PROTACs (Proteolysis Targeting Chimeras) are reshaping the landscape of drug discovery by harnessing the body’s own protein degradation machinery to eliminate disease-causing proteins. PROTAC molecules consist of three essential components: a ligand that binds the target protein of interest (POI), a linker that connects the two ends, and an E3 ligase ligand that recruits an E3 ubiquitin ligase.
Among these, the E3 ligase ligand plays a pivotal role in determining which E3 ligase is engaged, directly influencing the degradation efficiency, selectivity, and overall success of the PROTAC molecule. While more than 600 E3 ligases are known in the human genome, only a handful, such as MDM2, IAPs, VHL, and CRBN, have well-characterized, druggable ligands and are routinely used in PROTAC design. The choice of E3 ligase affects not only the cellular localization and expression profile but also the ability to form a stable and cooperative ternary complex with the POI. However, significant advancements in recent years have led to the discovery and characterization of several novel E3 ligase ligands, greatly expanding this initial repertoire.
Timeline of E3 Ligase Ligand Discovery in PROTACs design
(up to June 2025)
Classification of E3 Ligase Ligands
1. MDM2 Ligands
MDM2, an E3 ligase that often plays a role in cancer by degrading the tumor suppressor p53, was an early target in PROTAC development. Initial MDM2 inhibitors, like Nutlin-3, served as the foundation for the first MDM2-recruiting PROTACs. The SARM-Nutlin PROTAC, created in 2008, was a pioneer, demonstrating the ability to degrade target proteins. While later efforts also showed success in degrading proteins and reactivating p53’s anti-cancer functions, MDM2-recruiting PROTACs haven’t seen as much widespread use, likely due to challenges with their larger size and less ideal drug-like properties. Still, MDM2’s contribution remains an important part of the PROTAC story.
Representative MDM2 Ligands
2. IAPs Ligands
Inhibitor of Apoptosis Proteins (IAPs), a family of E3 ligases often overexpressed in cancer, were explored early in PROTAC development. In 2010, the first IAPs-recruiting PROTAC demonstrated the feasibility of degrading target proteins using these ligases. While subsequent efforts expanded the range of targets and improved potency with novel ligands like those derived from IAPs antagonists, IAPs are less favored today. This is primarily because IAPs ligands can trigger antoubiquitination of the ligase itself, leading to inefficient degradation of the intended target. Despite this limitation, the foundational work with IAPs provided valuable early insights that shaped the PROTAC field.
Representative IAPs Ligands
3. VHL Ligands
The von Hippel-Lindau (VHL) protein is the substrate recognition subunit of the CRL2VHL E3 ligase complex, best known for its role in regulating hypoxia-inducible factor 1α (HIF-1α) under normal oxygen conditions. VHL binds to hydroxylated HIF-1α at their proline residue, tagging it for ubiquitination and proteasomal degradation.
Building on the structural understanding of the VHL- HIF-1α interaction, researchers developed potent, high-affinity small-molecule ligands that mimic the hydroxyproline-containing HIF-1α peptide. These VHL ligands typically incorporate a central hydroxyproline core that anchors binding, flanked by substituents that allow linker attachment and improve physicochemical properties.
Compared to CRBN ligands, VHL-based ligands often yield PROTACs with greater structural rigidity and metabolic stability. More importantly, because these ligands are derived from a peptidic scaffold, they offer multiple sites for chemical modification. This structural flexibility allows for precise tuning of binding affinity, physicochemical properties, and pharmacokinetics, making VHL ligands both versatile and highly engineerable components in PROTAC design.
Representative VHL Ligands
4. CRBN Ligands
The development of cereblon (CRBN) ligands represents one of the most well-characterized and widely applied advances in the PROTAC field. Thalidomide, initially introduced in the 1950s to treat morning sickness, was later withdrawn due to severe birth defects. Decades later, it re-emerged as an effective treatment for multiple myeloma and B-cell malignancies.
In 2010, CRBN was identified as the direct target of thalidomide, revealing that the drug hijacks CRBN – a substrate receptor of the CRL4 E3 ligase – to degrade transcription factors like ASLL4. This finding introduced the concept of E3 ligase-mediated neosubstrate degradation. Thalidomide analogs such as lenalidomide and pomalidomide (IMiDs) were subsequently developed to enhance CRBN binding and improve pharmacological properties. These iMiD-based CRBN ligands have since become core components in PROTAC design, enabling the degradation of a broad range of disease-relevant proteins.
CRBN ligands are attractive due to their small size, drug-like properties, oral bioavailability, and the abundant structural knowledge available. Notably, the crystal structure of CRBN bound to thalidomide or its analogs has enabled rational optimization of CRBN ligands for PROTACs. However, despite their popularity, IMiD-based ligands come with potential off-target effects, as they can degrade endogenous neosubstrates such as IKZF1/3 or SALL4, which may lead to immune modulation or toxicity. To address these concerns, new CRBN ligands are being designed such as non-IMiD scaffolds, photo-controllable designs, etc.
Representative CRBN Ligands
5. Emerging Ligands
As PROTAC technology rapidly advances, the quest for novel E3 ligases beyond the well-established four is paramount to overcoming current limitations and unlocking the full therapeutic potential of targeted protein degradation.
AhR Ligands: A newly explored class of E3 ligase recruiters that use AhR (aryl hydrocarbon receptor) agonists to direct protein degradation, though ongoing optimization addresses challenges like self-degradation.
DCAF16 Ligands: This type of E3 ligase leverages the nuclear-expressing E3 ligase DCAF16 for targeted protein degradation.
RNF4 Ligands: Emerging as SUMO (small ubiquitin-like modifier) -dependent E3 ligase recruiters, they enable degradation with covalent binders like CCW 16.
RNF114 Ligands: Natural product nimbolide and its synthetic analogs are used as ligands to enable degradation with unique specificity profiles.
KEAP1 Ligands: An emerging class of E3 ligase recruiters, shifting from early covalent binders to more selective non-covalent compounds.
DCAF15 Ligands: These ligands enable PROTACs to recruit DCAF15 for target degradation.
DCAF11 Ligands: This class of E3 ligase recruiters enables PROTACs to degrade targets such as FKBP12 and AR.
DCAF1 Ligands: With covalent probes like MY-1B enabling PROTACs to selectively degrade targets like FKBP12 and BRD4.
L3MBTL3 Ligands: These ligands enable PROTACs to harness the emerging E3 ligase L3MBTL3 for highly selective protein degradation, showing distinct preferences among homologous targets.
FEM1B Ligands: Represent a newly explored class of E3 ligase recruiters, with the covalent ligand EN106 demonstrating the ability to guide PROTACs for target degradation.
FBXO22 Ligands: Discovered through an innovative CRISPR-based screening platform, these ligands enable electrophilic PROTACs to effectively degrade targets like FKBP12 and BRD4 via the newly identified FBXO22 E3 ligase.
KLHDC2 Ligands: These ligands enable PROTACs to recruit KLHDC2, a CUL2 E3 ligase known for recognizing C-terminal diglycine motifs, for the degradation of various targets like BRD4 and the androgen receptor.
SKP1 Ligands: A newly developed class of E3 ligase recruiters that enable PROTACs to target SKP1, a core component of the Skp1-Cullin1-F-box (SCF) complex, for protein degradation.
SPOP Ligands: A new class of E3 ligase recruiters that use a bridged PROTAC strategy, leveraging SPOP’s endogenous substrate (like GLP) for targeted protein degradation.
Representative Recent Disclosed E3 Ligase Ligands
Key Considerations in the Design of E3 Ligase Ligands
With the Rapid development of targeted protein degradation technologies such as PROTACs, the design and optimization of E3 ligase ligands have become a pivotal step toward achieving efficient and selective protein degradation. A successful E3 ligase ligand must not only exhibit strong affinity and specificity for the target E3 ligase, but also meet a series of criteria in terms of synthetic feasibility, metabolic stability, and conformational compatibility within the ternary complex. Below are the key dimensions to consider when designing E3 ligase ligands:
E3 Ligase Ligand Design: Key Aspects
1. Selection of the Target E3 Ligase
Different E3 ligases (e.g., CRBN, VHL, MDM2, IAPs, etc.) vary significantly in terms of tissue expression, substrate specificity, and structural plasticity. Choosing an appropriate E3 ligase requires balancing several factors:
· The ability to form a stable ternary complex with the target protein
· Expression levels of the E3 ligase in relevant tissues or disease models
· Availability and maturity of small-molecule ligands
· Potential immunogenicity or off-target toxicity
2. Binding Site and Interaction Mode of the Ligand
An effective E3 ligand should bind tightly and selectively to its E3 ligase while maintaining a favorable conformation within the ternary complex. Key design considerations include:
· Key binding interactions in known co-crystal structures (such as hydrogen bonds, hydrophobic contracts)
· Whether ligand binding induces conformational changes that enhance or disrupt ternary complex formation
· For novel E3 ligases, availability of structural information or the need for in silico binding pocket prediction
3. Medicinal Chemistry Properties
As one arm of a bifunctional PROTAC molecule, the E3 ligand is subject to stringent medicinal chemistry constraints:
· Ideally low molecular weight (<500 Da)
· Good solubility and membrane permeability
· Chemical modifiability: the ligand must offer a suitable “exit vector” for linker attachment that does not compromise binding affinity
4. Metabolic Stability and Toxicity
Many natural products or early scaffolds (e.g., thalidomide derivatives) used as E3 ligands suffer from metabolic liability or potential toxicity. Optimization strategies may include:
· Avoidance of moieties prone to CYP-mediated metabolism
· Replacement of metabolically unstable groups
· Evaluation of stereoisomers or regioisomers for improved in vivo performance
5. Compatibility with the Linker
E3 ligands may undergo significant conformational or steric changes upon linker attachment. Therefore, early evaluation of linker compatibility is critical:
· Whether the attachment site is distal from key binding motifs
· The need for rigid or conformationally restricted linkers to favor ternary complex formation
· Avoidance of steric clashed or unfavorable ternary complex geometry
Challenges and Future Directions
While the development of PROTACs has primarily centered around a few well-established E3 ligases, the future of targeted degradation will likely rely on expanding the ligandable E3 ligase repertoire and improving the design of ligands with better precision, safety, and tissue selectivity. Key challenges and opportunities include:
Challenges and Future Directions
1. Expanding E3 Ligase Diversity
Only a small fraction of the >600 human E3 ligases have been effectively harnessed for PROTAC design. Continued efforts in chemical proteomics, ligand discovery, and structural biology are essential to identify new druggable E3 Ligases.
2. Tissue-Selective Degradation
The ability to recruit tissue-specific or disease-specific E3 ligases could greatly enhance PROTAC selectivity, reducing systemic side effects and enabling degradation in previously inaccessible contexts.
3. Non-Canonical Mechanisms
Beyond class high-affinity ligands, alternative mechanisms such as allosteric binders or molecular glues that stabilize E3-substrate interactions without requiring tight binding are gaining attention. These approaches may enable access to otherwise “undruggable” targets.
4. Predictive Tools for Degrader Design
Advances in computational modeling, AI-driven ligand screening, and degrader-specific metrics (e.g., cooperativity, degradation kinetics) are beginning to reshape the way E3 ligase ligands are identified and optimized.
5. Synthetic and Pharmacokinetic Optimization
Balancing potency, selectivity, metabolic stability, and linker compatibility remains an ongoing challenge. Now chemotypes, scaffold hopping, and prodrug strategies may help improve developability.
Conclusion
E3 ligase ligands are a cornerstone of PROTAC design, directly influencing degradation efficiency, selectivity, and drug-like properties. While CRBN and VHL remain the most widely used ligases, the discovery of new E3 ligase ligands and the rational optimization of known scaffolds are expanding the possibilities of targeted protein degradation. The ability to fine-tune binding affinity, linker compatibility, and metabolic stability is essential to unlocking the full potential of this technology.
At Precise PEG, we specialize in the development and production of high-quality building blocks for PROTACs, including E3 ligase ligands, functionalized linkers, and bifunctional intermediates such as VHL ligand-linker conjugates, CRBN ligand-linker conjugates, and other ligand-linker conjugates. Our chemistry-driven approach ensures reliable structure optimization, scalable synthesis, and fast turnaround to support your degrader programs from hit generation to lead development.
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