The Expanding E3 Ligase-Ligand Landscape for PROTAC Technology

Abstract

Proteolysis-targeting chimeras (PROTACs) are a transformative therapeutic modality that co-opts the ubiquitin-proteasome system for selective protein degradation. To date, the development of PROTACs has been overwhelmingly dominated by the recruitment of four canonical E3 ligases: CRBN, VHL, MDM2, and IAP. This limited repertoire represents a critical bottleneck, restricting the scope of degradable proteins and potential therapeutic applications. Addressing this challenge, recent years have witnessed a surge in the successful recruitment of novel E3 ligases. This review provides a dedicated and comprehensive summary of this progress, focusing exclusively on the emerging E3 ligases and their cognate ligands reported for PROTAC technology outside of the well-established quartet. We detail their discovery and strategic application, highlighting how this rapidly expanding toolbox promises to overcome existing limitations and unlock the full potential of targeted protein degradation.

1. Introduction

The traditional “occupancy-driven” paradigm of pharmacology, which relies on competitive inhibitors or antagonists to modulate protein function, has been a cornerstone of drug discovery for decades. While immensely successful, this approach is fundamentally limited to proteins with well-defined, functional binding pockets and often requires high, sustained drug concentrations to achieve therapeutic efficacy. In recent years, a paradigm shift has occurred with the advent of event-driven pharmacology, most prominently represented by targeted protein degradation (TPD) [1]. This innovative strategy does not merely inhibit a target protein but eliminates it entirely from the cellular environment by hijacking the cell’s own protein disposal machinery [2,3].

At the forefront of TPD is the proteolysis-targeting chimera (PROTAC) technology [4,5]. PROTACs are heterobifunctional molecules comprising three key components: a ligand that binds to a protein of interest (POI), a ligand that recruits an E3 ubiquitin ligase, and a flexible linker connecting the two. By simultaneously engaging the POI and an E3 ligase, a PROTAC induces the formation of a productive ternary complex (POI-PROTAC-E3 ligase). This proximity-induced event triggers the transfer of ubiquitin from a charged E2 conjugating enzyme to lysine residues on the surface of the POI. The resulting polyubiquitinated POI is then recognized and degraded by the 26S proteasome, releasing the PROTAC molecule to engage in further catalytic cycles of degradation (Figure 1A). This catalytic nature allows for potent, sub-stoichiometric activity and can lead to a more profound and durable pharmacological response compared to traditional inhibitors. Furthermore, by targeting proteins for degradation rather than inhibition, PROTACs have opened the door to addressing the vast portion of the proteome previously considered “undruggable”, including scaffolding proteins and transcription factors that lack enzymatic activity [2,6,7,8,9].

The specificity and efficiency of this process are critically dependent on the E3 ubiquitin ligase, the terminal enzyme in the ubiquitin-proteasome system (UPS) cascade. The human genome encodes for an estimated 600–700 E3 ligases, each possessing a distinct substrate scope, expression profile, and regulatory mechanism [10,11]. This diversity represents a vast, largely untapped reservoir of potential for directing protein degradation with precision. However, the historical development and current clinical landscape of PROTACs have been overwhelmingly reliant on a very small fraction of these enzymes [10,11]. The inception and validation of this field were built upon the recruitment of four canonical E3 ligases: Cereblon (CRBN) [12,13,14], von Hippel-Lindau (VHL) [15,16,17,18], Mouse double minute 2 homolog (MDM2) [19,20,21,22], and the inhibitor of apoptosis protein (IAP) family, which was first harnessed through the development of specific and nongenetic IAP-dependent protein erasers (SNIPERs) by Naito and colleagues [23,24,25,26]. The availability of well-characterized, high-affinity small molecule ligands for these E3 ligases, such as thalidomide and its analogs for CRBN [27,28], and hypoxia-inducible factor (HIF) mimetics for VHL [29], provided a crucial starting point that enabled the rapid validation of the PROTAC concept and the advancement of multiple candidates into clinical trials. This trajectory is reflected in the clinic. This foundation has propelled numerous candidates into the clinic, culminating in a landmark achievement for the entire field [30,31], notably including two orally administered degraders, ARV-110 (an androgen receptor degrader) and ARV-471 (an estrogen receptor degrader) (Figure 1B). The oral estrogen receptor PROTAC, vepdegestrant (ARV-471), recently met its primary endpoint in the pivotal Phase 3 VERITAC-2 trial for ER+/HER2- advanced breast cancer [32]. Vepdegestrant demonstrated a statistically significant and clinically meaningful improvement in progression-free survival (PFS) over the standard-of-care, fulvestrant, particularly in patients with resistance-driving ESR1 mutations. With a favorable safety profile, regulatory submissions have been filed, positioning it to potentially become the first approved PROTAC degrader [32]. This success provides definitive clinical validation for the PROTAC platform, confirming that this modality can yield best-in-class oral medicines capable of overcoming key mechanisms of drug resistance.

This foundational reliance, while instrumental to the field’s success, has now become a significant bottleneck. The constraints imposed by this limited E3 ligase toolbox are manifold. Firstly, it restricts the degradable proteome; not all POIs can form stable and productive ternary complexes with CRBN or VHL, leaving many important disease-driving proteins inaccessible to current PROTACs. Secondly, it presents a clear avenue for acquired resistance. Clinical and preclinical studies have shown that mutations in the E3 ligase or its associated pathway components can abrogate PROTAC efficacy [33,34,35], a challenge that could be circumvented by having alternative E3 ligases to recruit. Thirdly, the ubiquitous expression of CRBN and VHL, while enabling broad applicability, complicates the development of tissue-selective degraders and can lead to on-target, off-tissue toxicities [36,37,38]. Recruiting an E3 ligase with a restricted expression pattern offers a compelling strategy to enhance the therapeutic window. Finally, the choice of E3 ligase can profoundly influence the degradation profile (e.g., kinetics, maximal degradation (D_max), and potency (DC₅₀)) [39,40,41], and the optimal E3 for a given POI may not be among the canonical four.

In response to these challenges, a concerted effort across academia and industry has been directed towards expanding the repertoire of E3 ligases available for PROTAC technology [42,43]. This endeavor has begun to bear significant fruit, with a recent surge in the discovery of novel E3 ligases and the development of cognate ligands capable of hijacking their degradative capacity. This expansion is poised to fundamentally reshape the future of TPD.

Therefore, this review will provide a focused and comprehensive overview of the progress made in this critical area. We will move beyond the well-trodden ground of CRBN, VHL, MDM2, and IAP to exclusively survey the emerging landscape of novel E3 ligases and their respective ligands that have been successfully deployed for PROTAC development (Figure 2). We will systematically detail the discovery strategies, chemical matter, and biological validation for these new systems. By charting this expanding frontier, we aim to illuminate the immense potential of these novel E3 ligases to overcome current limitations and realize the full therapeutic promise of TPD.

2. RNF Family

2.1. RNF4 E3 Ligase

In 2019, the Nomura group employed an activity-based protein profiling (ABPP) [44,45,46,47] covalent ligand screening platform to identify new chemical starting points for E3 ligase recruiters [48]. The screen identified TRH 1-23 as a hit that covalently binds to RING finger protein 4 (RNF4), an E3 ubiquitin ligase for which no chemical tools previously existed. Mass spectrometry analysis revealed that TRH 1-23 modifies the zinc-coordinating cysteines C132 and C135 within the RNF4 RING domain. Importantly, this binding was found to be non-functional, as it did not inhibit the autoubiquitination activity of RNF4, suggesting a non-functional binding mode suitable for ligase recruitment. Through structural optimization of this initial hit, they developed a more potent ligand, CCW 16, with an IC₅₀ of 1.8 μM. To demonstrate its utility, CCW 16 was incorporated into a PROTAC degrader, CCW 28-3, by linking it to JQ1, a known inhibitor of the BET bromodomain protein family (Figure 3) [48]. CCW 28-3 demonstrated higher binding affinity for RNF4 than CCW 16, with an IC₅₀ of 0.54 μM in competitive inhibition assays, successfully achieving degradation of bromodomain-containing protein 4 (BRD4) protein via both proteasome- and RNF4-dependent mechanisms. While acknowledging that the degradation efficiency and cellular target engagement of CCW 28-3 were modest compared to established degraders and that further optimization is needed, the study serves as a critical proof-of-concept. It validates the use of chemoproteomics-enabled covalent screening to successfully expand the arsenal of E3 ligase recruiters.

2.2. RNF114 E3 Ligase

In 2019, the Nomura group positioned RING finger protein 114 (RNF114) [49] as a novel E3 ubiquitin ligase for small-molecule PROTACs, recruited via covalent engagement by the Neem-derived natural product nimbolide (Figure 4A) at cysteine-8 (C8) in its disordered N-terminal region, as revealed by ABPP in triple-negative breast cancer models [50]. This modification disrupts substrate recognition, inhibiting ubiquitination of tumor suppressors like p21 and p57, thereby stabilizing them and inducing cell cycle arrest and apoptosis. Harnessing this, nimbolide-JQ1 conjugates XH2 (Figure 4B) enable RNF114-dependent, proteasome-mediated degradation of BRD4 with nanomolar potency and selectivity over other BET proteins, confirmed by proteomics and knockout studies, while also stabilizing additional substrates like PEG10 and CTGF. Cysteine-reactive screening identified simpler acrylamides that mimic nimbolide, offering tractable scaffolds to expand the limited E3 recruiter arsenal for targeted degradation.

In 2020, building on this concept, the Nomura group developed a series of novel PROTACs using nimbolide as a covalent recruiter for RNF114, designed to selectively degrade the oncogenic fusion protein BCR-ABL, which drives chronic myeloid leukemia (CML) [51]. By linking nimbolide with the BCR-ABL inhibitor dasatinib, they constructed a PROTAC BT1 (Figure 4B) capable of specifically degrading BCR-ABL. Experimental results demonstrated that, compared to PROTACs recruiting CRBN or VHL as E3 ligases, BT1 exhibited higher selectivity in degrading BCR-ABL and showed stronger anti-proliferative effects in leukemia cells. Additionally, BT1 treatment significantly increased levels of the tumor suppressor protein p21, suggesting a potential multi-target anticancer mechanism.

To overcome the inherent synthetic complexity of a natural product like nimbolide, the next logical step was to identify simpler, more tractable synthetic ligands. In 2021, the Nomura group screened 318 cysteine-reactive chloroacetic acid amine and acrylamide ligands using a gel-based competitive ABPP assay, and found that EN219 effectively inhibits the binding of the IA-rhodamine probe to the RNF114 protein, with an IC₅₀ value of 470 nM (Figure 4A) [52]. EN219 inhibits the self-ubiquitination of RNF114 and the ubiquitination of its substrate p21 by covalently binding to the N-terminal cysteine (C8) of RNF114. Further studies linked EN219 with the BET inhibitor JQ1 or the BCR-ABL inhibitor dasatinib to construct PROTAC molecules (such as ML 2-14 and ML 2-23), achieving selective degradation of BRD4 and BCR-ABL, with the degradation process dependent on the proteasome and RNF114 (Figure 4B). This study not only provides new chemical tools for E3 ligase-based TPD applications but also demonstrates the potential of chemical biology methods to discover synthetic molecules that mimic the functions of natural products, offering new insights into targeting “undruggable” proteins.

3. DCAF Family

3.1. DCAF16 E3 Ligase

DDB1 and CUL4-associated factor 16 (DCAF16) is a largely uncharacterized substrate receptor for the Cullin–RING ubiquitin ligase 4 (CRL4) family [53]. By simultaneously docking to CUL4–DDB1 and a client protein, DCAF16 nucleates the formation of a ternary CRL4–DCAF16–substrate complex, thereby licensing poly-ubiquitination and proteasomal degradation of nuclear clients. Beyond housekeeping roles in DNA-damage responses and cell-cycle control [54,55], DCAF16 E3 ligase had not been exploited for ligand-directed protein degradation.

Using a chemical-proteomic platform, the Cravatt group screened heterobifunctional electrophilic PROTACs in which a broad-spectrum cysteine-reactive fragment (e.g., chloroacetamide KB02) was tethered to established ligands for FKBP12 (SLF) or BRD4 (JQ1) (Figure 5) [56]. Chemical-proteomic mapping, including isotopic tandem orthogonal proteolysis-activity-based protein profiling (isoTOP-ABPP) and affinity enrichment experiments, revealed that these PROTACs covalently modify a cluster of cysteines (likely C177 and/or C179) within DCAF16. Remarkably, occupancy of only 10–40% of the total DCAF16 pool was sufficient to drive robust, nuclear-restricted degradation of FKBP12 and BRD4, while the bulk of unmodified DCAF16 remained available for endogenous substrates. The covalent engagement also endowed the PROTACs with washout-resistant activity, a feature distinct from classic reversible degraders. This study positions DCAF16 as the first electrophile-engaged nuclear E3 ligase for PROTAC applications and demonstrates the potential of electro-affinity PROTACs in precisely regulating protein function.

Building on the identification of DCAF16 as a nuclear-restricted E3 ligase recruitable by covalent ligands, the Li group advanced the development of DCAF16-based PROTAC degraders targeting poly(ADP-ribose) polymerase 2 (PARP2) and cyclin-dependent kinase 4/6 (CDK4/6) for triple-negative breast cancer (TNBC) therapy [57,58]. They first tethered the PARP1/2 inhibitor olaparib to the covalent DCAF16 ligand KB02 to afford PROTAC C8 (Figure 5). Structural modeling predicted that C8’s terminal α-chloroacetamide would self-react with Cys845 of PARP1, sterically preventing productive engagement of DCAF16 and thereby endowing selective PARP2 degradation. They next conjugated the CDK4/6 inhibitor palbociclib to KB02 to generate a series of DCAF16-based PROTACs with varied chains. Among these, the lead compound A4 induced concentration- and time-dependent CDK4/6 degradation in MDA-MB-231 cells via DCAF16 recruitment and proteasomal pathways.

3.2. DCAF15 E3 Ligase

In 2020, the Chen group reported a novel BRD4 degrader DP1 by conjugating the DDB1 and CUL4-associated factor 15 (DCAF15) ligand E7820 with the BRD4 inhibitor JQ1 (Figure 6), which can induce persistent degradation of the target protein and demonstrates therapeutic potential against hematological malignancies both in vitro and in vivo [59]. Although the current drug activity is not high, requiring μM levels to achieve better degradation, this study provides a series of evidence proving that DCAF15 can serve as a supplement to the limited E3 ligases in PROTAC design. In 2024, Lucas et al. optimized the SPLAM sulfonamide E7820 into the 53 nM DCAF15 ligand and generated a series PROTACs. Only one degraded BRD4, but competition and knockout assays showed the effect was DCAF15-independent [60]. Their study highlights that potent glue ligands do not guarantee functional PROTAC warheads and warns against using BRD4/JQ1 models without rigorous proof of mechanism.

3.3. DCAF11 E3 Ligase

In 2021, the Cravatt group identified DDB1 and CUL4-associated factor 11 (DCAF11) as a novel substrate adaptor capable of supporting ligand-induced protein degradation through covalent engagement [61]. Employing a cell-based screening strategy across diverse human cancer cell lines with a focused library of electrophilic PROTACs—bifunctional compounds featuring an FKBP12-binding ligand SLF linked to structurally varied α-chloroacetamide electrophiles—they discovered multiple hits that degraded cytosolic and nuclear FKBP12 in a proteasome- and Cullin-RING ligase-dependent manner, with compound 21-SLF exhibiting prostate cancer cell-specific activity (Figure 7). Chemical proteomics and CRISPR/Cas9 knockout experiments pinpointed DCAF11 as the key E3 component, revealing that 21-SLF covalently modifies conserved cysteines (notably C460, alongside C443 and C485) to form ternary complexes facilitating ubiquitination and degradation. Extending this to endogenous targets, they demonstrated DCAF11-dependent degradation of the androgen receptor using an analogous electrophilic PROTAC 21-ARL (Figure 7).

In 2024, the Li group introduced alkenyl oxindoles as novel PROTAC moieties capable of recruiting the DCAF11 E3 ubiquitin ligase complex for efficient protein degradation, thereby expanding the limited repertoire of E3 ligases available for small-molecule degraders [62]. Alkenyl oxindoles previously was identified as autophagosome-tethering compounds (ATTECs) for mutant huntingtin degradation [63]. They synthesized a series of heterobifunctional conjugates linking these moieties to the BRD4 inhibitor JQ1 via diverse linkers, conducting structure-activity relationship analyses to optimize degradation potency. Contrary to expectations of autophagy-lysosomal mediation, mechanistic investigations revealed that these conjugates, including the lead compound HL435 (with DC₅₀ values of 11.9 and 21.9 nM in MDA-MB-231 and MCF-7 cells, respectively), induced BRD4 ubiquitination and proteasomal degradation in a Cullin-RING ligase-dependent manner, unaffected by autophagy inhibitors but blocked by proteasome or neddylation inhibitors. Employing a pooled CRISPR interference (CRISPRi) screening, they identified CRL4^DCAF11 components (CUL4B, RBX1, DDB1, and DCAF11) as essential for this activity, with knockdown validations confirming DCAF11’s role in ternary complex formation. This work not only repurposes alkenyl oxindoles as versatile E3 recruiters but also highlights the value of unbiased screening in uncovering underutilized ligases. Building on prior findings that alkenyl oxindoles covalently engage DCAF11, they further modified the aryl ring, linker types, and oxindole substituents to optimize structure-activity relationships [64]. Employing a novel high-throughput screening system based on high-content imaging, they identified L134 as a highly potent BRD4 degrader (DC₅₀ = 7.36 nM, D_max > 98%), which operates via the ubiquitin-proteasome system in a DCAF11-dependent manner, as confirmed by mechanistic studies involving proteasome inhibitors and DCAF11 knockdown (Figure 7). L134 exhibited robust antitumor activity in cellular and in vivo models, underscoring its potential as a therapeutic candidate for BRD4-driven cancers.

In 2025, the Hansen group developed novel histone deacetylase (HDAC) degraders that recruit DCAF11, transforming a selective HDAC6 PROTAC into a pan-HDAC degrader [65]. They synthesized eleven PROTACs on solid-phase, incorporating an alkenyl oxindole-based DCAF11 ligand and a pan-HDAC inhibitor, with FF2039 identified as a potent degrader of HDAC1 and HDAC6, as well as other isoforms across classes I, IIa, and IIb (Figure 7). Mechanistic studies confirmed that FF2039 operates via the ubiquitin-proteasome system in a DCAF11-dependent manner, inducing significant antiproliferative effects, cell cycle arrest, and apoptosis in both hematological and solid cancer cell lines. Compared to their prior CRBN-recruiting HDAC6-selective PROTAC A6 [66], switching to DCAF11 altered the degradation profile, highlighting the impact of E3 ligase choice on selectivity and efficacy.

3.4. DCAF1 E3 Ligase

In 2024, the Thoma group leveraged a selective DDB1 and CUL4-associated factor 1 (DCAF1) binder to develop potent PROTACs, including DBr-1 (targeting BRD9) and DBt-10 (targeting BTK), which efficiently degrade their respective targets via the DCAF1 E3 ligase, as validated through chemical and genetic rescue experiments (Figure 8) [67]. Notably, DBt-10 effectively degraded BTK in cells with acquired resistance to CRBN-based PROTACs, while DBr-1 circumvented intrinsic resistance to VHL-based degraders. Additionally, a dasatinib-based DCAF1 PROTAC successfully degraded multiple tyrosine kinases, broadening the scope of targetable proteins. This work highlights DCAF1 as a promising E3 ligase for PROTAC design, offering a strategy to address both intrinsic and acquired resistance in clinical settings.

In a 2025 study, the Vedadi group advanced DCAF1-based PROTACs by targeting WDR5, a key cancer-associated protein [68], and provided structural insights into DCAF1’s substrate specificity through high-resolution crystal structures of the DCAF1-PROTAC-WDR5 ternary complex (PDB ID: 9B9H, 9B9T, 9B9W and 9DLW) [69]. Using potent DCAF1 ligand OICR-8268, they designed four PROTACs that effectively degrade both endogenous and exogenous WDR5 via the ubiquitin-proteasome system, mediated by the CUL4-DCAF1 E3 ligase complex. Among the four DCAF1-based PROTACs, PROTAC OICR-41114 achieves the most robust WDR5 degradation (Figure 8). The crystal structures reveal that DCAF1’s flexible loops confer surface plasticity, enabling diverse substrate interactions, which explains its broad substrate specificity.

4. AhR E3 Ligase

The Naito group broadens the E3 ligase repertoire by incorporating β-naphthoflavone (β-NF) as a ligand to recruit the aryl hydrocarbon receptor (AhR) E3 ligase complex, thereby creating a new class of degraders (Figure 9) [70]. Specifically, β-NF-ATRA, targeting cellular retinoic acid binding proteins (CRABPs), induced AhR-dependent degradation of CRABP-1 and CRABP-2 through the ubiquitin-proteasome pathway. Similarly, ITE-ATRA, employing an alternative AhR ligand ITE, also effectively degraded CRABPs. Extending this strategy, β-NF-JQ1 was developed to target BRD proteins, promoting AhR-BRD interactions and exhibiting potent anticancer activity that aligned with its protein knockdown efficacy.

5. KEAP1 E3 Ligase

Kelch-like ECH-associated protein 1 (KEAP1), a critical substrate-recognition subunit of the CUL3-based E3 ubiquitin ligase complex, plays an essential role in orchestrating protein ubiquitination and proteasomal degradation [71,72]. By regulating the degradation of nuclear factor erythroid 2-related factor 2 (Nrf2), KEAP1 maintains cellular redox homeostasis, making it a key regulator of oxidative stress responses [73,74]. In 2020, the Nomura group pioneered the application of KEAP1 as an E3 ubiquitin ligase in TPD [75]. They engineered a novel PROTAC (CDDO-JQ1) by conjugating the KEAP1-Nrf2 activator bardoxolone with the BRD4 inhibitor JQ1, achieving potent BRD4 degradation (Figure 10). In the 231MFP human breast cancer cell line, CDDO-JQ1 demonstrated dose-dependent BRD4 degradation, which was abolished by pretreatment with proteasome inhibitor bortezomib and E1-activating enzyme inhibitor MLN7243, confirming its proteasome-dependent degradation mechanism. Additionally, the NEDD8-activating enzyme inhibitor MLN4924 attenuated degradation, underscoring the involvement of CUL3-family E3 ligases, which require NEDDylation for activity. Despite these advances, mechanistic questions remain, including whether CDDO-JQ1 targets KEAP1’s BTB domain or other cysteine residues and whether additional E3 ligases contribute to the observed degradation. These findings lay a robust foundation for future chemoproteomic and genetic studies to elucidate the full scope of CDDO-JQ1’s mechanism and for the development of next-generation KEAP1-based PROTACs.

In 2021, the Jin group further explored the potential of KEAP1 in PROTAC applications by developing MS83, a novel PROTAC linking a highly potent, selective, non-covalent KEAP1 ligand KEAP1-L-OEt to the BET bromodomain inhibitor JQ1 (Figure 10) [76]. In MDA-MB-468 cells, MS83 effectively induced degradation of BRD4 (both long and short isoforms) and BRD3 but showed minimal effects on BRD2. Notably, MS83 exhibited more sustained degradation kinetics compared to CRBN-recruiting PROTAC dBET1. MS83 was specifically designed and synthesized as a prodrug of MS83A using the ethyl ester derivative KEAP1-L-OEt to enhance cell permeability. This design allows MS83 to effectively degrade BRD4 at lower concentrations. However, this process requires a longer time, as it depends on the in-cell hydrolysis of its ethyl ester group to release the active carboxylic acid group. Interestingly, in MDA-MB-231 cells, MS83 selectively degraded the oncogenic BRD4 short isoform while sparing the potentially tumor-suppressive long isoform.

The Lv group expanded KEAP1 applications into novel territory by identifying piperlongumine (PL), a natural product, as a novel covalent E3 ligase ligand [77]. By conjugating PL with the CDK9 inhibitor SNS-032, they developed PROTAC 955 capable of degrading CDK9 (Figure 10). Through systematic synthesis of PL-SNS-032 conjugate and evaluation in MOLT4 cells, 955 demonstrated potent CDK9 degradation via both proteasomal and CRL E3 ligase-dependent mechanisms. TurboID-based proteomics confirmed KEAP1 as the recruited E3 ligase. They further investigated PL as a novel covalent KEAP1 ligand by synthesizing PL-ceritinib conjugates designed to degrade the EML4-ALK fusion oncoprotein, a driver of ceritinib resistance in NSCLC. The successful degradation of EML4-ALK by conjugate 819 confirms the potential of PL as a versatile covalent ligand for building E3 ligase-based protein degraders.

6. FEM1B E3 Ligase

Fem-1 homolog B (FEM1B), a substrate-recognition component of the CUL2 E3 ubiquitin protein ligase, plays a pivotal role in cellular reductive stress response by recognizing the reduced cysteine residue C186 on its substrate FNIP1, thereby mediating FNIP1 ubiquitination and degradation to restore mitochondrial activity and redox balance [78,79,80]. This unique mechanism positions FEM1B as a potential therapeutic target. In 2022, the Nomura group reported the discovery of EN106, the first covalent ligand targeting FEM1B [81]. EN106 forms a covalent bond with C186 of FEM1B, disrupting FEM1B-FNIP1 recognition and inhibiting FNIP1 degradation. Experimental results demonstrated that EN106 effectively stabilizes FNIP1 and exhibits high target specificity and degradation efficiency in cells. Building on this discovery, the researchers conjugated EN106 with the BET bromodomain inhibitor JQ1 to develop FEM1B-based BET degraders [81]. Among these, NJH-1-106 achieved the most potent degradation of BRD4, with a DC₅₀ of 250 nM and a D_max of 94%. Additionally, the FEM1B-based kinase degrader NJH-2-142 successfully targeted the oncogenic BCR-ABL fusion protein, demonstrating the versatility of FEM1B as a recruiter in PROTAC applications (Figure 11).

7. KLHL20 E3 Ligase

In 2022, the Yeh group designed a synthetic macrocyclic ligand BTR2000 for KLHL20, a substrate adaptor of the CUL3 E3 ligase complex, and validated KLHL20 as an effective E3 ligase for PROTAC applications [82]. Through structure-guided design (PDB ID: 6GY5), the authors developed a bifunctional degrader BTR2003 that recruits KLHL20 to BET family proteins (Figure 12), achieving potent, dose-dependent degradation in cellular models via the ubiquitin-proteasome pathway, as confirmed by mechanistic studies involving ligase inhibitors and knockdown experiments. This macrocyclic PROTAC demonstrated improved efficiency over linear counterparts, highlighting the role of conformational constraints in enhancing ternary complex formation.

8. KLHDC2 E3 Ligase

In 2024, the Békés group expanded the repertoire of recruitable E3 ligases for PROTACs by identifying and characterizing small-molecule ligands for KLHDC2, a CRL2 substrate adapter that recognizes glycine-ending C-end degrons [83]. Through biochemical assays, the authors developed potent KLHDC2 binders and functionalized them into PROTACs targeting BRD2/3/4 (K2-B4-3e and K2-B4-5e) and the AR (K2-AR-1), demonstrating KLHDC2-dependent ubiquitination and proteasomal degradation in cellular models (Figure 13). Structural insights from X-ray crystallography and cryo-EM revealed that KLHDC2 assembles into a dynamic tetramer stabilized by its C terminus (PDB ID: 8SGE and 8SGF), with ligand and substrate engagement modulating this oligomeric state to facilitate ternary complex formation. This work not only validates KLHDC2 as a versatile E3 ligase for PROTAC design, but also provides mechanistic understanding of its substrate specificity, potentially enabling broader applications in overcoming resistance and targeting undruggable proteins.

In the same year, through ligand screening and structure-based optimization, the Schulman group developed compounds engaging the U-shaped degron-binding pocket of KLHDC2, which were conjugated to the BET inhibitor JQ1 via tailored linkers to optimize exit vectors and induce cooperative ternary complexes [84]. The lead PROTAC, SJ46421, selectively recruited and ubiquitinated BRD3BD2 in vitro, while prodrug variants (e.g., SJ46420) addressed cell permeability challenges imposed by acidic degron-mimicking moieties, enabling robust BRD3 degradation in cells without affecting other BET paralogs like BRD2 or BRD4 (Figure 13). Mechanistic insights revealed KLHDC2’s auto-inhibition overcome by neo-substrate engagement, ensuring ligase selectivity over related KLHDC1, KLHDC3, and KLHDC10. This work establishes a framework for designing selective PROTACs with C-degron E3 ligases, enhancing neo-substrate specificity and expanding the molecular toolkit to tackle challenges in drug discovery.

9. FBXO22 E3 Ligase

In 2024, the Zhang group first identified FBXO22 as a novel recruitable ligase through a CRISPR-based transcriptional activation screen of human E3 ligases [85]. Using a heterobifunctional PROTAC, 22-SLF, which links an FKBP12-binding ligand to an electrophilic warhead, they demonstrated FBXO22-dependent degradation of FKBP12, mediated by covalent interactions with cysteines C227 and/or C228 in FBXO22, as confirmed by mechanistic studies. Further, FBXO22-based PROTACs effectively degraded additional endogenous proteins, including BRD4 (22-JQ1) and the oncogenic fusion protein EML4-ALK (22-TAE), in cellular models (Figure 14). This work validates FBXO22 as a versatile E3 ligase for PROTAC design, offering a new avenue to diversify the limited pool of ligases.

10. GID4 E3 Ligase

The Dong group pioneered the use of glucose-induced degradation deficient complex 4 (GID4), a substrate receptor of the CTLH E3 ligase complex [86], for TPD using noncovalent small-molecule ligands [87]. By linking the E3 ligase GID4 antagonist PFI-7 [88,89] to warheads like JQ1, they developed PROTACs, NEP108 and the optimized NEP162 (Figure 15), which trigger ubiquitin-proteasome-dependent degradation of BRD4 and BRD2 (but not BRD3) in U2OS and SW480 cancer cell lines, demonstrating potent antiproliferative effects and tumor suppression in xenograft models with good tolerability. Crystal structures of GID4–PROTAC–BRD4 ternary complexes reveal plastic and dynamic interactions (PDB ID: 8X7G and 8X7H), providing crucial insights for rational design, while extending applicability to additional targets like ERα and SMARCA2 (Figure 15). GID4 is ubiquitously expressed in most tissue types, with notable enrichment in the testis, suggesting that future efforts can leverage GID4 recruiters to design additional PROTACs and develop tissue-specific degraders to enhance disease specificity or target selectivity.

11. Key Challenges and Future Perspectives

The successful recruitment of novel E3 ligases has broken new ground for PROTACs, yet it has also brought the complexity and scale of the remaining challenges into sharp relief. Moving from a handful of validated E3 Ligases to a truly versatile platform requires a concerted effort to bridge the gap between discovery and rational application.

11.1. The Ligand Discovery Bottleneck: From Binder to Functional Recruiter

The primary obstacle in TPD remains the discovery of high-quality ligands for E3 ligases. However, the core challenge extends beyond identifying mere binders; it involves the intricate and often unpredictable transformation of these binders into functional recruiters capable of inducing effective ubiquitination and degradation. Historical examples in the field abound where high-affinity ligands failed to translate into degraders due to issues like poor ternary complex formation or suboptimal exit vectors for linker attachment. To overcome this bottleneck, a multifaceted integration of technologies is imperative (Figure 16). High-throughput methods such as DNA-encoded libraries and phenotypic screening can generate initial hits efficiently, while chemoproteomics enables proteome-wide mapping of ligandable hotspots on the E3-ome. The pivotal step, however, is the optimization phase, where computational biology and machine learning emerge as transformative tools. These not only predict binding affinities but also simulate the dynamic conformational changes in ternary complexes that govern ubiquitination efficiency, thereby accelerating the design of potent, selective recruiters.

11.2. The Craft of Rational E3 Ligase Selection: A Paradigm Shift to Precision

With an expanding menu of E3 ligases, the paradigm of degrader design must shift from opportunistic discovery to rational, hypothesis-driven selection. This art of selection requires a multi-parameter calculus that weighs several critical factors:

(1)

Tissue-Specific Expression: This remains the key goal for enhancing the therapeutic index. Systematically mapping the E3 ligase proteome across healthy and diseased tissues will provide the foundational atlas for designing tissue-restricted degraders.

(2)

Subcellular Topography: A target protein’s efficacy is intimately tied to its location. Matching the E3 ligase’s subcellular address—be it nuclear, cytosolic, or membrane-associated—to that of the target is a non-negotiable prerequisite for success.

(3)

The Principles of Ternary Complex Cooperativity: The rules governing productive ternary complex formation are exquisitely specific to each E3-target pair. Deciphering these complex principles of protein–protein interactions is essential for predicting which pairings will be synergistic and which will be futile.

(4)

Exploiting Inherent Biology: Leveraging an E3’s native function offers a compelling pathway to create multi-functional medicines. The recruitment of cIAP1, for instance, provides a dual-action strategy that couples target degradation with the induction of apoptosis. Similarly, co-opting MDM2 in a p53-wildtype cancer could not only eliminate a primary oncoprotein but also therapeutically stabilize the p53 tumor suppressor by sequestering its key negative regulator. This strategic approach transforms the E3 ligase from a passive scaffold into an active, synergistic contributor to the overall therapeutic effect, adding a powerful dimension to rational degrader design.

To illustrate this calculus in action, consider the following case studies, which highlight how different criteria become the primary drivers of the decision depending on the therapeutic context:

Scenario 1: Targeting a Cytosolic Protein in a Neurodegenerative Disease

The primary challenge is to design a degrader for a pathogenic, cytosolic protein (e.g., a pathogenic kinase or a misfolded protein like Tau) expressed mainly in the central nervous system (CNS), while minimizing potential toxicities in peripheral organs. In this context, tissue expression becomes the paramount selection criterion. While the default E3 ligases, CRBN and VHL, are ubiquitously expressed and could be effective, they carry an inherent risk of on-target, off-tissue degradation in the periphery. A more rational, hypothesis-driven approach would prioritize the recruitment of a novel E3 ligase with enriched CNS expression, such as FEM1B or specific DCAF members, pending proteomic validation in relevant disease models. Although other criteria like subcellular localization are met by multiple candidates and serve as a necessary filter, the decision is ultimately driven by the goal of creating a brain-targeted degrader with a superior therapeutic index.

Scenario 2: Overcoming Acquired Resistance in Oncology

Consider a scenario where a potent, CRBN-based PROTAC against an oncogenic kinase shows initial clinical efficacy, but patients relapse with mutations in CRBN that preclude degrader binding. Here, the immediate problem is resistance risk, making the switch to a different E3 ligase family—such as VHL, cIAP1, or RNF114—the most logical step. However, the selection of the next E3 offers an opportunity for a more sophisticated strategy by exploiting inherent biology.

A simple switch to VHL might restore activity, but if the cancer type is known to be sensitive to apoptotic stimuli, recruiting cIAP1 becomes a highly compelling choice. A cIAP1-based degrader would provide a dual-action mechanism: it would not only degrade the primary oncogenic driver but also co-opt the intrinsic pro-apoptotic function of cIAP1 recruitment. This synergistic approach, which combines targeted degradation with apoptosis induction, could lead to deeper, more durable responses. Thus, while multiple E3 switches are possible, a rational selection informed by the tumor’s biological vulnerabilities represents a superior strategy that actively leverages the E3 ligase’s inherent function.

Mastering this art of selection is the gateway to unlocking true precision medicine in the TPD space, enabling the degradation machinery to be tailored to the specific context of the disease.

11.3. The Computational and Data-Driven Revolution in Degrader Design

While early degrader discovery relied heavily on empirical screening, the field is undergoing a computational revolution. In silico and data-driven tools are becoming indispensable for navigating the immense combinatorial complexity of E3-ligand-target space and are accelerating the entire design-build-test cycle.

The true challenge in degrader design lies in predicting the structure and dynamics of the ternary complex, which ultimately dictates degradation efficiency. Here, AI-based structure prediction tools like AlphaFold-Multimer and DiffDock are revolutionizing the field [90,91]. These models can now predict the three-dimensional structure of the entire POI-PROTAC-E3 complex with remarkable accuracy, enabling the in silico screening of thousands of potential E3-target pairings before any wet lab synthesis. This predictive power is further enhanced by molecular dynamics simulations, which model the flexibility of the linker and the subtle conformational rearrangements crucial for productive ubiquitination.

Looking ahead, the next frontier is the integration of these predictive AI/ML models with large-scale experimental data. For example, by combining chemoproteomics data that maps the “ligandable E3-ome” with tissue-specific proteomics and structural information, AI models could be trained to generate a predictive “E3-substrate compatibility matrix”. Such a tool would guide the rational selection of the optimal E3 for any given target in a specific disease context. Ultimately, the fusion of these powerful computational and data-driven approaches is transitioning degrader development from an empirical art to a more predictable engineering science.

11.4. The Next Frontier: Expanding into Untapped E3 Ligase Families

While this review has focused on the successful recruitment of novel RING-type E3 ligases, a vast and largely uncharted territory of the “E3-ome” remains to be explored. To truly unlock the full potential of TPD, the field must venture beyond the currently accessible E3 ligases. Major and biologically important E3 ligase families—including the HECT family, the TRIM family, the membrane-embedded MARCH ligases, and the majority of the ~70 F-box protein substrate receptors—remain largely unexploited due to unique challenges in their catalytic mechanisms, structural complexity, or subcellular localization. Overcoming the hurdles to drugging these untapped families represents a key long-term goal and a major future direction for the field.

11.5. Conditionally Activated and Inducible Degraders for Spatiotemporal Control

The current generation of PROTACs are “always-on”, which can lead to on-target, off-tissue toxicities. The next generation of degraders will be “smart”, activated only at the desired time and place. This can be achieved through several exciting strategies for conditional activation, including light-activated (photodegraders) that can be switched on with spatiotemporal precision, and degraders that are activated by the unique conditions of a tumor microenvironment, such as hypoxia or low pH. These inducible systems promise to unlock an unprecedented level of control, significantly widening the therapeutic window.

11.6. Scope and Limitations of the Current Landscape

Finally, it is crucial to frame the exciting progress detailed in this review within the context of the field’s current limitations. This review, by necessity, reflects the state of a rapidly evolving but still nascent area. Several key caveats should be acknowledged. First, while many novel E3 ligases have been successfully recruited, most of the cognate ligands discussed are tool compounds and have not yet been optimized for drug-like properties such as oral bioavailability or metabolic stability. Second, a significant portion of the literature on these emerging E3 ligases represents critical proof-of-concept work in cellular models. Rigorous and extensive in vivo validation of efficacy and safety for most of these new systems is a necessary next step that is still largely pending. Lastly, the scope of this review reflects the current focus of the TPD field, which has been predominantly concentrated on oncology. While immensely promising, the application of these novel E3 ligases in other key therapeutic areas, such as neurodegenerative and immune diseases, is still in its early stages and represents a major frontier for future research. Acknowledging these limitations highlights the immense opportunities that still lie ahead in translating this expanded E3 toolbox into broadly applicable, next-generation medicines.

12. Conclusions

The field of TPD stands at a pivotal juncture. The recent clinical success of vepdegestrant (ARV-471), a degrader recruiting a canonical E3 ligase, has provided definitive validation for the entire modality, proving that orally bioavailable, event-driven pharmacology can yield best-in-class medicines. This triumph, however, does not mark an endpoint but rather energizes the next great challenge: moving beyond the foundational reliance on CRBN and VHL. As this review has delineated, the E3 ligase toolbox is undergoing a dramatic and accelerating expansion, from the early explorations with MDM2 and cIAP1 to the recent breakthroughs in recruiting novel ligases such as RNF114, KEAP1, and members of the DCAF and CRL2 families (

Table 1. Summary of Novel E3 Ligases and the Corresponding PROTACs.

Emerging E3 Ligase	Ligand	Ligand Type	PROTAC	Target	Degradation Efficiency (DC50/Dmax)	Cell Lines	Ternary Complex (PDB ID)	Refs.
RNF4	CCW 16	Covalent	CCW 28-3	BRD4		231MFP, HeLa		[48]
RNF114	Nimbolide	Covalent	XH2	BRD4		231MFP		[50]
			BT1	BCR-ABL		K562		[51]
	EN219	Covalent	ML 2-14	BRD4	Long isoform: DC50 = 36 nM Short isoform: DC50 = 14 nM	231MFP		[52]
			ML 2-23	BCR-ABL		K562		[52]
DCAF16	KB02	Covalent	KB02-SLF	FKBP12		HEK293T, MDA-MB-231		[56]
			KB02-JQ1	BRD4		HEK293T		[56]
			C8	PARP2	DC50 = 2 μM, Dmax > 92%	MDA-MB-231		[57]
			A4	CDK4/6	CDK4: DC50 = 6.5 μM, Dmax > 89% CDK6: DC50 = 8 μM, Dmax > 83%	MDA-MB-231		[58]
DCAF15	E7820	Noncovalent	DP1	BRD4	DC50 = 10.84 ± 0.92 μM, Dmax = 98%	SU-DHL-4		[59]
DCAF11	DCAF11 ligand 1	Covalent	21-SLF	FKBP12		22Rv1		[61]
			21-ARL	AR		22Rv1		[61]
	DCAF11 ligand 2	Covalent	HL435	BRD4	DC50 = 11.9 nM (MDA-MB-231), DC50 = 21.9 nM (MCF-7), Dmax > 99%	MDA-MB-231, MCF-7		[62]
	DCAF11 ligand 3	Covalent	L134	BRD4	DC50 = 7.36 nM, Dmax > 98%	MDA-MB-231		[64]
	DCAF11 ligand 4	Covalent	FF2039	HDAC1, HDAC2, HDAC4, HDAC6	HDAC1: Dmax = 90%, HDAC2, 4, and 6: Dmax = 71–76%	MM.1S		[65]
DCAF1	DCAF1 ligand 1	Noncovalent	DBr-1	BRD9		HEK293		[67]
			DBt-10	BTK		TMD8		[67]
	OICR-8268	Noncovalent	OICR-41114	WDR5	DC50 = 40 ± 24 nM, Dmax = 49 ± 1.9%	MV4-11	9DLW	[69]
AhR	β-NF	Noncovalent	β-NF-ATRA	CRABP-1, CRABP-2		MCF-7, IMR-32		[70]
			β-NF-JQ1	BRD2, BRD3, BRD4		MCF-7		[70]
	ITE	Noncovalent	ITE-ATRA	CRABP-1, CRABP-2		MCF-7, IMR-32		[70]
KEAP1	CDDO	Covalent	CDDO-JQ1	BRD4		231MFP		[75]
	KEAP1-L-OEt	Noncovalent	MS83	BRD4, BRD3		MDA-MB-468, MDA-MB-231		[76]
	PL	Covalent	995	CDK9	DC50 = 9 nM	MOLT4		[77]
			819	EML4-ALK		NCI-H2228		[77]
FEM1B	EN106	Covalent	NJH-1-106	BRD4	DC50 = 250 nM, Dmax = 94%	HEK293T		[81]
			NJH-2-142	BCR-ABL		K562		[81]
KLHL20	BTR2000	Noncovalent	BTR2003	BRD2, BRD3, BRD4	BRD2: DC50 = 46 nM, BRD3: DC50 = 87 nM, BRD4: DC50 = 777 nM	PC3		[82]
KLHDC2	KDRLKZ-1	Noncovalent	K2-B4-3e	BRD4	DC50 = 66 nM, Dmax = 62%	PC3		[83]
	KDRLKZ-2	Noncovalent	K2-B4-5e	BRD4	DC50 = 6.2 nM, Dmax = 93%	PC3		[83]
			K2-AR-1	AR		VCaP		[83]
	SJ46418	Noncovalent	SJ46421	BRD3		U2OS		[84]
			SJ46420	BRD3		U2OS		[84]
FBXO22	FBXO22 ligand	Covalent	22-SLF	FKBP12	DC50 = 0.5 μM, Dmax = 89%	HEK293T		[85]
			22-JQ1	BRD4		A549		[85]
			22-TAE	EML4-ALK		H2228		[85]
GID4	PFI-7	Noncovalent	NEP108	BRD4	DC50 = 3.8 μM	U2OS	8X7G	[87]
			NEP162	BRD4	DC50 = 1.2 μM (SW480), DC50 = 1.6 μM (U2OS)	SW480, U2OS	8X7H	[87]
			NEP168	ERα		MCF-7		[87]
			NEP202	SMARCA2		U2OS		[87]

).

This strategic diversification is not merely a numerical addition of E3 ligases; it represents a fundamental leap towards precision medicine. Each new E3 ligase, discovered through innovative engines like chemoproteomics, phenotypic screening, and structure-based design, brings a unique set of biological properties to the table. This expanding repertoire offers tangible solutions to the field’s most pressing challenges: it provides the tools to achieve tissue-selective degradation, new avenues to overcome acquired resistance, and the potential to expand the degradable proteome to targets previously inaccessible to the canonical recruiters. The art of rational E3 selection is therefore becoming a central pillar of modern degrader design, enabling a hypothesis-driven approach to tailor the degradation machinery to the specific context of a disease.

Looking ahead, the true promise of this expanded toolbox will be realized through its convergence with the powerful computational and AI-driven platforms that are reshaping drug discovery. The ability to accurately model and predict ternary complex formation, combined with a growing arsenal of E3 recruiters, is transitioning degrader development from an empirical art to a more predictable engineering science. By continuing to explore this vast E3 ligase landscape and integrating our discoveries with these powerful new design tools, the field is poised to deliver a new generation of precision-engineered degraders, capable of selectively erasing the root causes of disease with unprecedented control.