PROTAC-mediated regulation of programmed cell death: From molecular mechanisms to therapeutic breakthroughs

Abstract

Proteolysis-targeting chimeras (PROTACs) represent a revolutionary therapeutic strategy that achieves selective protein degradation through the ubiquitin-proteasome system, offering transformative potential for modulating programmed cell death (PCD) pathways. This review comprehensively examines the central role of PROTACs in regulating critical PCD mechanisms, including ferroptosis induction via GPX4 degradation, pyroptosis regulation through stimulator of interferon genes (STING) targeting, necroptosis modulation by MLKL/RIPK1 degradation, apoptosis activation through BCL-2/MDM2 elimination, and autophagy regulation via dual ubiquitin-proteasome and lysosomal pathways. These approaches effectively address the limitations of traditionally “undruggable” targets while demonstrating unique mechanistic properties and clinical promise. Currently, over 30 PROTAC candidates have entered clinical trials, including the estrogen receptor (ER) degrader ARV-471 for breast cancer and the IRAK4 degrader KT-474 for inflammatory diseases, both showing remarkable efficacy in overcoming drug resistance. While challenges remain in delivery systems, E3 ligase selectivity, and toxicity management, innovative technologies, such as nanocarriers, covalent PROTACs, and novel E3 ligases (e.g., RNF114) are advancing PROTAC applications in oncology, neurodegenerative disorders, and immune-related diseases. Future research will focus on optimizing molecular design, expanding the E3 ligase repertoire, and developing combination therapies. These efforts will establish PROTACs as groundbreaking solutions for intractable diseases, with their precise control of PCD pathways opening new therapeutic avenues. The technology’s ability to selectively modulate cell death mechanisms positions it as a transformative approach in precision medicine.

Graphical abstract

Public summary

• •PROTACs degrade PCD-associated regulatory proteins, overcoming drug resistance and suppressing multiple cancer pathways.
• •Bridged PROTACs target undruggable transcription factors and overcome CRBN/VHL-dependent resistance.
• •PROTACs remodel tumor immune microenvironment by reversing immunosuppression and enhancing antitumor immunity.
• •Nanoparticle PROTACs boost tumor targeting and delivery overcoming barriers with optimized design.

Introduction

Ubiquitination is a post-translational modification mechanism widely present in eukaryotic cells and a key process for maintaining protein homeostasis and regulating various physiological functions within cells. Through a three-step cascade involving ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3), ubiquitination covalently links ubiquitin molecules to the lysine residues of target proteins.^1,2,3,4 This process, mediated by E1, E2, and E3 enzymes, can lead to the formation of either monoubiquitination or various types of polyubiquitin chains (linked via Lys48 or Lys63). Among them, the Lys48-linked chains typically mark proteins for degradation by the proteasome, while Lys63-linked chains are involved in non-degradative regulation, such as signal transduction or organelle localization. Deubiquitinating enzymes dynamically maintain protein homeostasis by hydrolyzing isopeptide bonds that link ubiquitin to substrate proteins, reversing ubiquitin modifications and fine-tuning cellular signaling pathways.^5,6 Ubiquitination regulates protein stability, activity, or interactions, influencing critical physiological processes such as the cell cycle, DNA repair, inflammatory responses, and autophagy and maintaining cellular homeostasis and stress adaptation capabilities.⁷

The ubiquitin-proteasome system (UPS) serves as the primary machinery for selective protein degradation in eukaryotic cells, orchestrating the turnover of approximately 80%–90% of intracellular proteins. Beyond its canonical role in eliminating misfolded, damaged, or obsolete proteins, this sophisticated pathway exerts precise control over fundamental biological processes, including cell cycle progression, signal transduction cascades, transcriptional regulation, neuronal plasticity, and memory formation.^8,9,10 In eukaryotic cells, the UPS serves as the principal mechanism for maintaining protein homeostasis by selectively removing defective and damaged proteins.¹¹ The UPS exhibits remarkable selectivity and regulatory precision, primarily governed by the specificity of E3 ubiquitin ligases in target protein recognition. With over 600 distinct E3 ligases in human cells, each capable of identifying unique substrate proteins, the system achieves unparalleled degradation specificity. Further modulation occurs through diverse post-translational modifications and regulatory proteins, which collectively fine-tune the spatiotemporal control of protein degradation to maintain both accuracy and efficiency.^12,13 Ubiquitin-mediated degradation begins with activation of ubiquitin by the E1 enzyme in preparation for its attachment. This activated ubiquitin is transferred to the E2 enzyme, and subsequently the E3 ligase facilitates its binding to the target protein, usually forming a polyubiquitin chain that signals degradation.¹⁴ The proteasome forms a barrel-shaped architecture composed of multiple subunits, enclosing a central proteolytic chamber. The 26S proteasome recognizes ubiquitinated substrates, unfolds them through ATP-dependent mechanisms, and processively degrades them into oligopeptides. These peptide fragments are released into the cytosol, where they undergo further processing by cellular peptidases into reusable amino acids, completing the protein turnover cycle.^5,15

The regulatory mechanisms of the UPS have inspired diverse therapeutic strategies, including proteasome inhibitors and E3 ligase modulators. Bortezomib, as the inaugural therapeutic agent targeting the UPS, triggers apoptosis through selective inhibition of the 26S proteasome. This inhibition disrupts proteostasis, resulting in the accumulation of misfolded proteins and subsequent proteotoxic stress that overwhelms cellular repair mechanisms.^16,17 Another paradigm is molecular glue degraders like lenalidomide, which reprograms the E3 ligase cereblon (CRBN) to recruit neo-substrates (e.g., IKZF1/3) for ubiquitination and degradation.^18,19 Both agents are now cornerstone therapies for multiple myeloma (MM), demonstrating the clinical viability of UPS modulation in oncology. Building upon this foundation, Proteolysis-Targeting Chimeras (PROTACs) hijack the UPS via heterobifunctional molecules to catalytically degrade pathogenic proteins traditionally deemed undruggable. This approach represents a therapeutic paradigm shift from mere target inhibition to complete protein elimination, fundamentally redefining drug action mechanisms.^10,20 PROTACs are heterobifunctional molecules comprising three essential components: the protein of interest (POI) binding moiety, a linker, and an E3 ubiquitin ligase binding moiety (Figure 1).²¹ PROTAC molecules can bind to both E3 ligases and the target protein, forming a ternary complex of POI-PROTAC-E3 ligase. The complex can trigger polyubiquitination and subsequent proteasomal degradation of the target protein, while the PROTAC molecule dissociates from the complex and enters a catalytic cycle for repeated use.^22,23,24

Figure 1.

Mechanism of PROTACs based on the UPS

(1) Ubiquitination cascade: ATP energizes ubiquitin (Ub) activation by E1, transfer by E2, and E3-catalyzed conjugation to the POI, forming a polyubiquitin chain marking it for degradation. (2) PROTAC complex formation: the bifunctional PROTACs bind the POI (via POI ligand) and recruit an E3 ligase (via E3 ligand), forming a POI-PROTAC-E3 ternary complex enabling POI ubiquitination. (3) Proteasomal degradation: the polyubiquitinated POI is recognized, unfolded, and degraded into peptide fragments by the proteasome. (4) Ub and PROTAC fate: ubiquitin is recycled. PROTACs dissociate intact for reuse, catalyzing further POI degradation.

The advent of PROTACs addresses the challenge of undruggable targets, as traditional small-molecule drugs typically inhibit protein activity by occupying their active sites, a strategy that is ineffective against proteins lacking such sites.^25,26 PROTACs extend the range of druggable targets by enabling the degradation of proteins that are traditionally difficult to target, and they also offer a means to circumvent resistance due to protein overexpression, providing a new therapeutic avenue.^27,28,29 Since its inception in 2001 by Crews and Deshaies, PROTAC technology has evolved over two decades, transitioning from academic research to industrial application and becoming a focal point in drug discovery and development.³⁰ It has demonstrated the ability to degrade undruggable targets such as MYC and KRAS and has shown promise in overcoming resistance mechanisms in oncogene-addicted diseases. This innovative approach offers distinct therapeutic benefits, such as circumventing resistance mechanisms, minimizing adverse reactions, and enabling precise modulation of traditionally intractable protein targets.³¹ To date, over 30 PROTAC candidates have entered clinical trials, spanning diverse therapeutic areas such as oncology, neurodegenerative diseases, and immune disorders (Table 1). These investigations evaluate disease-specific therapeutic strategies, methodological advancements, and clinical development stages. The compiled data offer critical insights into PROTAC efficacy and progress, underscoring clinical utility and future directions for research and therapeutic translation.

Table 1.

The summary of PROTACs in and approaching the clinical trials

Sponsor	Drug	Targets	Disease	Phase	NCT
Arvinas	ARV-110	AR	metastatic castration resistant prostate cancer	completed	NCT03888612
Hinova Pharmaceuticals	HP518	AR	metastatic castration resistant prostate cancer	completed	NCT05252364
Suzhou Kintor Pharmaceutical	GT20029	AR	androgenetic alopecia	completed	NCT06692465
Novartis Pharmaceuticals	ARV-766	AR	metastatic prostate cancer	I/II	NCT05067140
Celgene	CC-94676	AR	metastatic castration resistant prostate cancer	I	NCT04428788
Jiangsu HengRui Medicine	HRS-5041	AR	metastatic castration resistant prostate cancer	I	NCT05942001
Arvinas	ARV-471	ER	breast cancer	completed	NCT05549505
Hinova Pharmaceuticals	HP568	ER	ER+/HER2− advanced breast cancer	I/II	NCT06757335
Shandong Suncadia Medicine	HRS-1358	ER	metastatic or local advanced breast cancer	I	NCT05628870
Children’s Oncology Group	DT2216	BCL-xL	hematologic malignancy/solid tumor	I/II	NCT06620302
Bristol-Myers Squibb	BMS-986458	BCL6	non-Hodgkin lymphomas	I/II	NCT06090539
Arvinas	ARV-393	BCL6	non-Hodgkin lymphomas	I	NCT06393738
BeiGene	BGB-16673	BTK	B cell malignancies	I/II	NCT05006716
Nurix Therapeutics	NX-2127	BTK	B cell malignancies	I	NCT04830137
Nurix Therapeutics	NX-5948	BTK	B cell malignancies	I	NCT05131022
AbbVie	ABBV-101	BTK	B cell malignancies	I	NCT05753501
Chia Tai Tianqing Pharmaceutical Group	TQB3019	BTK	hematological tumors	I	NCT06943677
Ubix Therapeutics	UBX-303061	BTK	B cell malignancies	I	NCT06590961
C4 Therapeutics	CFT-1946	BRAFV600	BRAFV600 mutant solid tumors	I/II	NCT05668585
Ranok Therapeutics	RNK05047	BRD4	solid tumors/B cell lymphoma	I/II	NCT05487170
Mitsubishi Tanabe Pharma America	MT-4561	BRD4	solid tumors	I/II	NCT06943521
NiKang Therapeutics	NKT3964	CDK2	solid tumors	I	NCT06586957
Biotheryx	BTX-9341	CDK4/CDK6	HR+/HER2− breast cancer	I	NCT06515470
BeiGene	BG-60366	EGFR	EGFR-mutant non-small cell lung cancer	I	NCT06685718
Haisco Pharmaceutical Group	HSK40118	EGFRL858R	non-small cell lung cancer	I	NCT06050980
Betta Pharmaceuticals	CFT-8919	EGFRL858R	non-small cell lung cancer	I	NCT06641609
Astellas Pharma	ASP3082	KRAS	solid tumors	I	NCT05382559
Kymera Therapeutics	KT-413	IRAK4	relapsed or refractory B cell non-Hodgkin lymphoma	completed	NCT05233033
Kymera Therapeutics	KT-474	IRAK4	atopic dermatitis/hidradenitis suppurativa	completed	NCT04772885
Sanofi	SAR444656	IRAK4	atopic dermatitis	II	NCT06058156
BeiGene	BGB-45035	IRAK4	autoimmune dermatological diseases	I	NCT06342713
Leadingtac Pharmaceutical	LT-002-158	IRAK4	hidradenitis suppurativa/atopic dermatitis	I	NCT06082323
Prelude Therapeutics	PRT3789	SMARCA2	solid tumor/SMARCA4 gene mutation	I	NCT05639751
Prelude Therapeutics	PRT7732	SMARCA2	solid tumors with a SMARCA4 mutation	I	NCT06560645
Kymera Therapeutics	KT-621	STAT6	atopic dermatitis	I	NCT06945458

Data source: https://clinicaltrials.gov, updated September 10, 2025.

Programmed cell death (PCD) is an active, orderly suicide process controlled by intrinsic genetic programs under physiological or pathological conditions. As a fundamental biological phenomenon widely present in multicellular organisms, PCD plays crucial roles in embryonic development, tissue homeostasis maintenance, and immune system maturation.^32,33 Among various PCD types, apoptosis is the most extensively studied form, characterized by cell shrinkage, chromatin condensation, and DNA fragmentation. Other important PCD modalities include ferroptosis, pyroptosis, necroptosis, autophagic cell death, and cuproptosis, and each is activated under specific conditions through distinct signaling pathways and molecular mechanisms. Dysregulation of PCD is closely associated with the pathogenesis of numerous diseases, such as cancer, neurodegenerative disorders, and autoimmune diseases. Therefore, in-depth investigation of PCD mechanisms holds significant promise for understanding disease pathophysiology and developing novel therapeutic strategies.^34,35,36

This review provides a comprehensive overview of the application of ubiquitination and PROTACs in regulating PCD. It elaborates on the mechanisms and advantages of PROTACs as well as their applications in various types of PCD. Additionally, the challenges faced by PROTACs and future perspectives are discussed. PROTACs can degrade both wild-type and mutant proteins resistant to conventional inhibitors, thus offering new possibilities for treating drug-resistant diseases. In various disease models, PROTACs have demonstrated significant therapeutic efficacy and, compared with traditional small-molecule inhibitors and other therapeutic approaches, exhibit higher safety and specificity. As PROTAC molecules advance into clinical trials, they face challenges such as poor water solubility, low cellular permeability, and potential off-target effects, which necessitate the development of effective delivery systems to enhance their in vivo performance.³⁷ Researchers are actively optimizing PROTAC molecule design to enhance selectivity, reduce molecular weight, and improve drug delivery, aiming to refine the technology and expand its clinical applications. Integrating PROTACs with advanced delivery technologies, such as nanocarriers and antibody conjugation, has paved new pathways for overcoming biological barriers and achieving precise protein degradation.³⁸

PROTAC design and optimization

The design and optimization of PROTACs are systematic engineering challenges, centered on the rational selection of E3 ubiquitin ligases, precise modulation of linker architectures, and accurate prediction of ternary complex stability. This process integrates computational modeling, structural biology, and experimental validation to overcome resistance mechanisms, enhance tissue specificity, and improve druggability, bridging the gap between laboratory discovery and clinical translation.

E3 ligase ligand selection

In PROTAC design, CRBN and von Hippel-Lindau (VHL) remain the most classical and widely utilized E3 ligases. CRBN ligands, typically derived from thalidomide and its derivatives (e.g., lenalidomide and pomalidomide), exhibit small molecular weight (∼273 Da) and excellent cellular permeability.³⁹ However, their inherent immunomodulatory drug (IMiD) activity induces complex biological effects, including the degradation of critical transcription factors such as Ikaros (IKZF1) and Aiolos (IKZF3), which are essential for treating hematologic malignancies like MM.^40,41 This dual functionality is a double-edged sword; while effective for oncogenic targets, it risks off-target degradation of non-intended substrates like SALL4, potentially causing teratogenicity and complicating PROTAC-based therapies.⁴² Furthermore, tumor cells may develop resistance through CRBN downregulation or mutations, necessitating careful consideration in clinical applications.⁴³ By contrast, VHL ligands (e.g., VH032-based hydroxyproline derivatives) demonstrate higher binding affinity (K_d = 185 nM) but greater molecular polarity (topological polar surface area [TPSA] >140 Ų), which may impair oral bioavailability. VHL holds promise in solid tumors, such as prostate cancer PROTAC therapies, though its functional loss in ∼50% of clear cell renal cell carcinomas (due to VHL mutations) necessitates careful evaluation for specific cancer types.^44,45 Beyond CRBN and VHL, cIAP ligands like LCL161 (SMAC mimetics) are employed to degrade cIAP1/2, though their potential to aberrantly activate nuclear factor κB (NF-κB) pathways limits their standalone use, often requiring combination with chemotherapeutics.⁴⁶

Although CRBN and VHL currently dominate the PROTAC landscape, the human genome encodes approximately 600 E3 ligases, of which only a tiny fraction have been utilized for PROTAC design.³⁹ Overreliance on a limited set of E3 ligases poses challenges, such as potential drug resistance and restricted tissue distribution. Therefore, expanding the repertoire of E3 ligases is crucial for advancing PROTAC technology. Current research is actively exploring the potential applications of novel E3 ligases, such as RNF114, DCAF16, and MDM2. RNF114, a zinc-finger domain-containing E3 ligase, can be recruited by aryl sulfonamide-based ligands and shows promise for degrading nuclear hormone receptors.⁴⁷ DCAF16, a member of the CRL4 E3 ligase family, is uniquely characterized by its ability to engage covalent ligands, enabling specific degradation of nuclear proteins and offering a new strategy to overcome resistance conferred by CRBN or VHL mutations.⁴⁸ MDM2, a key negative regulator of the p53 tumor suppressor, can be harnessed using its ligands (e.g., Nutlin-3) to design PROTACs targeting oncoproteins like STAT3; however, this strategy generally requires a wild-type p53 background in tumor cells for efficacy.⁴⁹

Developing tissue-specific E3 ligases is another critical future direction, where the bridged PROTAC strategy demonstrates unique advantages. By using E3 ligase substrates specifically expressed in target tissues or tumors as “bridge proteins,” bridged PROTACs achieve precise activation within target tissues, significantly enhancing the therapeutic window and reducing systemic off-target toxicity. Their core mechanism involves recruiting endogenous substrate proteins to indirectly engage the E3 ligase complex, bypassing the dependency of traditional PROTACs on direct E3-targeting ligands, overcoming CRBN/VHL resistance mutations, and expanding the scope to previously undruggable targets.⁵⁰ For instance, the first SPOP-targeting PROTAC, MS479, was developed by Jian Jin’s team using the natural substrate GLP as a bridging ligand for this colorectal cancer-associated protein. This molecule connects BRD4 to SPOP via GLP, inducing ubiquitination and degradation of the short isoform (S isoform) of BRD4, the concentration at which 50% of the target protein is degraded (DC₅₀) is 6.2 μM, and demonstrating significant anti-proliferative activity in colorectal cancer cells the growth inhibition concentration at which 50% of the maximal inhibition (GI₅₀) is observed is 4.5 μM, with selectivity dependent on SPOP expression levels.⁵¹ For undruggable transcription factors like STAT3 and c-Myc, the team led by Chao Liang employed the nucleic acid aptamer AS1411, which targets nucleolin (NCL) on the surface of tumor cells (a partner protein of MDM2), to construct AS1411-NCL-MDM2-based PROTAC (ANM-PROTACs).⁵² These form an NCL-MDM2-PROTAC-POI quaternary complex, enabling rapid STAT3 degradation (DC₅₀ = 136 nM within 6 h) and significantly inhibiting tumor cell proliferation, the concentration at which 50% of the maximal inhibition (IC₅₀) is observed is 1.0 μM, while avoiding the systemic toxicity associated with traditional MDM2 ligands like Nutlin-3 through tumor-specific NCL expression. Furthermore, the team led by Huang innovatively utilized the natural diterpenoid Oridonin (Ori) as an MDM2 E3 ligase recruiter. By leveraging the formation of an Ori-NCL-MDM2 ternary complex, they developed bifunctional PROTACs targeting both BRD4 and EGFR (e.g., Ori-JQ1-1). These molecules efficiently degraded BRD4 in Hep3B cells (DC₅₀ = 950 nM) and inhibited tumor growth synergistically (reducing tumor volume by >50% in vivo) through dual MDM2/X degradation and activation of the p53 pathway, marking the first introduction of natural products into PROTAC design.⁵³ Collectively, these case studies validate the unique advantages of the bridged strategy in targeting undruggable proteins, enhancing tumor selectivity, and overcoming resistance, providing a new paradigm for the clinical translation of PROTAC technology.

Linker optimization

The linker, serving as the structural bridge connecting the E3 ubiquitin ligase ligand and the target protein (POI) ligand, requires rational design, as it is a core element for modulating the degradation efficiency, selectivity, and drug-like properties of PROTAC molecules. An ideal linker must not only facilitate the effective formation and spatial stabilization of the ternary complex but also balance the molecule’s cellular permeability, solubility, and metabolic stability. With the advancement of PROTAC technology, linker optimization has progressively evolved from initial empirical attempts to structure-based rational design.²³

The length and rigidity of the linker are critical parameters that govern the stability of the ternary complex. Research indicates that the optimal linker length typically falls within the range of 10–20 heavy atoms (approximately 5–15 Å). The length of the linker is critical for productive degradation complex formation. An overly short linker causes steric clashes between the E3 ligase and the target protein, while an excessively long one introduces high conformational flexibility, leading to an entropic penalty that destabilizes the complex. Furthermore, a long linker increases the risk of the “hook effect”; at high concentrations, the PROTAC molecule forms inert binary complexes with either protein, which competitively inhibits formation of the functional ternary complex.²⁹ Regarding rigidity, incorporating rigid structures such as alkynes, piperazine rings, or aromatic rings can effectively restrict the molecular conformational freedom, reducing the entropic penalty associated with ternary complex formation, consequently enhancing degradation activity and selectivity. This strategy has been successfully validated in clinical-stage PROTACs like ARV-110.⁵⁴ In contrast, flexible chains like polyethylene glycol (PEG), while beneficial for improving solubility and conformational adaptability, are often accompanied by a decrease in degradation efficiency. To integrate the advantages of both rigid and flexible linkers, emerging conformation-constrained strategies (e.g., macrocyclic linkers) pre-organize the active conformation of PROTACs, which has been demonstrated to significantly enhance binding affinity for the ternary complex and degradation potency.⁵⁵

The linker has evolved from a passive connecting unit into a regulatory switch capable of actively responding to biological or external cues. The design of stimulus-responsive linkers greatly expands the capacity for precise control over PROTACs. For instance, linkers containing disulfide bonds can be specifically reduced and cleaved by the high intracellular concentrations of glutathione (GSH) in tumor cells, whereas peptide-based linkers can be selectively cleaved by cathepsin B, which is often overexpressed in tumor tissues, achieving tumor microenvironment-specific protein degradation, enhancing therapeutic selectivity, and reducing systemic toxicity.⁵⁶ Photo-controllable PROTACs are another frontier breakthrough. By incorporating photoisomerizable moieties such as azobenzene into the linker, PROTAC activity can be reversibly regulated using light of specific wavelengths, providing a high spatiotemporal precision tool for protein function studies and opening new avenues for the precise treatment of localized diseases (e.g., skin diseases or solid tumors).⁵⁷

Currently, linker optimization strategies have fully transitioned toward structure-based rational design and digital empowerment. Advances in X-ray crystallography and cryo-electron microscopy (cryo-EM) now allow researchers to directly resolve the three-dimensional structure of PROTAC-induced ternary complexes, providing a precise blueprint for optimizing linker length, orientation, and rigidity.⁵⁸ On the other hand, computational chemistry and artificial intelligence are reshaping the linker development workflow. For example, computational tools like PROsettaC can simulate and predict the stability of ternary complexes under different linker designs, enabling large-scale virtual screening,⁵⁹ while models based on deep reinforcement learning, such as DRlinker, can simultaneously optimize multiple key linker attributes like length and lipophilicity, signaling the advancement of PROTAC development toward a new paradigm of digital and automated design.⁶⁰ The integrated application of these interdisciplinary tools will systematically accelerate the clinical translation of next-generation highly efficient and selective PROTAC degraders.

Prediction of ternary complex stability

In PROTAC drug development, the stability of the ternary complex is a core factor determining degradation efficiency and selectivity. Its study relies on the close integration of computational simulation prediction and experimental validation.²³

Computational simulation technologies provide a theoretical framework and predictive tools for ternary complex stability. Molecular dynamics (MD) simulations track the conformational dynamics of the POI, PROTAC, and E3 ligase, enabling precise calculation of the binding free energy (ΔG) and cooperativity coefficient (α), where α > 1 indicates a positive cooperative binding effect. For instance, in the MZ1-mediated BRD4-VHL ternary complex, α = 3.5 correlates with a degradation efficiency (DC₅₀ = 0.3 nM) approximately 20-fold higher than systems with α = 1.⁵⁸ Deep learning tools like AlphaFold 3 can rapidly generate ternary complex structures; however, their prediction error for flexible linkers or novel E3 ligases can still reach 4.9 Å, necessitating correction using experimental structures from cryo-EM.⁶¹ Among structural parameters, interfacial shape complementarity and electrostatic interaction energy are core regulatory factors. When the electrostatic complementarity (ESP) score for the VHL-SMARCA2-PROTAC ternary complex exceeds 0.7, degradation activity increases 2-fold, confirming the decisive role of interface optimization in stability.⁶²

Advances in experimental techniques provide direct and multidimensional validation for computational predictions. Surface plasmon resonance (SPR) and homogeneous time-resolved fluorescence (HTRF) assays offer high-precision methods for studying ternary complexes. SPR quantifies the interaction strength between the POI and E3 ligase by monitoring binding kinetics (K_a/K_d) in real time. For example, VHL-based ternary complexes exhibit a K_a >1 × 10⁶ M⁻¹, corresponding to a half-life (t₁/₂) >60 min, which is significantly superior to traditional inhibitors.⁶³ HTRF technology, utilizing lanthanide labeling and time-resolved detection (with a limit of detection of 0.1 nM), enables high-throughput screening. Clinical-stage PROTACs, for instance, induce HTRF signal saturation at concentrations around 10 nM, verifying efficient ternary complex formation.⁵⁵ Cryo-EM technology further reveals dynamic assembly mechanisms; the BRD4-MZ1-VHL ternary complex structure (at 3.2 Å resolution) shows that linker-induced conformational rearrangement allows precise matching between the E3 active site and the POI binding pocket, a finding that directly guided the optimization of macrocyclic PROTACs.⁶⁴

The stability parameters of the ternary complex are closely related to its drug-like properties and clinical translation potential. The cooperativity coefficient α shows a significant negative correlation with DC₅₀ (R² = 0.75), and for every 0.1 increase in the interfacial complementarity score, the complex t₁/₂ extends linearly.⁶⁵ In linker design, incorporating disulfide bonds can leverage the high GSH concentration in the tumor microenvironment (TME) for selective activation, achieving a tumor/normal tissue selectivity ratio of 5–10:1 and significantly reducing off-target toxicity.⁵⁶ Additionally, pH-responsive linkers triggered by the slightly acidic TME can induce degradation, enhancing the efficacy of BCL-XL degraders by 3-fold in solid tumor models.⁴⁹ Future directions include developing multimodal prediction platforms (e.g., integrating AlphaFold-Multimer with MD) and standardized validation workflows (e.g., combined SPR-HTRF assays) to accelerate the clinical translation of highly selective PROTACs.⁶¹

The typical application of PROTACs for targeting

PROTACs leverage their unique bifunctional design to achieve precise degradation of critical disease targets, primarily focusing on three protein classes: kinases, nuclear receptors, and transcription factors (Table 2). Historically, these targets have been challenging to modulate due to the absence of druggable binding sites or susceptibility to resistance mutations. PROTAC technology overcomes these limitations through an event-driven degradation mechanism, offering significant clinical potential in oncology and immune disorders. Below, we detail representative applications and mechanistic breakthroughs for each class of target.

Table 2.

Typical applications of PROTACs in targeted protein degradation

Classification	Drug	Targets	Structure	Disease	Reference
Protein kinases	MT-802	BTK		chronic lymphocytic leukemia	Buhimschi et al.66
	compound F2	FAK		breast cancer, colorectal cancer, lung cancer	Xu et al.67
	MS154	EGFR		lung cancer	Cheng et al.68
	BSJ-03-123	CDK		acute myeloid leukemia	Brand et al.69
	GMB-475	BCR-ABL		chronic myeloid leukemia	Ye et al.70
	Compound 10c	JAK1		acute lymphoblastic leukemia, rheumatoid arthritis	Zhang et al.71
	KT-474	IRAK4		atopic dermatitis, hidradenitis suppurativa	Ackerman et al.72
Nuclear receptors	ARV-110	AR		prostate cancer	Snyder et al.73
	ARV-766	AR		prostate cancer	Zhang et al.74
	ARV-471	ER		breast cancer	Gough et al.75
	ERD-308	ER		breast cancer	Hu et al.76
	compound 9	RAR		acute promyelocytic leukemia	Scheepstra et al.77
Transcription factors	SD-36	STAT3		solid tumors, hematologic malignancies	Zhou et al.78
	SD-436	STAT3		leukemia, lymphoma	Xu et al.79
	Compound 6b	KLF5		breast cancer	Kong et al.80
	MZ1	MYC		breast cancer, hematologic malignancies, neuroblastoma	Otto et al.81

PROTACs for targeting protein kinases

PROTAC technology is a hot topic in the field of drug development for targeting protein kinases. Protein kinases are important signaling molecules in cells, and their abnormal activation is closely linked to the onset and progression of various diseases, especially cancer.⁸² Although traditional small-molecule inhibitors have achieved certain successes in treating related diseases, they often come with problems such as drug resistance and low selectivity.^83,84 PROTAC technology provides a new approach by inducing the degradation of target proteins.

In the targeting of protein kinases, PROTAC technology primarily works through the following mechanism. First, a PROTAC molecule contains a ligand that binds to the target protein kinase and a ligand that binds to the E3 ubiquitin ligase, connected by a linker. When the PROTAC molecule binds simultaneously to the target protein kinase and the E3 ligase, it promotes ubiquitination of the target protein, marking it for degradation. This mechanism can degrade wild-type and mutant proteins resistant to traditional inhibitors, providing new possibilities for treating drug-resistant diseases.^85,86

For example, BCR-ABL is an abnormally activated protein kinase in chronic myeloid leukemia.⁸⁷ Researchers have developed various BCR-ABL PROTAC molecules that can induce the degradation of BCR-ABL, including mutant BCR-ABL resistant to traditional inhibitors.⁸⁸ BRAF is a critical component of the mitogen-activated protein kinase (MAPK) signaling pathway and serves as a core member of the RAS-RAF-MEK-ERK cascade, regulating cell proliferation, differentiation, and survival. Oncogenic BRAF mutations (e.g., V600E) lead to constitutive activation of the protein, driving tumorigenesis.⁸⁹ In contrast to conventional inhibitors, BRAF-targeting PROTACs (e.g., SJF-0628) demonstrate broad activity against various BRAF mutants, the ability to overcome drug resistance, and selective degradation while sparing wild-type RAF proteins, significantly expanding the therapeutic window.⁹⁰ In addition to applications in cancer treatment, PROTAC technology is also being studied in non-oncological fields. The IRAK4-targeting PROTAC molecule KT-474 shows potential in treating inflammatory skin diseases. It binds to IRAK4 and brings in E3 ubiquitin ligase, which tags IRAK4 with ubiquitin. This tag directs IRAK4 to be degraded by cellular proteasomes, blocking IRAK4-mediated signaling. As IRAK4 is key in Toll-like receptor (TLR) and interleukin-1R (IL-1R) pathways, whose abnormal activation underlies many immune-inflammatory diseases, blocking these pathways reduces pro-inflammatory cytokines and chemokines, naturally easing inflammation.⁷²

Overall, PROTAC technology has shown tremendous potential in targeting protein kinases. It can not only overcome the drug resistance issues of traditional inhibitors but also provide new strategies for treating various diseases. With in-depth research and technological advancements, more PROTAC molecules are expected to enter clinical trials in the future, providing new options for disease treatment.

PROTACs for targeting nuclear receptors

Nuclear receptors (NRs) are part of the transcription factor family. Unlike traditional transcription factors, their primary function is to convert external signals into transcriptional outputs.⁹¹ Typical NRs consist of three structural domains: two domains that bind DNA and ligands, respectively, and an N-terminal regulatory domain highly variable in both sequence length and structural size.⁹² Ligand agonist binding induces conformational changes, exposing the nuclear localization signal, which allows NRs to translocate to the nucleus and bind response elements. In prostate and breast cancer, abnormal activation or expression of NRs can drive cancer cell proliferation and survival. Conventional treatment strategies for androgen receptor (AR) involve suppression of androgen production or administration of AR antagonists, and estrogen receptor (ER)-targeted approaches employ estrogen depletion or selective estrogen receptor modulators to antagonize receptor activity. However, cancer cells may develop resistance due to NR mutations or overexpression.

Small-molecule ligands that bind to the ligand-binding domain are designed to either activate (agonists) or block (antagonists/inhibitors) the signal transduction function of NRs.^93,94 However, small-molecule inhibitors exhibit conceptual and functional ambiguity in NR-targeted therapy. For instance, AR antagonists may, paradoxically, function as agonists under conditions of AR overexpression or mutation, leading to therapeutic contradictions.⁹⁵ Moreover, approximately one-third of NRs (e.g., COUP-TFII) are orphan receptors with undefined ligand-binding mechanisms, complicating inhibitor design due to the lack of well-defined target sites. These factors collectively contribute to the complexity and unpredictability of NR-targeted drug development.⁹⁶

The emergence of PROTAC technology has enabled targeting a broader range of NRs. NRs such as AR and ER are involved in various important physiological processes in the body and are closely related to prostate and breast cancers.⁹⁷ In contrast, PROTACs can directly induce NR degradation, eliminating even mutated or overexpressed receptors, overcoming resistance. Consequently, a series of PROTACs targeting ER or AR has been developed. Arvinas has developed ARV-110, the first PROTAC drug to enter clinical trials. It completed its clinical trials this year and has demonstrated anti-tumor activity and safety in patients with metastatic castration-resistant prostate cancer. These findings provide significant data supporting the application of PROTAC technology in cancer treatment.⁷³ Another Arvinas product, ARV-766, is a second-generation AR-targeted PROTAC protein degrader designed to selectively degrade both wild-type and mutant ARs, including the L702H mutation associated with clinical resistance.⁷⁴ ARV-471 is an orally administered ER-targeted PROTAC protein degrader that can degrade not only the wild-type estrogen receptor but also ESR1 mutants associated with resistance, inhibiting the estrogen receptor-mediated signaling pathway and exerting anti-tumor effects.⁷⁵ It is currently in phase I clinical trials for the combined treatment of advanced or metastatic ER-positive/HER2-negative breast cancer with everolimus. Furthermore, MTX-23 is a PROTAC molecule capable of targeting both the AR-V7 splice variant and full-length AR protein, demonstrating promising anti-tumor activity.⁹⁸

PROTACs exhibit revolutionary capabilities for NRs (e.g., AR/ER) by eliminating both wild-type and mutant variants, circumventing resistance mechanisms inherent to conventional antagonists (e.g., ARV-766 targeting AR-L702H). Nevertheless, clinical advancement encounters obstacles such as restricted E3 ligase diversity (overreliance on CRBN/VHL) and organ-specific toxicity concerns stemming from NRs’ pleiotropic physiological functions.

PROTACs for targeting transcription factors

Transcription factors (TFs) are proteins that bind to specific DNA sequences to regulate gene transcription. By recognizing and binding to specific sequences in gene promoters or enhancers, TFs modulate the expression levels of genes.⁹⁹ Apart from NRs, directly targeting TFs has been particularly challenging for small-molecule inhibitors, leading to their reputation as undruggable proteins for decades.^100,101 Inducing protein degradation has emerged as a potential approach for TFs. TFs can be classified into dozens of families based on the structural characteristics of their DNA-binding domains. Among these, C2H2 zinc-finger proteins, homeodomains, and basic-helix-loop-helix domains represent the three major families that dominate transcriptional regulation in eukaryotes.¹⁰²

Signal transducer and activator of transcription 3 (STAT3) is a key nuclear transcription factor phosphorylated on tyrosine 705 and integrates signals from cytokines and growth factors to regulate a range of cellular processes.¹⁰³ Targeting STAT3 is a common therapeutic strategy for various cancers, inflammatory, and autoimmune diseases. Phosphorylation of STAT3 at the Tyr705 site triggers its dimerization, which is closely associated with the transcriptional regulation of target genes.¹⁰⁴ Conventional approaches focus on inhibiting the SH2 domain to block STAT3 dimerization. However, these inhibitors often lack sufficient selectivity due to high structural homology among STAT family members. Moreover, monomeric STAT3 retains partial transcriptional activity, challenging complete functional inhibition.¹⁰⁵ PROTACs mediate therapeutic effects by inducing STAT3 protein degradation rather than direct activity inhibition. Zhou et al. developed SD-36, the first STAT3-targeting PROTAC molecule.⁷⁸ SD-36 consists of the STAT3 inhibitor SI-109 conjugated to lenalidomide, where SI-109 specifically binds the STAT3 SH2 domain to recruit E3 ubiquitin ligase, promoting STAT3 ubiquitination and degradation. This strategy not only potently suppresses STAT3 transcriptional activity but also overcomes the limitations of conventional small-molecule inhibitors, including poor selectivity and residual function of monomeric STAT3. SD-36 demonstrates remarkable therapeutic potential for leukemia and lymphoma treatment.

The PROTAC molecule threose nucleic acid (TNA)-E box-pomalidomide (TEP) is a bivalent chimera comprising TNA and DNA elements, designed to specifically target and degrade c-Myc protein by binding to c-Myc/Max heterodimers. As a master transcriptional regulator governing numerous oncogenic proteins, c-Myc is overexpressed in over 50% of human cancers.^106,107 The TEP architecture integrates a TNA aptamer with an E-box DNA sequence, which cooperatively engages distinct epitopes on c-Myc/Max, achieving markedly enhanced binding affinity (K_d = 13–22 nM). In triple-negative breast cancer (TNBC) cells, TEP effectively degrades c-Myc with an IC₅₀ of ∼53 nM, demonstrating ∼50-fold greater potency than the conventional inhibitor MYCi975 (IC₅₀ = 2.6 μM). Mechanistically, TEP-mediated c-Myc depletion not only suppresses TNBC proliferation but also sensitizes cells to the CDK4/6 inhibitor palbociclib. In 4T1 TNBC murine models, TEP/palbociclib combination therapy significantly inhibited tumor growth while reducing Ki67 and c-Myc expression. This nucleic acid-based PROTAC represents a novel therapeutic strategy for TNBC, particularly demonstrating synergistic anti-tumor efficacy in combination regimens.¹⁰⁸

JQAD1 is a PROTAC compound targeting EP300 (E1A-binding protein, also known as p300), which suppresses tumor growth by specifically degrading EP300. JQAD1 was designed based on the EP300 inhibitor A485, utilizing its A485 moiety to bind EP300 while recruiting the E3 ubiquitin ligase CRBN via an IMiD moiety, forming a ternary complex that induces ubiquitination and proteasomal degradation of EP300. In high-risk neuroblastoma cells, JQAD1 effectively degrades EP300, reducing histone H3 lysine 27 acetylation (H3K27ac) modification, particularly at super-enhancer regions. This results in the suppression of key oncogenic genes, including MYCN. Treatment with JQAD1 decreases H3K27ac levels and promotes MYCN protein degradation, ultimately inducing apoptosis and inhibiting tumor cell proliferation. This study not only elucidates the critical role of EP300 in neuroblastoma but also provides a novel strategy for developing PROTACs targeting TFs. JQAD1 demonstrates significant anti-tumor efficacy, particularly in tumors with high CRBN expression, highlighting its therapeutic potential.¹⁰⁹

The targets of PROTACs, such as kinases (e.g., RIPK1, HK2, and EGFR) and NRs (e.g., AR and ER), are themselves important nodes regulating PCD pathways, including apoptosis and pyroptosis. Through their unique event-driven mode of action, PROTACs exhibit significant advantages over traditional inhibitors in targeting three classes of historically challenging targets: protein kinases, NRs, and transcription factors. By degrading these targets, PROTACs can precisely regulate key steps of PCD pathways, inhibit tumor growth, and enhance anti-tumor immunity, providing a novel strategy for cancer therapy. In the future, with the further advancement of PROTAC technology, its application in PCD pathway regulation will become more extensive.

The advantages of PROTACs

Innovative therapeutic approaches, including small-molecule inhibitors, monoclonal antibodies, RNA interference (RNAi), gene editing, and molecular glues, have become fundamental tools in drug development. Small-molecule inhibitors exert their effects by occupying the active site of their target, competing with native ligands to inhibit the function of the target protein or enzyme. However, long-term clinical use of these inhibitors is often hampered by the emergence of resistance and the potential for off-target effects.¹¹⁰ Monoclonal antibodies offer the advantage of high-affinity binding to target proteins, modulating cellular responses by interrupting extracellular protein-protein or protein-ligand interactions. Despite their precision, they are limited by poor cellular permeability, the inability to be administered orally, and high production costs.¹¹¹ RNAi targets gene silencing by reducing mRNA levels, with the potential to degrade multiple mRNA transcripts due to the catalytic nature of RNAi. However, challenges such as off-target effects, poor oral bioavailability, and inadequate tissue penetration complicate the development of effective drug delivery systems.¹¹² CRISPR-Cas9 gene editing enables precise DNA modification through single guide RNA (sgRNA) targeting, and it can even mediate mitochondrial DNA (mtDNA) editing, but off-target effects caused by sequence similarity or cellular environmental differences remain a safety concern. Clinical translation of this technology, which balances its high precision potential with current limitations (e.g., ethics controversies about germline editing), necessitates ongoing optimization of sgRNA/Cas9 designs (e.g., high-fidelity Cas variants) and robust regulatory oversight.^113,114 Molecular glues can modulate cellular signaling, reprogram the cell cycle, or reduce pathogen infectivity by inducing or stabilizing protein-protein interactions, demonstrating high specificity and efficacy with significant therapeutic potential for various diseases. However, the discovery and development of molecular glues face challenges, including the need for precise mechanistic studies to elucidate their mode of action and the high costs and complexities associated with large-scale screening and validation processes.¹¹⁵

As a promising therapeutic modality, PROTACs offer distinct advantages over traditional small-molecule inhibitors, monoclonal antibodies, and other therapeutic strategies (Table 3).¹¹⁶ First, traditional small-molecule drugs rely on the active sites or binding pockets of target proteins; however, approximately 80% of disease-related proteins, such as transcription factors, RNA-binding proteins, and scaffold proteins, are considered undruggable due to the absence of well-defined binding sites. PROTACs overcome these limitations through modular designs involving small-molecule ligands or peptide motifs that directly recognize key functional domains of target proteins (e.g., DNA-binding motifs or spatial conformations), enabling targeted degradation of proteins previously deemed inaccessible to conventional therapeutics. The unique pharmacokinetic profile of PROTAC technology confers a superior safety advantage compared to traditional occupancy-driven inhibitors. PROTACs promote the degradation of target proteins, and their efficacy does not depend on maintaining high drug concentrations. This means that effective degradation of target proteins can be achieved even at lower drug concentrations. This event-driven pharmacology reduces the potential toxic risks associated with long-term high-concentration drug exposure, enhancing the safety of treatment.¹²⁸ Traditional small-molecule inhibitors require sustained high doses to maintain target occupancy, whereas PROTACs achieve prolonged efficacy through the catalytic degradation of target proteins. Additionally, after degrading the target protein, the slow resynthesis rate prolongs the reduction in protein levels. This delayed effect reduces the frequency of drug administration and further decreases the risk of cumulative toxicity.²⁹

Table 3.

Comparisons of PROTACs with other therapeutic strategies

Strategy	Mechanism	Advantages	Limitations	Applications	Reference.
PROTACs	Bifunctional molecules recruit E3 ligases to induce target protein degradation via the ubiquitin-proteasome system.	①targets undruggable proteins	①high molecular weight (>700 Da)	cancer, neurodegenerative diseases, immune disorders	Martín-Acosta et al.116; Qi et al.117
		②high selectivity	②susceptibility to the hook effect”
		③long-term catalytic effect	③high development costs and complexity
LYTAC	Bifunctional molecules induce degradation of extracellular and membrane proteins by simultaneously engaging the target and a lysosome-targeting receptor.	①degrade extracellular and membrane proteins	①high molecular weight (∼150 kDa) and rapid hepatic clearance	cancer, Alzheimer’s disease, autoimmune diseases, lysosomal storage disorders	Banik et al.118; Ahn et al.119
		②significantly expands the scope of targetable proteins	②potential immunogenicity
			③complex manufacturing process
AUTAC	Bifunctional molecules induce selective autophagy by recruiting the autophagic machinery to target proteins via a biomimetic degradation signal.	①capable of degrading large intracellular complexes and damaged organelles	①poor chemical stability of the S-guanylate tag and short in vivo half-life	cancer, mitochondrial diseases, pathogen infections, protein aggregation diseases	Takahashi et al.120; Takahashi et al.121
		②potential for clearing intracellular pathogens	②degradation efficiency requires optimization for many targets
			③complex design and synthesis
Molecular glues	monofunctional compounds that mediate target protein-E3 ubiquitin ligase interactions	①low molecular weight and favorable drug-like properties	①complex design	cancer, immune diseases	Dewey et al.115; Li et al.122
		②typically exhibit excellent oral bioavailability	②limited target range
Small-molecule inhibitors	bind to and inhibit the activity of target proteins	①well-established technology	①inhibit the function and cannot degrade the protein	cancer, cardiovascular diseases, infectious diseases	Liu et al.123; Moon et al.124
		②good oral bioavailability	②ineffective against undruggable targets
			③prone to resistance via target overexpression or mutation
Monoclonal antibodies (mAbs)	bind to extracellular targets, blocking function or marking for immune clearance	①high specificity	①limited to extracellular targets	cancer, autoimmune diseases, infectious diseases	Tsao et al.111; Kaur et al.125
		②long residual action	②expensive production
		③engage immune responses	③potential immunogenicity
RNA interference (RNAi)	siRNA/shRNA degrades target mRNA, reducing protein expression.	①target undruggable proteins	①significant in vivo delivery challenges	genetic disorders, viral infections, cancer	Won Lee et al.112; Jadhav et al.126
		②high specificity	②possible off-target effects
			③risk of innate immune activation
Gene editing (e.g., CRISPR)	directly modifies DNA to correct or knock out disease-causing genes	①permanent genetic correction	①risk of unpredictable off-target editing	genetic diseases, rare disorders, cancer	Vaghari-Tabari et al.113; Wang et al.127
		②broad therapeutic potential	②immunogenicity and safety concerns of delivery vectors

Furthermore, PROTAC technology also excels in specificity. PROTAC molecules consist of three parts: a target protein-binding domain, an E3 ubiquitin ligase-binding domain, and a linker connecting the two.^58,129 This design allows PROTAC molecules to precisely bring the target protein and E3 ubiquitin ligase together, triggering ubiquitination and degradation of the target protein. This mechanism not only increases the degradation efficiency of the target protein but also reduces interference with non-target proteins, thus enhancing the specificity of treatment.¹³⁰ Moreover, PROTAC technology has the potential to target proteins that are difficult to drug with traditional small-molecule inhibitors, such as transcription factors and scaffold proteins, which typically lack suitable active sites for small-molecule binding.¹¹⁷

PROTACs demonstrate significant anti-tumor efficacy in preclinical studies by selectively degrading key oncoproteins, effectively suppressing tumor proliferation or metastasis. The CDK4/6-targeting PROTAC Pal-pom inhibits TNBC cell proliferation by degrading cell cycle regulators and blocking the Rb-E2F signaling pathway.¹³¹ Another example is PROTAC-3, which degrades epithelial-mesenchymal transition (EMT)-related proteins (e.g., FAK and β-catenin), disrupting scaffold functions critical for tumor cell migration and reducing metastatic potential in TNBC and prostate cancer.¹³² Notably, PROTACs overcome drug resistance, as evidenced by BTK-targeting PROTACs that effectively degrade C481S mutant proteins, providing an innovative therapeutic approach for B cell malignancies.¹³³

PROTACs have achieved several groundbreaking clinical advances in the field of oncology. ARV-471 (vepdegestrant), the first-in-class PROTAC drug targeting the ER, developed through a collaboration between Arvinas and Pfizer, successfully completed its phase III VERITAC-2 trial in 2025. In patients with ESR1-mutated ER⁺/HER2⁻ metastatic breast cancer, it demonstrated a median progression-free survival (PFS) of 5.0 months, significantly superior to the 2.1 months observed with fulvestrant (hazard ratio = 0.57). The objective response rate (ORR) was 18.6% compared to 4.0% in the control group. Furthermore, ARV-471 exhibits favorable oral bioavailability and a manageable safety profile, making it the first PROTAC to reach the new drug application (NDA) submission stage.¹³⁴ In the prostate cancer therapy landscape, ARV-110, an AR-targeting PROTAC, has completed phase II clinical trials, focusing on patients with metastatic castration-resistant prostate cancer (mCRPC) who have developed resistance to AR inhibitors such as enzalutamide and abiraterone. By depleting intracellular AR protein levels, ARV-110 shows potential to overcome resistance to existing treatment regimens (ClinicalTrials.gov: NCT03888612 and NCT05177042).¹³⁵ KT-474 is an orally available heterobifunctional degrader targeting the IRAK4 protein. Its first-in-human clinical trial (ClinicalTrials.gov: NCT04772885) has been completed. This randomized, double-blind, placebo-controlled phase I study was conducted in healthy subjects and patients with hidradenitis suppurativa and atopic dermatitis. Results demonstrated that KT-474, at once-daily doses ranging from 50 to 200 mg, achieved up to 98% degradation of IRAK4, effectively inhibited downstream inflammatory cytokines, and exhibited a favorable safety profile. Based on these positive phase I outcomes, KT-474 has advanced to phase II clinical development for the treatment of moderate to severe autoimmune diseases.¹³⁶ In addition to the aforementioned PROTAC molecules, more than 20 other PROTAC drugs have entered clinical trials, demonstrating immense therapeutic potential and a revolutionary outlook.

Beyond PROTAC technology, the field of targeted protein degradation has witnessed the continuous emergence of innovative strategies. Notably, breakthroughs in novel technologies such as lysosome-targeting chimeras (LYTACs) and autophagy-targeting chimeras (AUTACs) have significantly expanded the spectrum of degradable targets by harnessing endogenous cellular degradation pathways, providing a richer toolbox for disease treatment.⁸⁵

LYTACs function as bifunctional molecules formed by covalently linking a target protein ligand (e.g., an antibody) to a lysosome-targeting receptor-recruiting moiety (e.g., M6P). Their mechanism of action involves the ligand binding to the target protein, followed by the M6P moiety recruiting the cell-surface CI-M6PR receptor. This initiates internalization via clathrin-mediated endocytosis, resulting in the formation of an endosome. Acidification of the endosome triggers receptor dissociation, and the target protein is efficiently degraded upon fusion of the endosome with the lysosome.^118,119 LYTACs break the limitation of traditional PROTACs, which primarily degrade intracellular proteins, by enabling the targeted clearance of membrane proteins and secreted proteins for the first time. They thus complement PROTACs, collectively building a protein degradation network covering intracellular, membrane, and extracellular spaces.

In contrast, AUTACs are bifunctional modules comprising a target protein ligand connected via a linker to an autophagy-recruiting tag. After the ligand binds to the target protein, the tag induces K63-linked polyubiquitination of the target protein. This modification recruits the autophagy receptor p62/SQSTM1, leading to encapsulation within the autophagosome membrane. Subsequently, the autophagosome fuses with the lysosome, and the target protein is degraded by acidic hydrolases.^120,121 Compared to LYTACs, AUTACs can target protein aggregates and damaged organelles (e.g., mitochondria), demonstrating potential for clearing aberrant proteins/organelles in pathological contexts such as neurodegenerative and metabolic diseases.

Beyond LYTACs and AUTACs, other lysosome-based degradation technologies are also advancing. Autophagosome tethering compounds (ATTECs) directly link the target protein to LC3, a key autophagy protein, forming a complex that is engulfed into the autophagosome and degraded via the lysosome. This process is ubiquitination independent; ATTECs have relatively small molecular weights and can even target non-protein macromolecules like lipid droplets, showing significant drug development potential.¹³⁷ Antibody-based PROTACs (AbTACs) are bispecific antibodies. One end binds a membrane protein, while the other recruits a membrane-associated E3 ligase (e.g., RNF43), facilitating degradation of the membrane protein through the lysosomal pathway. Leveraging antibody specificity, AbTACs can precisely target membrane proteins that are traditionally difficult for PROTACs to address.¹³⁸ These emerging lysosomal pathway degradation technologies, together with the proteasome-based PROTAC technology, constitute a diversified targeted protein degradation toolbox. This collective arsenal enables the degradation of extracellular proteins, membrane proteins, protein aggregates, and even organelles, greatly expanding the application prospects of targeted protein degradation.

PROTACs demonstrate the core advantages of high selectivity and efficacy by achieving complete degradation of disease-causing proteins rather than merely inhibiting their activity, establishing them as a pivotal strategy in cancer therapy. Importantly, this unique protein degradation capability exhibits intrinsic molecular synergy with PCD pathways. Research indicates that, through the degradation of key regulatory proteins such as GPX4 and RIPK1, PROTACs can directly intervene in cell death processes, including ferroptosis and necroptosis. This multi-level regulatory capacity over cell death pathways provides a unique perspective for developing novel anti-tumor strategies. As clinical trials advance, the application prospects of PROTAC technology in cancer treatment are becoming increasingly clear. The following section will focus on the specific molecular mechanisms and research progress regarding the regulation of PCD processes such as ferroptosis and apoptosis.

Application of PROTACs in different PCD pathways

PCD pathways, including ferroptosis, pyroptosis, necroptosis, apoptosis, autophagic cell death, and cuproptosis, play crucial roles in maintaining cellular homeostasis and responding to various physiological and pathological conditions.^139,140 Their dysregulation is associated with a wide range of diseases, such as cancer, neurodegenerative diseases, and autoimmune diseases.^35,141 Traditional therapeutic approaches targeting these pathways often encounter challenges due to drug resistance, off-target effects, and poor bioavailability. In recent years, PROTAC technology has emerged as a promising solution with significant potential to overcome the limitations of conventional therapies. Below is an overview of the applications of PROTAC technology in different PCD pathways.

Applications of PROTACs in ferroptosis

Ferroptosis is an iron-dependent, non-apoptotic form of PCD characterized by the accumulation of lipid reactive oxygen species (ROS), leading to oxidative damage and disruption of antioxidant defense balance.¹⁴² This unique form of PCD is distinct from apoptosis, necrosis, and autophagic cell death and is primarily driven by the peroxidation of polyunsaturated fatty acids (PUFAs) in the presence of iron and lipoxygenases.^143,144 The execution of ferroptosis is intricately linked to the phospholipid peroxidation of PUFAs, which is catalyzed by iron and results in cell death.^145,146,147 Ferroptosis is also associated with the depletion of the antioxidant enzyme GPX4, which is crucial for reducing lipid peroxides and maintaining cellular redox homeostasis (Figure 2).¹⁴⁸ The process is influenced by various metabolic pathways, including those involving iron, lipids, and amino acids as well as degradation pathways such as macroautophagy/autophagy and the UPS.¹⁴⁹ The morphological hallmarks of ferroptosis comprise shrunken mitochondria with ruptured outer membranes, loss of cristae, and an intact plasma membrane while maintaining normal nuclear morphology without chromatin condensation.^150,151

Figure 2.

Molecular mechanisms of ferroptosis and PROTAC-based therapeutic targeting

This figure illustrates the core ferroptosis pathways and potential PROTAC-mediated intervention strategies. (1) The cystine/glutamate antiporter system Xc⁻ (composed of SLC3A2 and SLC7A11) imports cystine for GSH synthesis. GSH is essential for GPX4 activity, which converts lipid peroxides (L-OOH) into non-toxic alcohols (L-OH). Inhibition of system Xc⁻ or GPX4 leads to lethal lipid peroxidation. (2) Labile iron (Fe²⁺) drives the Fenton reaction, generating ROS. Ferritinophagy, mediated by NCOA4, releases Fe³⁺, which is reduced to Fe²⁺, further promoting lipid peroxidation. (3) NRF2 dissociates from Keap1 under oxidative stress and translocates to the nucleus, activating antioxidant genes. (4) PROTACs can be designed to degrade key ferroptosis regulators: NCOA4-targeting PROTACs inhibit ferritinophagy and reduce iron overload. GPX4-targeting PROTACs deplete GPX4 and induce ferroptosis in cancer cells. Keap1-targeting PROTACs stabilize NRF2 and enhance antioxidant defense. PARP1-targeting PROTACs degrade PARP1 and enhance anticancer efficacy or overcome PARP inhibitor (PARPi) resistance.

Ferroptosis holds broad therapeutic potential across various diseases, particularly in cancer treatment.¹⁵² Targeting GPX4 is a crucial strategy in cancer therapy. RSL3 directly inhibits GPX4 activity to induce ferroptosis, demonstrating superior efficacy against non-small cell lung cancer (NSCLC) cells compared to erastin while exhibiting minimal toxicity to normal cells.¹⁵³ EMP1 enhances RSL3-induced downregulation of GPX4, increasing intracellular ROS and lipid peroxidation levels, thereby inducing ferroptosis in head and neck squamous cell carcinoma (HNSCC) cells.¹⁵⁴ Several PROTAC-based GPX4 degraders have been developed, including GDCNF-11, DC-2, compound 18a, ZX703, and NC-R17. Among these, GDCNF-11 is an HSP90-based HIM-PROTAC that facilitates GPX4 ubiquitination and degradation via the HSP90 chaperone complex, effectively reducing endogenous GPX4 levels to induce ferroptosis in HT-1080 cells with a DC₅₀ of 0.08 μM.¹⁵⁵ Similarly, DC-2 demonstrates potent ferroptosis-inducing activity in HT-1080 cells with a lower DC₅₀ of 0.03 μM.¹⁵⁶ Compound 18a, another GPX4-targeting PROTAC, exhibits a DC₅₀ (48 h) of 1.68 μM. All of these PROTAC molecules induce characteristic ferroptotic events, including lipid peroxide accumulation and mitochondrial depolarization.¹⁵⁷ Notably, NC-R17 represents a distinct class of noncovalent GPX4-targeting PROTACs derived from the RSL3 scaffold, demonstrating promising anti-tumor activity.¹⁵⁸ In contrast to conventional PROTACs, ZX703 (compound 5I) uniquely employs dual degradation pathways, both the UPS and autophagy-lysosome pathway, to achieve dose- and time-dependent GPX4 degradation (DC₅₀ = 0.135 μM).¹⁵⁹

Li et al. developed a novel PARP1-targeting PROTAC (NN3) to overcome PARP inhibitor (PARPi) resistance caused by PARP1 mutations, demonstrating anti-tumor efficacy in p53-mutant TNBC through ferroptosis induction. Mechanistically, NN3-mediated PARP1 degradation activates both wild-type and mutant p53, leading to significant downregulation of SLC7A11 and GPX4 expression. This results in GSH depletion and a subsequent lipid peroxidation increase. Notably, NN3 exhibits superior therapeutic efficacy and reduced toxicity compared to conventional PARPi in both in vitro and in vivo models while maintaining effectiveness against tumor cells with intact homologous recombination repair capacity.¹⁶⁰

NRF2-mediated regulation of ferroptosis primarily functions through its antioxidant defense mechanisms to suppress ferroptosis initiation.¹⁶¹ Under oxidative stress, NRF2 dissociates from Keap1 and translocates to the nucleus, activating downstream target genes (including NQO1 and HO-1) to enhance cellular resistance against lipid peroxidation.^162,163 In a groundbreaking approach, ARE-PROTACs were designed based on antioxidant response elements to achieve coordinated degradation of both the transcription factor NRF2 and its heterodimeric partner MafG. While NRF2 is frequently hyperactivated in cancers, its classification as an undruggable target stems from the absence of conventional small-molecule binding pockets. The chimeric molecule C2 was developed by chemically linking NRF2’s DNA-binding sequence (ARE) with a CRBN E3 ubiquitin ligase ligand. Through the UPS, C2 selectively degrades the NRF2-MafG heterodimer, effectively suppressing NRF2 transcriptional activity. This intervention disrupts the antioxidant defense system in cancer cells, rendering them more susceptible to both ferroptosis and conventional chemotherapeutic agents.¹⁶⁴

NR coactivator 4 (NCOA4) functions as a selective cargo receptor that directly binds ferritin heavy chain (FTH) and mediates its delivery to autolysosomes for degradation, a process termed ferritinophagy.¹⁶⁵ This degradation pathway liberates intracellular iron ions for recycling, elevating labile iron levels. These released iron ions catalyze the Fenton reaction to generate highly reactive hydroxyl radicals, which subsequently induce lipid peroxidation and compromise membrane integrity, ultimately triggering ferroptotic cell death.¹⁶⁶ The PROTAC-based NCOA4 degrader-1 (compound V3) demonstrates potent NCOA4 degradation activity (DC₅₀ = 3 nM in HeLa cells). Treatment with compound V3 effectively reduces both NCOA4 expression and intracellular Fe²⁺ levels, demonstrating therapeutic efficacy in ameliorating CCl4-induced acute liver injury in animal models.¹⁶⁷

As more clinical studies are conducted and new targets are discovered, PROTAC technology is expected to play a greater role in ferroptosis therapy, providing new strategies and potential targets for cancer treatment (Table 4). Nanoparticle delivery systems can significantly enhance the bioavailability and tumor targeting of PROTAC technology. By protecting the drug from degradation and improving its solubility, nanoparticles (NPs) prolong the circulation time of PROTACs in the body. Their passive targeting leverages the enhanced permeability and retention (EPR) effect to accumulate in tumor tissues, while active targeting uses surface ligands to precisely locate cancer cells. For instance, the multifunctional nanoplatform BPNpro achieves potent ferroptosis-mediated cancer therapy through synergistic inhibition of dihydroorotate dehydrogenase and GPX4, two key ferroptosis defense proteins.^168,169,170 Another innovative approach involves GSH-depleting nanoengineered PROTACs (nano-PROTACs), wherein the BRD4 degrader ARV-771 is encapsulated within GSH-responsive poly(disulfide amide) (PDSA) polymeric nanoparticles. This strategy not only enhances tumor accumulation and intracellular release through nanocarrier delivery but also amplifies ARV-771-induced c-Myc-dependent ferroptosis and cell-cycle arrest via PDSA-mediated GSH depletion.^171,172 Consequently, this dual mechanism enables superior anti-tumor efficacy at reduced dosage while significantly improving the bioavailability and targeted protein degradation efficiency of PROTACs.

Table 4.

Representative PROTACs applied in ferroptosis

PROTACs	Targets	E3 ligase	Activity		Mechanism	Diseases	Reference
			DC50	Dmax
GDCNF-11	GPX4	HSP90 complex	HT-1080 DC50 = 0.08 μM	–	HIM-PROTAC chaperone-mediated degradation	RAS-mutant tumors	Dong et al.155
DC-2	GPX4	CRBN	HT-1080 DC50 = 0.03 μM	>80%	CRBN-dependent ubiquitination	solid tumors	Wang et al.156
Compound 18a	GPX4	CRBN	HT-1080 DC50 = 1.68 μM	85%	reduces GPX4 levels, accumulates lipid ROS	fibrosarcoma	Song et al.157
ZX703	GPX4	VHL	HT-1080 DC50 = 0.135 μM	86%	dual UPS/autophagy degradation	liver cancer	Hu et al.159
NC-R17	GPX4	CRBN	–	–	non-covalent degradation	non-small cell lung cancer	Zheng et al.158
NN3	PARP1	CRBN	TNBC DC50: <100 nM	–	degrades PARP1, downregulates SLC7A11/GPX4, depletes GSH	p53-mutant triple-negative breast cancers	Li et al.160
C2 (ARE-PROTAC)	NRF2-MafG	CRBN	A549 DC50 (NRF2) = 1.85 nM DC50 (MafG) = 66 nM	–	DNA-binding domain targeting	non-small cell lung cancer	Ji et al.164
Compound V3	NCOA4	VHL	HeLa DC50 = 3 nM AML12 DC50 = 202 nM	–	ferritinophagy inhibition	acute liver injury	Ji et al.167

Applications of PROTACs in pyroptosis

Pyroptosis is a lytic and inflammatory form of PCD, primarily executed by gasdermin-family proteins (e.g., GSDMD and GSDME). These proteins are cleaved and activated by either inflammatory caspases (e.g., caspase-1/4/5/11) in canonical/non-canonical pathways or apoptosis-associated caspases (e.g., caspase-3/8) under specific stimuli, leading to membrane pore formation and cell rupture. It is characterized by the formation of pores in the cell membrane via the N-terminal structural domain of gasdermin proteins, leading to cell swelling, rupture, and the release of large amounts of pro-inflammatory factors (e.g., IL-1β and IL-18), which trigger an intense inflammatory response.^173,174

The molecular mechanism of pyroptosis involves three key pathways. In the classical pathway, pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) activate inflammatory vesicle complexes, such as NLRP3 or AIM2, which, in turn, activate caspase-1-mediated cleavage of GSDMD. The non-classic pathway, on the other hand, involves the direct activation of caspase-4/5/11 by intracellular lipopolysaccharide (LPS), which also leads to proteolytic activation of GSDMD. In addition, specific stimuli, such as chemotherapeutic agents or granzymes (GzmB/GzmA) released by cytotoxic lymphocytes, can selectively shear GSDME or GSDMC through the activation of caspase-3/8. All of these pathways lead to the formation of pores in the cell membrane by the N-terminal structural domain of the gasdermin protein, which induces osmotic lysis of the cell and the release of proinflammatory factors (Figure 3).^175,176

Figure 3.

Molecular mechanisms of pyroptosis and PROTAC-based therapeutic targeting

This illustration demonstrates how death ligands (TNF-α/FasL/TRAIL) bind to their respective receptors (TNFR/Fas/DR4/DR5), initiating a caspase cascade (caspase-8/3/1) via Bid and BAK/BAX activation and corresponding PROTACs targeting approaches. (1) Receptor ligation triggers caspase-8 activation, which cleaves Bid to tBid. tBid activates BAK/BAX, inducing mitochondrial outer membrane permeabilization (MOMP). (2) Mitochondrial damage: MOMP releases cytochrome c and accumulates ROS. (3) Iron dysregulation: ferritin degradation via NCOA4-mediated ferritinophagy, increasing labile iron pool (LIP). (4) Inflammasome activation: ASC oligomerization triggers NLRP3 inflammasome assembly to activate caspase-1 (promoting IL-1β/IL-18 maturation). (5) cGAS-STING pathway: cytosolic DNA sensors (cGAS) activate STING, amplifying inflammatory responses. (6) PROTAC intervention nodes: HK2-targeting PROTACs block glycolysis, cause mitochondrial damage, and then induce GSDME-dependent pyroptosis. Ferritin-targeting PROTACs suppress ferritinophagy to limit iron-driven ROS. STING-targeting PROTACs terminate aberrant inflammatory signaling. BRD4-targeting PROTACs attenuate pro-inflammatory transcription (e.g., NF-κB targets).

In clinical applications, the dual regulatory role of pyroptosis has become a hot research topic. On the one hand, anti-tumor immune response can be significantly enhanced by inducing tumor cell pyroptosis. Studies have shown that chemotherapeutic drugs such as cisplatin trigger tumor cell pyroptosis by activating the caspase-3/GSDME pathway and that the released cellular contents can promote dendritic cell maturation and T cell activation.¹⁷⁷ Chimeric antigen receptor (CAR)-T cell therapy enhances tumor immunogenicity by releasing granzyme B to cleave the GSDME, converting apoptosis into pyroptosis.¹⁷⁸ On the other hand, excessive pyroptosis can exacerbate pathological injury, such as GSDMD-mediated cytokine storm in sepsis, which can lead to multi-organ failure. At this point, pathological inflammatory injury can be effectively attenuated by using the caspase-1 inhibitor VX-765 or MCC950, which targets the NLRP3 inflammatory vesicle.¹⁷⁹ Importantly, radiotherapy-induced focal damage to normal tissues also needs to be controlled by regulating gasdermin protein activity.¹⁸⁰ Future studies should focus on the design of gasdermin-activating prodrugs for the TME as well as the selection of reversible pyroptosis inhibitors for systemic inflammation, which will provide new targets for the treatment of cancer, infectious diseases, and autoinflammatory syndromes.

Here, we summarize representative therapeutic applications of PROTAC drugs in modulating pyroptosis pathways (Table 5). The stimulator of interferon genes (STING) pathway plays a crucial role in immune responses and has emerged as a compelling target in cancer therapy.¹⁸⁶ STING drives pyroptosis via dual pathways: activation of NLRP3 inflammasomes and direct promotion of GSDMD cleavage, both of which represent critical immune responses triggered by infection, ischemia, or metabolic stress.^187,188 In ischemic stroke and myocardial injury, STING detects mtDNA leakage via the cGAS-STING axis, subsequently triggering NLRP3-dependent caspase-1 activation.^189,190,191 This cascade culminates in GSDMD-mediated plasma membrane pore formation and the release of pro-inflammatory cytokines (e.g., IL-1β and IL-18).¹⁹² STING further aggravates ferroptosis-associated pyroptosis via non-canonical pathways (e.g., autophagy-dependent degradation of GPX4), amplifying tissue injury in pathological contexts.¹⁹³ Abnormal activation of the STING signaling pathway plays a critical role in the pathogenesis of acute kidney injury (AKI).¹⁹⁴ STING-PROTAC (SP23) is the first STING protein degrader designed based on PROTACs for treating diseases caused by overactivation of the STING signaling pathway. Liu et al. designed a series of PROTAC molecules by linking the STING inhibitor C-170 to the CRBN ligand pomalidomide, among which SP23 exhibited optimal STING-degrading activity (DC₅₀ = 3.2 μM). In a mouse model of cisplatin-induced AKI, SP23 showed significant anti-inflammatory effects.¹⁸¹ STING is highly expressed in inflammatory bowel disease (IBD) and positively correlates with disease activity. SP23 inhibits activation of the NLRP3 inflammasome through degradation of STING, reduces pro-inflammatory factor release, attenuates macrophage M1 polarization, and restores tight-junction protein expression, which protects intestinal barrier integrity.¹⁹⁵ This degradation mechanism significantly alleviated dextran sulfate sodium (DSS)-induced colitis, suggesting potential application of STING-PROTAC in IBD treatment.¹⁹⁵ STING protein is also highly expressed in renal cell carcinoma (RCC) and promotes tumor survival by inhibiting endoplasmic reticulum stress-dependent pyroptosis. Wu et al. employed SP23 to degrade STING to lift its inhibition of endoplasmic reticulum stress, activate the PERK/eIF2α/ATF4/CHOP pathway, and prompt caspase-8 to shear GSDMD to form membrane pores. HMGB1 and ATP released by pyroptotic cells promote CD8⁺ T cell infiltration, making the tumor more sensitive to PD-1 inhibitors. The elucidation of this mechanism provides a novel therapeutic target for RCC, as restoring the pyroptotic capacity of tumor cells can enhance anti-tumor immune responses.¹⁸² This suggests that STING-PROTAC has potential in inflammatory diseases and shows promising applications in tumor therapy.

Table 5.

Representative PROTACs applied in pyroptosis

PROTACs	Targets	E3 ligase	Activity		Mechanism	Diseases	Reference
			DC50	Dmax
SP23	STING	CRBN	THP-1 DC50 = 3.2 μM	–	inhibits NLRP3 inflammasome via STING degradation	acute kidney injury, inflammatory bowel disease, renal cell carcinoma	Zhong et al.100; Liu et al.181; Wu et al.182
L@NBMZ	BRD4	CRBN	MCF-7 DC50 = 50.87 nM	>90%	light-activated BRD4 degradation and ROS-induced GSDME cleavage	breast cancer	Huang et al.183
DeFer-2	ferritin	VHL	–	–	triggers caspase-3/GSDME pyroptosis via iron dysregulation	melanoma (B16F10)	Chen et al.184
C-02	HK2	CRBN	4T1 DC50 = 2.56 μM MDA-MB-231 DC50 = 0.79 μM	–	reprograms glycolysis, induces pyroptosis, and downregulates PD-L1	breast cancer	Sang et al.185

Conventional PROTACs suffer from poor membrane permeability and low bioavailability due to their high molecular weight (>700 Da). BRD4-PROTAC MZ1 co-loaded liposomes (L@NBMZ) with the photosensitizer NBSEt remained highly active under hypoxic conditions (IC₅₀ = 153.2 nM), and its degradation efficiency (DC₅₀ = 50.87 nM) was 3-fold higher than that of free PROTACs. For the first time, light-driven cellular pyroptosis of breast cancer cells was realized. This strategy blocked gene transcription by degrading BRD4 protein, and photodynamically generated ROS synergistically enhanced the shearing of GSDME by caspase-3, resulting in a “dual activation” effect of colocalization, which significantly inhibited the growth and lung metastasis of breast cancer.¹⁸³ This study not only demonstrates the potential of PROTACs in breast cancer treatment, but also provides a new direction for photodynamic therapy. Ferritin-PROTAC (DeFer-2), a ferritin degrader, induces cancer cells to undergo pyroptosis instead of the expected ferroptosis by targeting the degradation of ferritin through PROTACs. A research team led by Chen elucidated the mechanistic link between dysregulated iron metabolism and pyroptosis activation via the caspase 3-GSDME pathway. Their study employed albumin nanoparticle-encapsulated DeFer-2 (aDeFer-2; drug loading: 1.86%) to leverage the EPR effect for improved tumor targeting. aDeFer-2 significantly inhibited tumor growth in a mouse model of B16F10 melanoma with no major organ toxicity or hematological abnormalities, indicating good safety and therapeutic efficacy.¹⁸⁴

HK2-PROTAC (C-02) is a novel HK2-targeted PROTAC molecule that enhances immunogenic cell death (ICD) in breast cancer cells by blocking glycolysis through the degradation of HK2 and inducing GSDME-dependent cellular pyroptosis. C-02 was optimized for degradation activity compared to the parent compound through optimization of the linker chain structure (lonidamine-thalidomide chimera), and it significantly reduced resistance to the parent compound by 30-fold. Optimizing the linker structure (lonidamine-thalidomide chimera) resulted in a 30-fold increase in degradation activity compared to the parent compound and significantly reduced toxicity to normal cells. For the first time, the study combines reprogramming of tumor energy metabolism with immunotherapy, providing a new strategy for breast cancer treatment. C-02 not only significantly inhibits tumor growth but also reverses the immunosuppressive microenvironment by downregulating membrane PD-L1, demonstrating a favorable anti-tumor effect. These innovations addressed the bottleneck of PROTAC druggability and enhanced the specificity of pyroptosis induction through spatiotemporally controlled release.¹⁸⁵

Although promising, PROTAC-mediated pyroptosis treatment still faces challenges. First, the degradation efficiency is limited by the tissue distribution of E3 ligase, which may affect the organ specificity of PROTACs.¹⁹⁶ Second, the inflammatory response triggered by pyroptosis needs to be accurately regulated, and overactivation may lead to cytokine storm.¹⁹⁷ Third, the selectivity of the existing PROTAC for GSDMB/E isoforms is insufficient.¹⁰⁰ Future studies could focus on developing tissue-specific E3 ligands, constructing activatable PROTAC prodrugs, and optimizing synergistic therapeutic regimens in combination with immune checkpoint inhibitors, enhancing the clinical application of PROTAC technology.

Applications of PROTACs in necroptosis

Necrosis is a form of cell death characterized by cellular swelling, plasma membrane rupture, and accompanying inflammatory response.¹⁹⁸ Despite its morphological resemblance to conventional necrosis, necroptosis is orchestrated by distinct molecular pathways. It serves as an “alternative” cell death pathway, activated when apoptosis is impaired, such as during caspase inhibition. Necroptosis is particularly crucial in inflammation, infection, and tissue injury.¹⁹⁹

Necroptosis is primarily mediated through the RIPK1-RIPK3-MLKL signaling axis. When apoptosis is suppressed (e.g., by caspase-8 inhibition), activation of death receptors (TNFR/Fas) triggers RIPK1 to recruit and phosphorylate RIPK3 via its RIP homotypic interaction motif (RHIM) domain, forming the necrosome complex. This complex then phosphorylates MLKL, inducing its oligomerization and subsequent insertion into the plasma membrane to form pores, ultimately leading to membrane rupture. Beyond death receptor signaling, viral infection (via ZBP1) and TLR3/4 activation (through TRIF) can also initiate this pathway (Figure 4). Additionally, excessive PARP-1 activation exacerbates necroptosis by depleting cellular nicotinamide adenine dinucleotide (NAD⁺). Upon membrane rupture, DAMPs, such as HMGB1 and ATP, are released, triggering a robust inflammatory response. This tightly regulated cascade is governed by kinase activity and cellular metabolic state, playing a critical role in various pathological conditions.^200,201,202

Figure 4.

Molecular mechanisms of necroptosis and PROTAC-based therapeutic targeting

This figure depicts the molecular mechanisms of necroptosis and corresponding PROTAC intervention strategies. (1) Ligation of death receptors (TNFR/FAS) by TNF-α/FASL/TRAIL or TLR3/4 activation by viral dsRNA/bacterial LPS recruits RIPK1. When caspase-8 activity is suppressed, RIPK1 recruits RIPK3 to form the necrosome via RHIM domain interaction, enabling RIPK3 autophosphorylation and activation. (2) Z-RNA/DNA binds ZBP1, triggering RIPK3 activation independent of RIPK1. ADAR1 antagonizes ZBP1 to limit this pathway. (3) Activated RIPK3 phosphorylates MLKL, inducing MLKL oligomerization, membrane translocation, and pore formation, culminating in plasma membrane rupture and inflammatory cell death. (4) PROTAC intervention strategies: ZBP1-targeting PROTACs suppress virus-induced necroptosis by degrading ZBP1 and blocking RIPK3-MLKL activation independent of RIPK1. ADAR1-targeting PROTACs unleash ZBP1-dependent PANoptosis by abrogating Z-RNA editing. RIPK1-targeting PROTACs block RIPK1-dependent signaling initiation. MLKL-targeting PROTACs inhibit pore-forming execution by degrading oligomerized MLKL.

Currently, modulation of key proteins involved in necroptosis has emerged as a promising therapeutic strategy. Inhibiting RIPK1 or RIPK3 has been shown to reduce necroptosis-mediated tissue damage in ischemic stroke.²⁰³ The small-molecule compound necrosulfonamide (NSA) can bind to MLKL and block its phosphorylation and activation, inhibiting the occurrence of necroptosis.²⁰⁴ In the field of cancer therapy, strategies that induce necroptosis have shown potential in apoptosis-resistant cancers. For instance, combining second mitochondrial activator of caspases (SMAC) mimetics with caspase-8 inhibitors has been used to induce necroptosis in preclinical models of acute myeloid leukemia (AML).²⁰⁵ Epigenetic regulation of necroptosis-related genes is also being explored as a therapeutic strategy. For example, the histone deacetylase inhibitor chidamide enhances necroptosis in FLT3-ITD positive AML by inhibiting RIPK1.²⁰⁴ In colorectal cancer, necroptosis can trigger an inflammatory cascade that enhances antimicrobial defense, which may be harnessed to boost the immune response against cancer.²⁰⁶ Furthermore, machine learning-based analysis of necroptosis-related gene signatures has identified potential prognostic markers and therapeutic targets in breast cancer.²⁰⁷

Clinical case studies have revealed a significant correlation between MLKL and the development of liver fibrosis. Genetic ablation of MLKL has been shown to markedly reduce liver fibrosis in mice subjected to carbon tetrachloride and bile duct ligation.²⁰⁸ In the HT29 cell model, compound 36 effectively inhibited TSZ (tumor necrosis factor alpha [TNF-α]/SMAC-mimetic/ZVAD-FMK)-induced necroptosis by degrading MLKL, the terminal effector protein of necroptotic signaling. Degradation of MLKL (DC₅₀ = 2.4 μM) directly suppressed its phosphorylation and subsequent membrane pore formation. In contrast, monovalent MLKL ligands, which lack E3 ligase-recruiting capability, failed to exhibit this inhibitory effect.²⁰⁹

PROTAC technology employs a covalent binding strategy to degrade MLKL protein, effectively inhibiting necroptosis. This approach not only diminishes toxicity but also enhances the selectivity and efficacy of therapeutics, offering promising treatment outcomes for associated diseases.²¹⁰ Led by Wang’s team at China Pharmaceutical University, a series of covalent PROTAC degraders targeting MLKL were developed through computer-aided modeling, MD simulations, and structure-activity relationship (SAR) studies. MP-11, a degrader of MLKL, selectively targets Cys86 through covalent binding, achieving potent degradation efficacy with a half-maximal effective concentration (EC₅₀) of 17 nM (nanomolar range). Compared to conventional covalent inhibitors such as TC13172, MP-11 significantly reduces cytotoxicity while maintaining high degradation efficiency. In xenograft models, MP-11 effectively degrades MLKL, demonstrating in vivo safety and highlighting its therapeutic potential for hepatitis, psoriasis, and related diseases.²¹¹ Grohmann et al. developed a novel NanoLuc-targeted protein degradation system (NanoTACs). The study established NanoTACs as a catalytic degradation platform employing CRL4CRBN to target NanoLuc-fused proteins. Successful inhibition of MLKL-mediated necroptosis by NanoTACs underscores their potential for probing necroptotic pathways.²¹²

Another key regulatory protein of necroptosis, RIPK1, promotes activation of the RIPK3/MLKL pathway through its kinase activity. RIPK1-PROTAC (R1-ICR-5) degrades RIPK1, relieving its suppression of the TNFR1 and TLR3/4 signaling pathways and promoting RIPK3 activation and necroptosis. This process enhances the immunostimulatory and anti-tumor effects of radiotherapy (RT) and immune checkpoint blockade (ICB). In a breast cancer model, the combination of R1-ICR-5 and anti-PD-1 achieved an 80% complete response rate, a mechanism that relies on RIPK1 deficiency-induced ICD and increased TNF/interferon γ (IFN-γ) secretion.²¹³ By regulating upstream signaling pathways in necroptosis, studies have revealed the redundant roles of TRAF2 and RIPK1 in the canonical NF-κB signaling pathway mediated by TNFR1 and CD95 death receptors. Complementary studies using TRAF2/RIPK1 double knockout models demonstrated that RIPK1-PROTAC (LD4172) completely abrogates NF-κB signaling via selective RIPK1 degradation, underscoring RIPK1’s pivotal role in inflammatory and antitumor immunity.²¹⁴

Emerging PROTAC technologies, such as Z-PROTAC and C-PROTAC, rapidly advance and offer new approaches to modulate necroptosis by targeting key proteins like ADAR1 and ZBP1. Z-PROTAC leverages the unique left-handed Z-DNA conformation to specifically bind ADAR1 and employs a VHL E3 ubiquitin ligase ligand to induce ADAR1 ubiquitination and degradation. This process releases the inhibition of ZBP1, activating the RIPK3-MLKL pathway.²¹⁵ ZBP1 is a critical protein for sensing pathogens, such as viral Z-DNA/Z-RNA, and its activation triggers the necroptosis signaling cascade involving RIPK3-MLKL phosphorylation, leading to robust inflammatory responses and tissue damage. Traditional methods inhibiting downstream molecules (e.g., RIPK3 or MLKL) are often insufficient for controlling inflammation, whereas directly targeting and degrading ZBP1 can block the upstream signaling more effectively. C-PROTAC, on the other hand, covalently binds to ZBP1 (with a K_d of 2.71 nM) to block virus-induced inflammatory storms. In an H1N1 infection model, C-PROTAC has increased survival rates to 80%.²¹⁶

PROTACs represent an innovative approach to therapeutic intervention through targeted degradation of core necroptotic effectors (MLKL, ZBP1, and RIPK1), enabling precise control of this ICD pathway for treating inflammation-related pathologies and malignancies (Table 6). Future development could focus on PROTACs directed against upstream regulators (RIPK3/ZBP1) to enhance spatial precision. At the same time, strategic combinations with immunotherapy or RT may synergistically augment tumor-specific immune responses by harnessing necroptosis-induced immunogenicity.

Table 6.

Representative PROTACs applied in necroptosis

PROTACs	Targets	E3 ligase	Activity		Mechanism	Diseases	Reference
			DC50	Dmax
Compound 36	MLKL	VHL	HT-29 DC50 = 2.4 μM	>90%	targeted degradation of MLKL blocks TNF-α/SMAC-mimetic/ZVAD-FMK	liver fibrosis	Rathje et al.209
MP-11	MLKL	CRBN	HT-29 DC50 = 12 nM	95.06%	irreversible covalent binding Cys86	hepatitis, psoriasis	Li et al.211
R1-ICR-5	RIPK1	CRBN	–	–	enhances TNF/IFN-γ signaling	breast cancer	Mannion et al.213
LD4172	RIPK1	VHL	DC50: 4–400 nM (cell-dependent)	–	targeted degradation of RIPK1 blocks NF-κB signaling	melanoma (B16F10)	Wagner et al.214; Yu et al.217
Z-PROTAC	ADAR1	VHL	–	–	Z-DNA interacting protein degradation	melanoma (A375 cells)	Wang et al.215
C-PROTAC	ZBP1	VHL	A549 DC50 = 25.69 nM	–	blocks viral Z-RNA sensing	H1N1 infection	Huang et al.216

Applications of PROTACs in apoptosis

Apoptosis is a form of PCD essential for maintaining an organism’s homeostasis and serves as a defense mechanism in pathological conditions. It involves a series of precisely regulated biochemical processes, including nucleation and cytoplasm changes. Characteristic changes in apoptosis include chromatin condensation (pyknosis) and nuclear and DNA fragmentation (karyorrhexis).²¹⁸ Following the initiation of apoptosis, cellular junctions are lost, the cell membrane shrinks, and cytosolic components are organized into apoptotic bodies. The presence of phosphatidylserine on the outer leaflet of the cell membrane marks apoptotic bodies for phagocytic elimination.²¹⁹ Apoptosis has two pathways: intrinsic and extrinsic. The intrinsic pathway is triggered by mitochondrial dysfunction, which releases cytochrome c, activates caspase-9, and initiates the caspase cascade, activating effector caspases. The extrinsic pathway is initiated by binding death ligands to cell surface death receptors, which activate caspase-8 and its downstream caspases. Executioner caspase-3 induces morphological and functional changes in apoptosis by cleaving key cellular proteins and condensing chromatin (Figure 5).^220,221,222

Figure 5.

Molecular mechanisms of apoptosis and PROTAC-based therapeutic targeting

This figure depicts core apoptotic mechanisms and PROTAC interventions targeting key regulatory nodes. (1) Extrinsic pathway: death ligands (TNFα, FASL, and TRAIL) bind receptors (TNFR, FAS, and TRAILR), recruiting adaptors (TRADD/FADD) to activate caspase-8. (2) Intrinsic pathway: cellular stress induces p53 activation, disrupting BCL-2/BAX balance. BAK/BAX oligomerization causes cytochrome c release, forming the APAF1-procaspase-9 apoptosome. (3) Execution phase: activated initiator caspases (−8/−9) cleave effector caspases-3/7, executing apoptosis. (4) PROTAC intervention strategies: BCL-xL-targeting PROTACs degrade anti-apoptotic BCL-xL to antagonize BAK/BAX suppression, promoting cytochrome c-dependent caspase-9 activation and intrinsic apoptosis execution. BCL-2-targeting PROTACs degrade the anti-apoptotic protein BCL-2 to antagonize pro-apoptotic protein sequestration, restoring intrinsic apoptosis execution via caspase-9 activation. MDM2-targeting PROTACs degrade MDM2, stabilizing p53 protein and activating intrinsic apoptosis.

Apoptosis plays a role in various physiological and pathological processes, including normal cell turnover, development and function of the immune system, hormone-dependent atrophy, embryonic development, and chemically induced cell death.²²³ Apoptosis offers new therapeutic insights into various diseases. In chronic lymphocytic leukemia (CLL), apoptosis is often suppressed. The BH3 mimetic navitoclax (ABT-263) was the first orally bioavailable BCL-2/BCL-xL inhibitor, but its clinical utility is limited by BCL-xL inhibition-induced thrombocytopenia. Venetoclax competitively binds to the BH3-binding groove of BCL-2, relieving its inhibition of the pro-apoptotic proteins BAK/BAX and reversing apoptosis resistance in tumor cells.²²⁴ BCL-2-family proteins play a crucial role in regulating apoptosis, including BCL-2, BCL-xL, and MCL1, which inhibit the mitochondrial pathway to prevent apoptosis.^225,226 DT2216 is a BCL-xL-targeting PROTAC that induces apoptosis by simultaneously binding to BCL-xL and the VHL E3 ligase, forming a ternary complex that triggers BCL-xL ubiquitination and subsequent proteasomal degradation. This degradation relieves BCL-xL-mediated inhibition of BAK/BAX, activating the caspase-3-dependent apoptotic pathway. A key advantage of DT2216 lies in its tissue-selective mechanism. While VHL is highly expressed in tumor cells, it is nearly absent in platelets. Consequently, DT2216 selectively degrades BCL-xL in cancer cells while sparing platelets, circumventing the dose-limiting thrombocytopenia commonly associated with navitoclax. This selective degradation profile enhances its therapeutic potential while minimizing off-target toxicity.^227,228

Both 753B and WH244 are heterobifunctional PROTAC degraders that simultaneously target BCL-xL and BCL-2 for degradation, enhancing antitumor activity by overcoming apoptosis inhibition. Similar to DT2216, both compounds recruit the E3 ubiquitin ligase VHL to induce polyubiquitination and subsequent degradation of the target proteins. WH244 replaces the flexible alkyl linker in 753B with a rigid 1,4-dimethylpiperazine moiety, which stabilizes the ternary complex and significantly improves degradation efficacy and selectivity. As a structurally optimized successor of 753B, WH244 demonstrates superior degradation efficiency and reduced off-target effects, making it a promising candidate for therapeutic applications. Notably, WH244 demonstrates potent efficacy in eliminating chemotherapy-induced senescent tumor cells, which may prevent tumor relapse. In preclinical studies, WH244 exhibited superior activity over single-target inhibitors in AML models and synergized with chemotherapeutic agents such as cytarabine. Its dual degradation mechanism not only overcomes resistance to venetoclax (a BCL-2-selective inhibitor) but also mitigates the thrombocytopenia associated with navitoclax, positioning WH244 as a promising next-generation BH3 mimetic.^225,226,228

In cancer treatment, the inactivation of the p53 gene is a common mechanism by which cancer cells evade apoptosis. Nutlin-3, an MDM2 antagonist, binds to MDM2 to block its interaction with p53, stabilizing and activating p53 to induce apoptosis and exert anti-tumor effects.²²⁹ KT-253 is a p53 stabilizer and a PROTAC degrader for MDM2 (DC₅₀ = 0.4 nM). KT-253 demonstrates potent anti-tumor activity in the acute lymphoblastic leukemia (ALL) RS4;11 cell line by inhibiting proliferation (IC₅₀ = 0.3 nM), inducing G2/M phase cell-cycle arrest, and triggering apoptosis.²³⁰ Studies have demonstrated that KT-253 exhibits superior efficacy compared to conventional MDM2 small-molecule inhibitors across multiple hematologic malignancies (AML and ALL) and solid tumor (adenoid cystic carcinoma) models. The MDM2-PROTAC (YX-02-030) molecule specifically recognizes its target protein through its MDM2-binding domain and recruits the E3 ubiquitin ligase via a VHL ligand, forming a stable MDM2-PROTAC-VHL ternary complex. This process induces polyubiquitination of MDM2, leading to its degradation by the 26S proteasome and consequently relieving its inhibitory effect on TAp73, a member of the p53 family. The released TAp73 subsequently undergoes Tyr-99 phosphorylation mediated by c-Abl and acetylation mediated by CBP/p300, acquiring transcriptional activity that upregulates the expression of pro-apoptotic genes such as BAX and PUMA. Ultimately, this cascade activates the caspase pathway, resulting in apoptosis.²³¹ Specifically, ganoderic acid A (GAA)-based PROTACs, such as V10, recruit the E3 ubiquitin ligase VHL to MDM2, facilitating its ubiquitination and proteasomal degradation. In p53 wild-type cells (e.g., MCF-7), degradation of MDM2 could stabilize p53, leading to p21-mediated cell-cycle arrest (G1/S phase) and activation of intrinsic apoptosis via BCL-2/BAX imbalance. In p53-mutant TNBC cells, MDA-MB-231, MDM2 degradation upregulates p21 independent of p53, inhibiting CDK1-cyclin B1 to induce G2/M arrest while concurrently suppressing anti-apoptotic BCL-2 and promoting BAX-dependent mitochondrial apoptosis. Clinically, these PROTACs overcome resistance mechanisms associated with conventional MDM2 inhibitors, demonstrating potent antitumor activity in zebrafish xenograft models with favorable safety profiles, positioning them as promising candidates for TNBC therapy.²³² He et al. designed a series of homo-PROTAC molecules based on the derivative of the MDM2 inhibitor Nutlin-3 (compound 2), aiming to induce autodegradation of MDM2 and treat NSCLC.²³³ Compared with conventional PROTACs, homo-PROTACs achieve self-degradation by recruiting MDM2 itself as an E3 ubiquitin ligase, circumventing the off-target risks associated with introducing exogenous E3 ligases. Among these, compound 11a and its enantiomer, 11a-1, exhibited excellent MDM2 binding affinity and degradation efficiency. In A549 lung cancer cells, compound 11a induced proteasome-dependent MDM2 degradation (DC₅₀ = 1.01 μmol/L), upregulated p53 expression, and inhibited cell proliferation (IC₅₀ = 1.0 μmol/L). In an A549 xenograft mouse model, compound 11a-1 achieved 52.4% tumor growth inhibition (TGI) at a dose of 30 mg/kg with good tolerability. This work addresses the dose-limiting toxicity of traditional MDM2 inhibitors and provides a novel strategy for the treatment of NSCLC and other p53 wild-type tumors.

PROTACs effectively induce tumor cell apoptosis by targeting the degradation of anti-apoptotic proteins (such as BCL-2 family members or MDM2) (Table 7). Despite their promise, PROTACs face pharmacological hurdles similar to those of other protein degraders, including restricted blood-brain barrier penetration and limited tissue-specific delivery.

Table 7.

Representative PROTACs applied in apoptosis

PROTACs	Targets	E3 ligase	Activity		Mechanism	Diseases	Reference
			DC50	Dmax
DT2216	BCL-xL	VHL	MOLT-4 DC50 = 63 nM	90.8%	selectively degrade BCL-xL	solid tumors	Khan et al.227
WH244	BCL-xL/BCL-2	VHL	Jurkat DC50 (BCL-xL) = 0.6 nM DC50 (BCL-2) = 7.4 nM	–	rigid linker and bridged morpholine enhance ternary complex stability	acute myeloid leukemia	Jia et al.225
753B	BCL-xL/BCL-2	VHL	Jurkat DC50 (BCL-xL) = 3.7 nM DC50 (BCL-2) = 50 nM KG-1 (AML) DC50 (BCL-xL) = 0.01 μM DC50 (BCL-2) = 0.06 μM	–	spares platelets, avoiding thrombocytopenia	acute myeloid leukemia	Nayak et al.225; Jia et al.226
KT-253	MDM2	CRBN	RS4;11 DC50 = 0.4 nM	>95%	stabilizes p53, induces G2/M arrest	hematologic cancers, solid tumors	Chutake et al.230
YX-02-030	MDM2	VHL	–	–	activates p53/TAp73 pathway	p53-inactivated triple-negative breast cancer	Adams et al.231
V10	MDM2	VHL	–	–	stabilizes p53, induces p21-mediated cell-cycle arrest	triple-negative breast cancer	Li et al.232
Homo-PROTAC	MDM2	MDM2	A549 DC50 = 1.01 μM	>95%	induces MDM2 self-degradation, stabilizes p53, induces p21-mediated cell-cycle arrest	non-small cell lung cancer	He et al.233

Applications of PROTACs in autophagic cell death

Autophagic cell death is a form of PCD that depends on the autophagy mechanism. Its defining feature is that cell death must be initiated or executed by autophagic activity rather than merely occurring alongside it.²³⁴ Autophagy is an evolutionarily conserved cellular self-degradation process whereby damaged or superfluous proteins, organelles, and pathogens are selectively targeted for lysosomal degradation. This essential quality control mechanism maintains cellular homeostasis, facilitates adaptation to environmental stressors, and participates in diverse physiological and pathological processes. Based on distinct degradation mechanisms, autophagy can be categorized into three main types: macroautophagy, microautophagy, and chaperone-mediated autophagy.^235,236

Initiation of autophagy is marked by the formation of the autophagosome, a process primarily dependent on the ULK1 complex, which includes ULK1, ATG13, FIP200, and ATG101. Under nutrient-rich conditions, mTORC1 binds to ULK1, inhibiting its kinase activity and suppressing autophagy. Autophagy is regulated by a complex interplay of signaling pathways, with the mechanistic target of rapamycin (mTOR) pathway as a central negative regulator and AMP-activated protein kinase (AMPK) as a positive regulator under energy stress conditions (Figure 6).^237,238

Figure 6.

Molecular mechanisms of autophagy and PROTAC-based therapeutic targeting

This diagram systematically delineates reciprocal regulation of EGFR signaling and autophagy pathways alongside PROTAC-based targeting strategies. (1) Ligand-bound EGFR initiates downstream PI3K-AKT-mTORC1 signaling, consequently suppressing autophagy initiation. (2) Inhibition of mTORC1 results in activation of the ULK1-FIP200-ATG13 complex, triggering autophagy induction. (3) The VPS34/Beclin-1 complex produces PI3P, which enables phagophore membrane nucleation. (4) LC3 lipidation progresses from LC3-I to LC3-II via the ATG5-ATG12-ATG16L1 ubiquitin-like system, facilitating autophagosome maturation. (5) Mature autophagosomes undergo fusion with lysosomes to generate autolysosomes, executing cargo degradation and nutrient recycling. (6) PROTAC intervention strategies: EGFR-targeting PROTACs degrade oncogenic EGFR to block downstream PI3K-AKT/mTOR and RAF-MEK-ERK signaling cascades, suppressing uncontrolled proliferation and metastasis in tyrosine kinase inhibitor-resistant cancers. VPS34-targeting PROTACs degrade the VPS34/Beclin-1 complex to abrogate PI3P production, consequently inhibiting phagophore nucleation and autophagic vesicle maturation in inflammation-driven pathologies.

Autophagy holds significant therapeutic potential in disease treatment. In cancer therapy, autophagy exhibits a dual role; it suppresses early-stage tumors by clearing oncogenic proteins but may also promote the survival of advanced tumors. Targeting the RAS-induced non-canonical autophagy via ATG8ylation (RINCAA) pathway or PI4KB phosphorylation can specifically inhibit RAS-mutated tumors.^239,240,241 Autophagy is central to the pathogenesis of neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis.²⁴² Autophagy facilitates the clearance of β-amyloid plaques and neurofibrillary tau tangles, slowing Alzheimer’s disease progression.²⁴³ For cardiovascular diseases, moderate autophagy protects cardiomyocytes, though its regulation must be stage specific.^244,245 In infectious diseases, autophagy enhances host defense by degrading pathogens and modulating immune responses, though certain pathogens evade this mechanism.²⁴⁶

From a clinical perspective, PROTAC technology’s high specificity and rapid degradation capability make it a potential tool for studying autophagy-related diseases. VPS34, a class III phosphatidylinositol 3-kinase (PI3K), is involved in the generation of phosphatidylinositol 3-phosphate (PtdIns3P) on endoplasmic reticulum membranes, which is crucial for autophagosome formation. HaloPROTAC-E, a specific PROTAC molecule belonging to the HaloPROTAC family, is designed to rapidly degrade VPS34. It covalently binds to HaloTag-fused proteins via a halogen tag and recruits E3 ligases to induce targeted protein degradation. The incorporation of halogen atoms enhances binding affinity and improves cellular permeability. Additionally, the halogen tag optimizes PROTAC stability and catalytic efficiency while minimizing off-target effects. HaloPROTAC-E not only efficiently degrades Halo-tagged VPS34 but also affects its regulatory subunits, including VPS15, Beclin1, ATG14, and UVRAG. This degradation may inhibit autophagosome formation and, consequently, impact cellular autophagy function.²⁴⁷

The autophagy pathway offers PROTAC drugs an additional degradation route, which is particularly important for degrading proteins that are difficult to target via the UPS.²⁴⁸ SIAIS125 and SIAIS126 degrade EGFR mutants (L858R+T790M) by simultaneously engaging both the UPS and autophagy-lysosomal pathways. These PROTACs, composed of the EGFR inhibitor canertinib and the CRBN ligand pomalidomide, form an EGFR-PROTAC-CRBN ternary complex that induces EGFR ubiquitination for proteasomal degradation. Concurrently, a subset of ubiquitinated EGFR is recognized by the autophagy receptor p62/SQSTM1 and degraded via LC3-II-mediated autophagosome formation and subsequent lysosomal processing, potentially through mTOR inhibition or AMPK activation. The optimized linker design ensures selective degradation of mutant EGFR while sparing the wild-type protein. This innovative dual-pathway degradation approach significantly broadens the spectrum of targetable substrates, encompassing challenging targets such as membrane-associated and aggregated proteins, while effectively circumventing the therapeutic resistance commonly associated with conventional single-target kinase inhibitors.^249,250

Structural refinement of the PROTAC molecules MS9449 and MS9427 confers mutant-selective EGFR Del19/L858R degradation through enhanced formation of productive ternary complexes with E3 ubiquitin ligases. The structural divergence between mutant and wild-type EGFR accounts for differential ubiquitination efficiency, with oncogenic variants demonstrating greater conformational adaptability for PROTAC-mediated degradation. Significantly, pharmacological inhibition of PI3K signaling overcomes the degradation resistance of wild-type EGFR, suggesting a combinatorial approach for comprehensive EGFR targeting. The molecular basis for this selectivity lies in mutant-specific protein conformations that facilitate optimal engagement with both the PROTAC molecule and downstream autophagy-associated ubiquitination machinery.²⁵¹

The therapeutic scope of PROTAC-mediated protein degradation has transcended cancer to address neurodegenerative pathologies characterized by protein aggregation. In Alzheimer’s disease models, aberrant tau accumulation has been shown to compromise autophagosome biogenesis through dysregulation of the TIA1-amino acid-mTORC1 signaling cascade, exacerbating proteostatic imbalance. The rationally designed PROTAC C004019 exhibits multimodal functionality by simultaneously targeting both α-synuclein and tau for ubiquitin-dependent degradation while restoring autophagic capacity, effectively breaking the cycle of pathological protein accumulation. This dual-pathway intervention paradigm provides a transformative strategy for neurodegenerative disorders where conventional pharmacology has failed, particularly for targets lacking traditional binding pockets.^252,253

Table 8 summarizes representative applications of PROTAC-based strategies in therapeutic contexts involving autophagy modulation. PROTAC-mediated targeting of autophagy pathways remains constrained by several fundamental limitations. The predominant reliance on UPS activation provides insufficient engagement of autophagic degradation machinery, particularly for clearing amyloidogenic protein aggregates or damaged organelles. Target scope restrictions become apparent with membrane-associated proteins, higher-order protein assemblies, and non-protein biomolecules, often mandating synergistic use with autophagy-specific degraders (AUTACs/ATTECs).²⁵⁵ The field’s current dependence on a limited subset of E3 ligases (CRBN/VHL) introduces potential vulnerabilities through tissue-specific expression variability and evolving resistance mechanisms. Strategic modifications addressing these limitations could significantly expand PROTAC utility in autophagy-modulated disease contexts.

Table 8.

Representative PROTACs applied in autophagy

PROTACs	Targets	E3 ligase	Activity		Mechanism	Diseases	Reference
			DC50	Dmax
HaloPROTAC-E	VPS34	HaloTag-VHL	293T DC50 = 21.6 nM	77%	degrades PI3K complex	neurodegenerative diseases	Grohmann et al.212; Tovell et al.247
SIAIS125	EGFRL858R+T790M/EGFREx19del	CRBN	H1975 DC50(EGFRL858R+T790M): 30–50 nM PC9 DC50 (EGFREx19del) = 100 nM	>95% 40%	dual UPS/autophagy degradation	non-small cell lung cancer with EGFR TKI resistance	Qu et al.249
SIAIS126	EGFRL858R+T790M/EGFREx19del	CRBN	H1975 DC50(EGFRL858R+T790M) < 30 nM PC9 DC50(EGFREx19del): 30–100 nM	>95%	dual UPS/autophagy degradation	non-small cell lung cancer with EGFR TKI resistance	Qu et al.249
MS9449(31)	EGFRL858R	VHL	HCC-827 DC50 = 7.1 nM	–	dual UPS/autophagy degradation	non-small cell lung cancer (EGFR-mutant)	Yu et al.251
MS9427(72)	EGFRL858R	CRBN	HCC-827 DC50 = 82 nM	–	dual UPS/autophagy degradation	non-small cell lung cancer (EGFR-mutant)	Yu et al.251
C004019	α-synuclein/tau	VHL	–	–	restores autophagic flux	Alzheimer’s disease	Wang et al.254

Applications of PROTACs in cuproptosis

Cuproptosis, first defined in 2022 by Tsvetkov et al., is a novel form of PCD directly triggered by excessive copper ions (Cu²⁺/Cu⁺).²⁵⁶ It is characterized by copper dependence, mitochondrial targeting, and a non-apoptotic nature. Unlike established cell death pathways, such as apoptosis or pyroptosis, cuproptosis strictly requires mitochondrial respiratory activity and can be inhibited solely by copper chelators.^257,258

Cuproptosis is driven by mitochondrial metabolic dysfunction initiated through the specific binding of copper ions to lipoylated enzymes such as DLAT in the tricarboxylic acid (TCA) cycle. This interaction induces abnormal oligomerization of lipoylated proteins and simultaneously promotes the degradation of iron-sulfur cluster proteins like FDX1.²⁵⁹ This process exerts a dual effect; the aggregation of lipoylated proteins directly disrupts the continuous operation of the TCA cycle, while the loss of iron-sulfur clusters further exacerbates mitochondrial oxidative phosphorylation defects, ultimately leading to cell death via irreversible proteotoxic stress. Mechanistically, FDX1 acts as a pivotal regulator by reducing Cu²⁺ to Cu⁺, which facilitates copper binding to lipoylated targets, whereas GSH antagonizes this process via copper chelation (Figure 7).^260,261

Figure 7.

Molecular mechanisms of cuproptosis and PROTAC-based therapeutic targeting

This figure depicts core cuproptosis mechanisms and PROTACs interventions targeting key regulatory nodes. (1) Copper ions (Cu²⁺) are taken up by SLC31A1 and effluxed via ATP7A/B to regulate intracellular copper homeostasis. (2) Mitochondrial collapse: Cu²⁺ inactivates FDX1, disrupting the TCA cycle, generating ROS via copper-dependent redox reactions, depleting GSH. (3) DLAT aggregation: Copper binds lipoylated DLAT, inducing its oligomerization, triggering proteotoxic stress. (4) p53-MDM2 axis: nuclear MDM2 constitutively ubiquitinates p53 for degradation, suppressing tumor surveillance. (5) PROTAC intervention strategies: MD-224 (CRBN-based PROTAC) degrades MDM2 to stabilize p53, which transcriptionally represses SLC7A11, amplifying copper accumulation and cuproptosis.

Targeted cuproptosis therapy has emerged as a transformative approach with broad therapeutic implications spanning genetic metabolic disorders, neurodegenerative pathologies, and oncological interventions. In oncology, copper ionophores (e.g., elesclomol) and disulfiram-copper complexes (DSF-Cu) directly trigger cuproptosis by inducing mitochondrial lipoylated protein (e.g., DLAT) aggregation and iron-sulfur cluster protein degradation.^262,263 Acid-responsive nanocarriers (e.g., CuO NPs) and multifunctional platforms (e.g., ES@CuO) enhance tumor-specific copper accumulation through targeted delivery, while their synergy with immunotherapies (PD-1 inhibitors or CAR-T cells) activates antitumor immunity by remodeling the immunosuppressive microenvironment.^264,265 For inherited metabolic disorders like Wilson’s disease, copper chelators and zinc agents alleviate hepatic/neurological damage by restoring copper homeostasis. In neurodegenerative diseases, copper modulators (e.g., PBT2) and delivery agents (e.g., CuII(atsm)) mitigate Aβ deposition or neuronal degeneration by balancing cerebral copper levels.²⁶⁶ Current challenges involve developing precision copper-targeted delivery systems and specific biomarkers, while future directions emphasize combination therapies (e.g., cuproptosis and immunotherapy/RT) and personalized strategies for regulating copper metabolism to overcome therapeutic resistance and toxicity limitations.²⁶⁷

Wang et al. innovatively developed a PROTAC-based cuproptosis sensitizer (CuS-MD@CS) that enhances tumor cell sensitivity to copper-dependent cell death through coordinated MDM2 degradation and metabolic reprogramming.²⁶⁸ The PROTAC component MD-224 induces MDM2 ubiquitination and subsequent degradation via CRBN E3 ligase recruitment, liberating p53 from suppression and activating its transcriptional regulatory functions. Activated p53 mediates a profound metabolic shift by transcriptionally repressing glucose transporter 1 (GLUT1) and SLC7A11, which concurrently inhibits glycolysis, depletes intracellular GSH reserves, and promotes glutaminolysis to generate α-ketoglutarate (α-KG), which fuels mitochondrial TCA cycle activity. This metabolic reprogramming creates conditions favorable for cuproptosis induction, as GSH depletion increases the bioavailability of copper ions, while enhanced mitochondrial respiration facilitates copper binding to lipoylated proteins, such as DLAT. This ultimately triggers pathological protein oligomerization and degradation of iron-sulfur cluster proteins. The nanoplatform employs copper sulfide nanoparticles loaded with MD-224 and surface-functionalized with chondroitin sulfate (CS) to achieve CD44 receptor-mediated tumor targeting. Under the acidic conditions of the TME (∼pH 5.6) and near-infrared laser irradiation, the system exhibits controlled synchronous release of both copper ions and MD-224, enabling spatiotemporal coordination of cuproptosis induction and Fenton reaction-mediated apoptosis. This integrated approach represents a significant advancement in targeted cancer therapy, combining PROTAC technology with metabolic modulation to overcome treatment resistance in aggressive malignancies.

Although PROTACs have not yet been extensively applied in cuproptosis-related therapies, their potential in this emerging field is highly promising. The catalytic degradation mechanism of PROTACs can synergize with copper-dependent cell death pathways while circumventing systemic copper toxicity. Future research directions may include (1) designing bifunctional PROTACs capable of concurrently degrading oncoproteins and modulating copper metabolism or (2) combining PROTAC-mediated protein degradation with copper ionophores (e.g., elesclomol) to amplify tumor-selective cuproptosis. As mechanistic studies on both PROTAC technology and cuproptosis advance, their strategic integration is expected to pave the way for novel therapeutic avenues for copper-dysregulated cancers and other diseases.

Molecular mechanisms and clinical potential of PROTACs in targeting multiple PCD pathways

The interplay between PCD pathways and metabolic remodeling represents a central node in tumor progression and therapeutic response. PROTAC technology, leveraging its unique event-driven catalytic degradation mechanism, transcends the limitations of conventional single-target inhibition and provides an unprecedented tool for systematically intervening in these complex cell death networks. This chapter explores how PROTACs facilitate multi-pathway cross-activation and metabolic intervention, induce ICD, and reprogram the tumor immune microenvironment. Furthermore, it discusses how these actions integrate targeted therapy, immune modulation, and intelligent nano-delivery into synergistic strategies, bridging their substantial potential from molecular mechanisms to clinical translation.

Multi-pathway cross-activation and metabolic intervention mechanisms

Although ferroptosis, pyroptosis, and apoptosis are distinct PCD pathways, they do not operate in isolation within the cell. Instead, they form an intricate signaling network characterized by extensive crosstalk. This interplay forms the basis for achieving synergistic therapeutic effects.²⁶⁹

Key molecular nodes act as “switches” between different cell death pathways. For example, caspase-3 and GSDME exemplify this role; in the canonical regulatory pathway, activated caspase-3 is the core executor of apoptosis, initiating the programmed apoptotic process through the specific cleavage of downstream apoptotic substrates, a hallmark event of apoptosis. However, in cells with high expression of GSDME, activated caspase-3 undergoes a switch in substrate specificity. It cleaves GSDME within its linker region, releasing the N-terminal domain. This domain oligomerizes and inserts into the plasma membrane to form non-selective pores, compromising membrane integrity and ultimately “reprogramming” the apoptotic signal into pyroptosis.²⁷⁰ Similarly, core regulatory proteins like p53 exhibit functional pleiotropy. Under specific contexts like DNA damage stress, p53 can initiate apoptosis by transcriptionally activating pro-apoptotic genes such as PUMA and NOXA.²⁷¹ In other specific contexts, such as oxidative stress or metabolic disturbances, its function depends on post-translational modifications and the intracellular microenvironment. Under these conditions, p53 can promote ferroptosis by inhibiting SLC7A11 expression or activating ALOX12-mediated lipid peroxidation, acting as a crucial “molecular switch” in determining cellular fate.²⁷² This cross-talk between pathways suggests the potential for PROTACs to overcome the limitations of targeting a single pathway.

Metabolic stress serves as a critical bridge connecting distinct cell death pathways. For example, mitochondrial damage, a key event in apoptosis, directly causes dysfunction of the electron transport chain, leading to a burst of ROS. Excessive ROS accumulation is a core driver of ferroptosis, as it not only exacerbates chain reactions of lipid peroxidation but also depletes GSH and inhibits GPX4 activity, promoting ferroptotic death.²⁷³ Consequently, apoptosis-inducing therapies targeting mitochondrial injury often simultaneously enhance cellular susceptibility to ferroptosis through ROS-mediated mechanisms. Furthermore, lipid peroxidation products generated during ferroptosis can attack mitochondrial membrane phospholipids, disrupt membrane potential, and impair respiratory chain function. This establishes a vicious cycle of “lipid peroxidation-mitochondrial damage, ” amplifying apoptotic signals (such as cytochrome c release and caspase activation).²⁷⁴ Such metabolic cross-activation interlinks apoptosis and ferroptosis, enabling mutual reinforcement and forming a complex metabolism-dependent network in cell fate regulation.

Finally, this interplay holds significant therapeutic implications, particularly for PROTACs. By degrading specific target proteins, PROTACs can precisely disrupt intracellular homeostasis. For instance, the PROTAC molecule DeFer-2 has been shown to degrade ferritin, leading to iron overload and increased sensitivity to ferroptosis; however, it unexpectedly activated caspase-3/GSDME-mediated pyroptosis.¹⁸⁴ This demonstrates the capability of PROTACs to achieve “switching” between cell death pathways. Such synergistic and switching effects not only enhance tumor cell killing but also activate the immune system through mechanisms like pyroptosis, yielding a “1 + 1 > 2” therapeutic outcome. In summary, understanding and leveraging these interactions, especially via precise interventions using PROTACs, opens new avenues for developing more effective combination cancer therapies.

Immunogenic cell death and remodeling of the immune microenvironment

The capacity of PROTAC technology to reshape the tumor immune microenvironment extends beyond its ability to induce Immunogenic cell death (ICD). It also manifests in its capacity to degrade key immune regulatory proteins, activating anti-tumor immunity through multidimensional mechanisms. ICD is a form of regulated cell death that can potently activate adaptive immunity. Its core mechanism involves the release of DAMPs during the cell death process. These DAMPs consist of pre-stored canonical DAMPs (cDAMPs), such as calreticulin (CRT) and ATP, as well as inducible DAMPs (iDAMPs) that are de novo synthesized and induced through the NF-κB and IFN signaling pathways. These molecules promote dendritic cell antigen presentation and cross-priming of CD8⁺ T cells, eliciting an anti-tumor immune response. Unlike apoptosis, ICD depends on RIPK3-mediated phosphorylation and oligomerization of MLKL, leading to plasma membrane rupture and content release. This process occurs without caspase-mediated suppression of immune signaling, thus possessing stronger immunostimulatory potential.^275,276

Nanoparticle assemblies of PROTACs targeting indoleamine dioxygenase (IDO) degrade IDO protein, blocking the conversion of tryptophan to kynurenine. This relieves the suppression on dendritic cells and cytotoxic T cells while reducing regulatory T cell activation. When combined with photodynamic therapy (PDT), these PROTACs are specifically activated at the tumor site, generating ROS to induce ICD while continuously degrading IDO to prevent adaptive immune resistance. This dual action leads to DAMP release, promoting dendritic cell (DC) phagocytosis, antigen presentation, subsequent CD8⁺ T cell activation, and regulatory T cell (Treg) suppression. Transcriptome analysis revealed that PROTAC treatment enriches antigen processing and presentation pathways and downregulates immunosuppressive signals like transforming growth factor β (TGF-β), collectively reshaping the immunosuppressive TME into an immune-permissive state.²⁷⁷ Similarly, PROTACs targeting protein tyrosine phosphatase 1B (PTP1B) can specifically degrade PTP1B. This degradation indirectly downregulates the expression of immune checkpoint molecules such as PD-1 and Tim-3, reverses T cell exhaustion, and ultimately enhances anti-tumor immunity. Concurrently, the combination of PDT and PROTACs induces potent ICD, overcoming apoptosis resistance in glioblastoma multiforme (GBM).²⁷⁸ Furthermore, BET-targeting PROTAC degraders induce endoplasmic reticulum stress, triggering the transcription factor CHOP to activate death receptor 5 (DR5) expression, initiating ICD. DR5 activation then initiates extrinsic apoptosis via the FADD-caspase-8 axis and amplifies intrinsic apoptosis through the Bid-BAX-mitochondrial pathway, forming an ER stress-CHOP-DR5 signaling axis that synergizes apoptosis with immune activation.²⁷⁹ Self-delivering nano-PROTAC systems, formed by self-assembly of the chemotherapeutic drug doxorubicin (DOX) and a BRD4-targeting PROTAC degrader, exemplify synergistic action. The PROTAC degrades BRD4 to enhance ICD immunogenicity, downregulates c-Myc to suppress glycolysis, and downregulates PD-L1 to reverse immune evasion. DOX directly induces apoptosis and DAMP release. Together, they synergistically amplify anti-tumor immunity and inhibit primary and metastatic colorectal cancer progression.²⁸⁰ Additionally, PROTACs targeting pyruvate dehydrogenase kinase 1 (PDK1) reverse the Warburg effect, reduce lactate accumulation, alleviate microenvironmental acidosis, and activate oxidative phosphorylation (OXPHOS), inducing tumor cell apoptosis and ICD with concomitant DAMP release.²⁸¹

The aforementioned examples, including HK2-PROTAC, demonstrate that PROTAC technology regulates the tumor immune microenvironment through a multidimensional synergistic network. It can degrade key immunomodulatory proteins to activate anti-tumor immune responses; induce ICD, releasing DAMPs to promote dendritic cell maturation and T cell activation; and target immunosuppressive pathways to reverse TME suppression. Its synergistic effects with modalities like PDT inhibit tumor growth and remodel the TME into an immune-permissive state, offering a novel, precise strategy for cancer immunotherapy.

Synergistic therapeutic strategies in translational applications

PROTAC technology demonstrates synergistic potential beyond monotherapies through deep integration with existing treatment modalities, opening new avenues for cancer therapy. In the context of combination with targeted therapies, the oral estrogen receptor degrader vepdegestrant (ARV-471) achieves more thorough and durable ER degradation compared to the current selective estrogen receptor degrader (SERD) fulvestrant (≥90% vs. 50%–60%). Not only does it demonstrate favorable tolerability as a single agent in patients with advanced breast cancer, but, when combined with CDK4/6 inhibitors such as palbociclib, mTOR inhibitors like everolimus, or PI3K inhibitors including alpelisib, it induces tumor regression in preclinical models. Analysis using the Bliss synergy model further validates the synergistic effects of these combination regimens, providing mechanistic rationale for PROTACs as foundational endocrine therapy alongside multi-pathway targeted agents.⁷⁵ Furthermore, nicotinamide phosphoribosyltransferase (NAMPT), a key enzyme in NAD⁺ synthesis within tumor cell energy metabolism, is a metabolic target of considerable interest. However, therapeutic strategies targeting NAMPT (including inhibitors and PROTACs) are associated with substantial toxicity due to concurrent disruption of energy supply in normal cells, limiting their clinical application. An innovative approach involves using an NAMPT-PROTAC to degrade this protein and disrupt the NAD⁺ salvage synthesis pathway in tumor cells while co-administering nicotinic acid (vitamin B3) to provide normal cells with an alternative NAD⁺ synthesis route via the Preiss-Handler pathway. This combination produces a synergistic effect characterized by synthetic lethality in tumor cells with NAPRT gene defects that prevent effective utilization of nicotinic acid. The combination strategy enables selective tumor cell killing while protecting healthy cells capable of using the alternative pathway, significantly expanding the therapeutic safety window.

In the realm of immunotherapy synergy, PROTAC technology shows promise in transitioning from intracellular regulation to microenvironment remodeling. For instance, the epigenetic factor bromodomain PHD finger transcription factor (BPTF), functioning as a crucial epigenetic reader, is overexpressed in various cancers (including liver, lung, and breast cancer) and is associated with tumor progression and poor prognosis. The BPTF-targeting PROTAC molecule 8d enhances natural killer (NK) cell-mediated killing in hepatocellular carcinoma by degrading BPTF, suppressing its downstream target heparanase (HPSE), which alleviates the inhibition of NK cell immune activity. This approach transcends the conventional scope of PROTACs focused solely on direct tumor cell killing, reveals the BPTF-HPSE axis as a novel mechanism of immune evasion in HCC, and provides a new target for immunotherapy.²⁸² On the other hand, AbTAC technology leverages antibody-mediated target engagement to degrade cell-surface proteins by bridging them to E3 ligases, expanding PROTAC target space beyond the intracellular proteome. Although traditional PROTACs can degrade intracellular proteins, their action depends on small-molecule ligands binding to intracellular domains of target proteins, rendering them ineffective against membrane proteins lacking such ligands (e.g., PD-L1). Given that membrane proteins constitute approximately 23% of encoded genes and are targeted by 70% of FDA-approved drugs, novel degradation strategies are urgently needed. AbTACs address this by employing bispecific antibodies that simultaneously engage a membrane-bound E3 ligase (e.g., RNF43) and a cell surface target protein (e.g., PD-L1), achieving degradation of cell-surface proteins without requiring small molecule ligands, expanding the applicability of PROTACs. Its fully recombinant nature, high specificity, and reversible degradation characteristics establish a new paradigm for targeting membrane proteins.^138,283

To achieve more precise and efficient drug delivery, various delivery systems have been developed to enhance the tumor targeting and safety of PROTACs. Among these, the antibody-PROTAC conjugate (Ab-PROTAC) developed by Maneiro et al. conjugates a HER2-targeting monoclonal antibody (trastuzumab) with a BRD4 degrader (derived from MZ1) via a cleavable linker, enabling HER2-dependent targeted protein degradation. This conjugate undergoes specific internalization in HER2-positive breast cancer cells, where it is activated in lysosomes to release active PROTACs, successfully inducing proteasomal degradation of BRD4. In contrast, no response was observed in HER2-negative cells. This strategy leverages specific antibody-antigen binding to minimize off-target toxicity, achieving precise delivery of PROTACs.²⁸⁴ As mentioned previously, a lipid nanodisc (LND)-based nano-delivery system was developed, which employs a dual strategy combining a prodrug design with an LND carrier to systematically address the core challenges in the clinical translation of PROTACs. The prodrug design involves chemical modification to block PROTAC activity, ensuring activation only within the high-GSH environment of tumor cells, significantly reducing systemic toxicity. The LND carrier substantially improves water solubility and tumor targeting, achieving a high drug loading capacity of 48% (compared to <15% for traditional carriers). This system utilizes a disulfide bond-responsive mechanism for spatiotemporally controlled release, and when combined with the LND’s unique deep tumor penetration capability (1,600 μm) and prolonged retention properties (>24 h), it synergistically enhances therapeutic efficacy. This platform is highly versatile and can be extended to other PROTAC molecules or hydrophobic drugs, providing an efficient, safe, and scalable solution for the clinical application of targeted protein degradation technologies.²⁸⁵

In prostate cancer therapy, a prostate-specific membrane antigen (PSMA)-targeted self-assembling nano-PROTAC has successfully achieved dual degradation of the AR and its molecular chaperone, HSP90, overcoming resistance in CRPC. This strategy is applicable to PSMA-positive patients resistant to enzalutamide, offering an efficient and low-toxicity therapeutic approach for CRPC.²⁸⁶ Furthermore, a novel self-assembling, dual-targeting nano-platform (berberine–hypericin–PROTAC [BHP] NPs) was designed for TNBC treatment by combining PDT with PROTAC-mediated metabolic starvation in a synergistic manner. Formed via the self-assembly of natural products without requiring additional carriers, this platform achieves an exceptionally high drug loading capacity of 92%. Based on its dual targeting to mitochondria and necrotic regions, it significantly enhances tumor penetration depth and therapeutic outcome. Its dual-targeting capability and deep penetration properties overcome the delivery limitations of traditional nanomedicines, showing promise for promoting the clinical translation of PROTAC-PDT combination therapy.²⁸⁷ Additionally, a photo-responsive nano-PROTAC (NanoTAC) also integrates PDT with PROTAC technology. It achieves high drug loading (78.5%) via supramolecular self-assembly, avoiding the toxicity associated with traditional nanocarriers. This system successfully overcomes metabolic barriers in the TME and enables efficient pyroptosis-mediated immune activation. In TNBC models, it demonstrated significant capabilities in inhibiting primary tumor growth, preventing metastasis, and inducing immune memory, providing an innovative solution for the immunotherapy of refractory cancers.²⁸⁸

Leveraging its unique event-driven catalytic degradation mechanism, PROTAC technology is progressively expanding its application depth and breadth in cancer therapy through multidimensional integration with targeted therapies, immune modulation, and advanced nano-delivery systems. From precise degradation of pathogenic proteins to synergistic activation of the immune microenvironment and spatiotemporally controlled drug release enabled by intelligent carriers, PROTACs represent a new generation of protein degradation strategies that are driving the evolution of cancer treatment paradigms toward greater efficacy and safety. This progression is providing sustained momentum for the future clinical implementation of precision oncology.

Challenges and future perspectives

PROTAC technology, as an emerging protein degradation strategy, has demonstrated significant potential in the study of PCD. By leveraging the cellular UPS, PROTAC technology can specifically degrade target proteins, modulating processes such as ferroptosis, pyroptosis, necroptosis, apoptosis, autophagic cell death, and cuproptosis. Currently, PROTAC technology has made remarkable progress in various fields, including targeting key proteins such as BCL-2 family proteins, GPX4, GSDME, MDM2, MLKL, RIPK1, and VPS34, showing promising applications in cancer treatment, neurodegenerative diseases, and autoimmune diseases.^38,289,290

In clinical applications, PROTAC technology offers several unique advantages. First and foremost, PROTACs can target proteins traditionally considered undruggable, such as transcription factors and scaffold proteins, significantly expanding the range of druggable targets. For instance, by degrading NRs like AR and ER, PROTACs can overcome the drug resistance often arising from target mutations or overexpression, a common issue with conventional inhibitors. Moreover, unlike traditional small-molecule inhibitors that merely inhibit the function of target proteins, PROTACs achieve a more complete blockade of signaling pathways by degrading the target proteins themselves, reducing the likelihood of drug resistance. Additionally, the “event-driven” mechanism of PROTACs allows them to be effective at lower concentrations, minimizing the toxicity risks associated with prolonged exposure to high drug concentrations.

PROTAC technology currently faces several significant challenges.^{291,292,293,294} Although developed to overcome resistance associated with conventional inhibitors, its clinical application may inadvertently give rise to novel resistance mechanisms. Downregulation or mutation of E3 ligases is a key mechanism contributing to resistance against PROTACs. In the phase III VERITAC-2 trial (ClinicalTrials.gov: NCT05654623) of the ER-targeting PROTAC ARV-471, while a significant extension in median PFS was observed in the ESR1-mutant subgroup (5.0 months vs. 2.1 months with fulvestrant), the intent-to-treat population did not reach statistical significance (median PFS 3.7 months vs. 3.6 months).¹³⁴ This outcome suggests that resistance may involve mechanisms beyond E3 ligase dysfunction, such as alterations in proteasome activity or mutations within the target protein itself that impair PROTAC binding and subsequent degradation. Furthermore, tumor cells may evade PROTAC-induced cell death by activating compensatory survival signaling pathways or enhancing drug efflux mechanisms.^295,296,297 PROTAC technology advances through iterative design strategies that recruit diverse E3 ubiquitin ligases to enhance degradation efficiency and overcome resistance. For instance, dual-ligand PROTACs simultaneously engaging multiple E3 ligases have demonstrated up to 10-fold higher degradation efficiency than monovalent counterparts by forming extended ternary complexes.²⁹⁸ This innovation proves critical for targeting drug-resistant mutants, as exemplified by CDK6-selective PROTACs that restore sensitivity to immunomodulatory drugs in lenalidomide-resistant MM cells.²⁹⁹ To further augment therapeutic efficacy, PROTACs are rationally combined with proteasome inhibitors. Notably, BET-PROTACs have shown synergistic anti-MM activity when combined with bortezomib, overcoming resistance to conventional proteasome inhibitor therapy.³⁰⁰

Many PROTACs exhibit inherent pharmacokinetic limitations that restrict their clinical application, primarily due to their typically large molecular weight (often exceeding 700 Da), high polarity, and strong hydrophobicity. These properties hinder effective cellular membrane penetration via passive diffusion, leading to rapid systemic clearance and manifesting as low oral bioavailability, poor cell membrane permeability, and suboptimal tissue distribution.^301,302 Nanoparticle delivery systems (e.g., liposomes, gold nanoparticles, exosomes) provide efficient solutions for PROTAC delivery. PEG-modified liposomes prolong circulation time and enhance tumor targeting, while exosomes demonstrate superior biological barrier penetration, achieving tumor internalization efficiency up to 10-fold that of liposomes with lower immunogenicity.³⁰³ Notably, exosome-inspired lipid nanoparticles (ELVs) incorporating anionic lipids, cholesterol, and aquaporin-1 increase the effective diffusion coefficient of drugs within tumor tissue by 80%, significantly improving delivery homogeneity.³⁰⁴ Similar to prodrug strategies, such biomimetic designs exploit TME characteristics to enable controlled release, offering a novel approach to overcoming the druggability challenges of PROTACs.³⁰⁵ At the molecular design level, rational structural optimizations, such as introducing cyclopropyl groups to enhance metabolic stability or developing miniaturized PROTACs to improve membrane permeability, constitute key strategies for boosting delivery efficiency.

The development of precision-engineered PROTACs critically addresses two major toxicological challenges. The first is on-target, off-tissue degradation, where PROTACs induce unwanted protein loss in healthy tissues due to ubiquitous E3 ligase expression. The second is off-target degradation that inadvertently recruits neo-substrates.¹⁹⁶ To mitigate these risks, researchers implement conditional activation strategies such as ROS-responsive linkers that exploit tumor oxidative stress for selective PROTAC release and enzyme-triggered designs that leverage overexpressed tumor proteases like cathepsin B to achieve spatiotemporal control.³⁰⁶ Additionally, tissue-specific E3 ligase ligands targeting BIRC2 or RNF114, which exhibit restricted expression profiles, enhance tumor selectivity while sparing normal cells.^307,308 Complementary to these molecular designs, targeted delivery systems such as PSMA-targeted nano-PROTACs employ antibody-drug conjugate principles to concentrate degraders specifically within prostate cancer tissues, reducing systemic exposure and associated toxicity.²⁸⁶

The scarcity of reliable biomarkers poses a significant obstacle to the clinical translation of PROTAC technology in precision medicine. A well-established biomarker system is paramount for accurately predicting therapeutic response, enabling patient stratification, and objectively evaluating pharmacodynamic effects. The current core challenge lies in the lack of effective means to systematically assess E3 ligase expression levels, the efficiency of ternary complex formation, and the successful activation of downstream PCD pathways. First, detecting the expression levels of relevant E3 ligases in tumor tissue serves as a fundamental predictive indicator of PROTAC activity.^309,310 Furthermore, determining the mutational status of the target protein is crucial. Specific target mutations are not only key for informing treatment selection but are also closely associated with PROTAC efficacy. The application of novel technologies like proteomics facilitates the discovery of new biomarkers. For example, the basal level of STAT1 protein correlates strongly with cellular sensitivity to STAT3 PROTAC degraders; higher STAT1 levels are associated with increased sensitivity, providing a novel rationale for screening potential responder populations via STAT1 quantification.³¹¹ Additionally, exploring pharmacodynamic biomarkers, such as directly monitoring target protein degradation levels, constitutes a direct method for demonstrating PROTAC pharmacological action in vivo. In preclinical studies, a multiplexed proteome dynamics profiling approach has been utilized to detect PROTAC-mediated target protein degradation in vitro, and this method theoretically translates to a pharmacodynamic monitoring tool in clinical trials.³¹²

Future research on PROTAC technology should focus on five key areas: molecular design optimization, delivery system innovation, combination therapy strategies, clinical translation acceleration, and therapeutic application expansion.³¹³ Primary efforts will concentrate on refining linker and ligand design to enhance PROTAC stability and target selectivity.²⁹¹ The field will continue exploring novel E3 ubiquitin ligases (such as RNF114 and DCAF16) to improve tissue specificity while developing smaller PROTAC molecules with enhanced membrane permeability.^314,315 This expansion will enable targeting of disease-relevant transcription factors and cell death regulators, broadening PROTACs’ therapeutic potential. Notably, the development of novel targets within the PCD pathway has emerged as a research hotspot, encompassing key regulatory factors of ferroptosis, such as GPX4, and mediators of necroptosis, e.g., RIPK1/RIPK3. PROTAC technology may induce novel modes of PCD by degrading these critical regulatory proteins. Theoretically, PROTACs could specifically degrade GLUT1 to block extracellular glucose influx. This metabolic perturbation further impairs the pentose phosphate pathway (PPP)-driven production of nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH), which is essential for maintaining intracellular redox homeostasis by regenerating GSH. The consequent decline in NADPH levels promotes the accumulation of intracellular disulfide bonds and ultimately triggers disulfidptosis.^316,317 These investigations not only open up unprecedented avenues for cancer therapy but also offer novel insights into the mechanisms of cell death.

Nanocarrier technologies promise to improve PROTAC solubility, cellular uptake efficiency, bioavailability, and targeting precision.³⁰⁵ Strategic combinations with other therapeutic approaches, including nanocarrier systems and antibody-PROTAC conjugates, could significantly enhance treatment accuracy and efficacy.^318,319 Accelerating clinical translation requires the parallel development of predictive biomarkers and comprehensive studies on resistance mechanisms. Expanding PROTAC applications beyond oncology to neurodegenerative and autoimmune disorders, along with exploring novel degradation pathways like lysosome-targeting chimeras, represents a transformative frontier.^85,255 This integrated approach of “precision design-intelligent delivery-combination therapy” optimization may ultimately enable PROTAC technology to transition from cancer treatment to broader clinical applications.

Funding and acknowledgments

This research was supported by the Construction Fund of Key Medical Disciplines of Hangzhou (2025HZGF04 and 2025HZZD01), National Natural Science Foundation of China (82202429), Zhejiang Provincial Natural Science Foundation of China (LBY23H080001), and Medical and Health Science and Technology Project of Zhejiang Province (2022KY236 and 2023RC131).

Author contributions

H.H., writing – original draft; A.D., data curation; F.P., writing – original draft; Y. Lu, writing – original draft; L.C., visualization; J.C., methodology; Q.T., writing – original draft; Y. Liu, visualization; Y.W., resources; J.D., writing – review and editing and conceptualization; Y. Li, funding acquisition and supervision; X.T., funding acquisition and supervision.

Declaration of interests

The authors declare no competing interests

Published Online: March 28, 2026