROTACs target E3 ligases as their POIs. Scientists examined VHL E3 ubiquitin ligase for self-degradation potential and developed CM11 which showed rapid protein knockdown results. Ciulli and colleagues published their work on dual CRBN-VHL-PROTACs in 2019 to explore how CRBN and VHL E3 ligases induce degradation of each other. The study revealed that among hetero-PROTACs the novel PROTAC-based compound demonstrated strong CRBN ligase degradation reaching profound levels. Recent publications cover both homo-PROTACs targeting MDM2 and hetero-PROTACs designed for MDM2 and CRBN. This event enabled researchers to discover new effective methods for targeting E3 ligases through this technique.
2. Trivalent PROTACs
In 2019, research teams engineered trivalent PROTAC structures that combined bivalent BET inhibitors with an E3 ligand connected by a branched linker because they believed higher binding valency in PROTACs would result in better degradation. SIM1 functions as the best degrader by linking to VHL ligand while intramolecularly binding BET protein in a cis configuration to both BD1 and BD2 resulting in the formation of a 1:1:1 complex through conformational alteration. SIM1 demonstrated improved efficacy and potency over bivalent PROTACs because of better pharmacodynamic characteristics such as avidity and cooperativity combined with extended residence time. Lately, Huang et al. Huang et al. synthesized trivalent PROTACs featuring a tert-butyl ester unit on the benzene ring to direct further functionalization and they based their designs on the MZ1 structure bound to human VHL and BRD4BD2. Based on the examples illustrated above researchers have observed that VHL ligands exhibit greater utilization than CRBN which may stem from the variable expression levels of E3 ligase in different target proteins across cells and tissues.
3. Treatment of viral infections
The rapid global spread of SARS-CoV-2 led to the massive COVID-19 pandemic which devastated healthcare infrastructures and fundamentally altered everyday life. During 2020 researchers introduced a new anti-COVID peptide-based PROTAC developed through computational design to attack the viral spike protein receptor-binding domain (RBD) using TRIM21. The recent scientific development of ribonuclease-targeting chimeras (RIBOTACs) binds an RNA-binding molecule to a latent ribonuclease (RNase L) ligand in order to trigger RNA degradation. Scientists discovered a RIBOTAC molecule that targets SARS-CoV-2 FSE RNA to degrade it for therapeutic purposes.
4. Treatment of rheumatoid arthritis and neurodegenerative diseases
The field of TPD continues to expand beyond oncology because it can degrade any targeted molecule. Preclinical experiments show that the BTK degrader NX-5948 combined with the interleukin-1 receptor-associated kinase 4 (IRAK4) degrader KT-474 can provide treatment options for various immuno-inflammatory diseases including rheumatoid arthritis. Development efforts for Tau-targeting degraders are underway at present. Tau serves as a critical protein within neuronal cells that stabilizes microtubules (MTs) while also facilitating the transport of cargo proteins along these tracks and maintaining the cell's structural shape. Scientists introduced a peptide PROTAC that relies on Keap1 to eliminate Tau through UPP and demonstrated promising results for neurodegenerative disease treatment while advancing the study of "undruggable proteins."
5. Cancer treatment
The PROTAC protein degrader Bavdegalutamide uses a cyclohexyl moiety to connect with the AR ligand-binding domain and then links to the CRBN-containing E3 ubiquitin ligase through an IMiD-based moiety which initiates AR polyubiquitination. AR degradation by bavdegalutamide showed high potency in both VCaP and LNCaP prostate cancer cell lines with a DC50 value of approximately 1 nM. The proteomic analysis of VCaP cells that contained approximately 4,000 detectable proteins showed that bavdegalutamide at 10 nM for 8 hours caused selective androgen receptor breakdown reaching a maximum degradation of 85%. Bavdegalutamide degraded both wild-type AR and important AR mutants like T878A, H875Y, F877L and M896V which show resistance to modern hormonal treatments such as abiraterone and enzalutamide.
References:
- Image retrieved from Figure 1 " Cellular protein degradation pathways and chemical-mediated targeted protein degradation methods," Hyun S., et al., 2021, used under [CC BY 4.0]. The original image was not modified.
- Hyun S., et al., "Chemical-mediated targeted protein degradation in neurodegenerative diseases." Life 11.7 (2021): 607.
- Yang N., et al., Recent advances in targeted protein degraders as potential therapeutic agents, Molecular Diversity, 2024, 28(1): 309-333.
- Qin L., et al., Key considerations in targeted protein degradation drug discovery and development, Frontiers in Chemistry, 2022, 10: 934337.
- Sincere N I., et al., PROTACs: emerging targeted protein degradation approaches for advanced druggable strategies, Molecules, 2023, 28(10): 4014.
- Salama A K A A., et al., Targeted protein degradation: clinical advances in the field of oncology, International Journal of Molecular Sciences, 2022, 23(23): 15440.
