Proteolysis Targeting Chimeras (PROTACs): Mechanisms, Design Strategies, and Therapeutic Applications

Conventional small-molecule inhibitors (SMIs) rely on occupancy-driven pharmacology, necessitating high-affinity binding to well-defined active sites^[1]. This approach renders an estimated 70-80% of human proteins “undruggable,” including numerous scaffolding proteins and transcription factors ^[17]. Targeted Protein Degradation (TPD), implemented via PROTACs, offers a solution by fundamentally shifting the therapeutic strategy from inhibition to elimination.

A PROTAC is a heterobifunctional molecule that utilizes a POI ligand and an E3 ligase ligand connected by a linker ^[1]. The function is dictated by proximity-induced degradation. The PROTAC recruits an E3 ligase (part of the UPS cascade) to the POI, forming a ternary complex. This complex catalyzes the transfer of ubiquitin chains (specifically K48-linked polyubiquitin chains) onto the POI, marking it for irreversible destruction by the 26S proteasome. Crucially, the PROTAC is then released and catalytically recycled. This event-driven mechanism means that PROTACs achieve their functional outcome through the complete removal of the target protein, thereby eliminating all its associated functions, including non-enzymatic roles ^[26]. The stability and cooperativity of the ternary complex, often mediated by neocontacts, determine efficiency and can promote highly selective degradation even when using a broadly binding warhead.

Figure 1. Schematic Representation of the PROTAC- Proteolysis-targeting chimeras.

Mechanism of Action: Hijacking the Ubiquitin-Proteasome System (UPS)

1. Molecular Architecture and Components

A PROTAC is a hetero bifunctional molecule composed of three parts:

1. POI Ligand (Warhead): Binds selectively to the protein of interest (POI) and determines target specificity.
2. E3 Ligase Ligand (Recruiter): Engages an E3 ubiquitin ligase.
3. Linker: Connects the two ligands and positions the POI and E3 ligase in the correct orientation to enable ubiquitination ^[18].

PROTACs often fall into the beyond Rule-of-Five (bRo5) chemical space due to their structural complexity ^[1].

Importantly, the warhead does not need to bind to the active site of the POI; any binding site with sufficient affinity that allows formation of the POI-PROTAC-E3 ligase ternary complex is enough to induce ubiquitination and subsequent degradation.

2. The Degradation Cascade

PROTACs induce targeted protein degradation by redirecting the ubiquitinproteasome system (UPS) ^[1].

1. Ubiquitination Enzyme Cascade:

• E1 activates ubiquitin using ATP.
• E2 carries the activated ubiquitin.
• E3 ligase transfers ubiquitin to the substrate; more than 600 E3 ligases exist in humans.

2. Ternary Complex Formation:
   The PROTAC brings the POI and E3 ligase into proximity, forming a stable POI-PROTAC-E3 ternary complex.
3. Ubiquitination:
   The E3 ligase ubiquitinates lysine residues on the POI. Successive additions generate a K48-linked polyubiquitin chain, marking the POI for degradation ^[22].
4. Proteasomal Degradation and Recycling:
   The 26S proteasome recognises the tagged POI and degrades it. The PROTAC is released intact and can repeatedly initiate new degradation cycles.

Figure 2. PROTAC Mode of Action: Ternary Complex Formation, Ubiquitination, and Catalytic Recycling.

3. Pharmacological Distinction

PROTACs function through an event-driven mechanism, unlike traditional small-molecule inhibitors (SMIs) that rely on occupancy-driven activity ^[28].

Table 1. Comparative Overview of Event-Driven PROTACs and Occupancy-Driven Small-Molecule Inhibitors.

Because PROTAC activity depends on inducing ubiquitination rather than sustained binding, even transient or low-abundance ternary complexes can drive robust protein degradation.

4. Structural Basis of Function: Ternary Complex Dynamics and Neocontacts:

The stability and cooperativity of the ternary complex (TC) largely determine PROTAC efficiency, often outweighing the affinities of the individual ligands.

1. Structural Insights:

The first atomic-level PROTAC TC structure, MZ1, bound to VHL and the BRD4 bromo domain, showed that PROTACs induce new protein-protein and protein-ligand interactions at the interface.

• These interfaces exhibit strong surface complementarity (e.g., ~688 Aº) and are stabilized by hydrophobic and electrostatic contacts.
• Such “neocontacts” are critical for TC stability and overall degradation efficiency ^[10].

2. Cooperativity and Selectivity :

The binding of one protein (POI) can enhance PROTAC affinity for the E3 ligase, creating positive cooperativity. This promotes TC formation and allows highly selective degradation, even when using a broadly binding warhead.

3. Role of the Linker:

The linker controls the orientation, conformation, and dynamics of the TC.

• Proper alignment ensures that key lysine residues on the POI are positioned toward the E2-ubiquitin catalytic site ^[18].
• Linker length, rigidity, and topology directly influence ubiquitination efficiency and overall degradation potency.

Advantages in Drug Discovery and Efficacy:

1. 1. 1. Targeting the “Undruggable” Proteome

A major advantage of PROTACs is their ability to dsegrade proteins traditionally considered “undruggable” significantly expanding the druggable proteome.

Figure 3. The Future of Drug Discovery with PROTAC Technology.

Limitations of Traditional SMIs:

• SMIs depend on occupancy-driven pharmacology and require well-defined active pockets for high-affinity binding.
• Approximately 70-80% of human proteins lack such pockets, making them inaccessible to conventional small molecules ^[2].

How PROTACs Overcome These Barriers:

• PROTACs rely on proximity-induced degradation, not functionalsite inhibition.
• They require only any binding site on the POI, not a catalytic pocket.
• Even moderateor low-affinity ligands can be effective if they support ternary complex formation.
• Degradation results in the complete removal of the protein and all associated functions.

Examples of Previously Undruggable Targets Now Accessible:

• Transcription Factors: Proteins like MYC and STAT3 (e.g., SD-36 degrader) can now be selectively degraded ^[30].
• Scaffolding/Regulatory Proteins: Non-enzymatic proteins such as FAK are now tractable targets ^[2].
• Oncogenic Mutants: PROTACs have enabled new strategies against mutant oncoproteins, including KRAS^{G12C [18].}
  1. 2. Enhanced Selectivity and Specificity

PROTACs exhibit superior selectivity compared to traditional SMIs, an important advantage for reducing off-target effects typically associated with higher drug exposures.

Dual-Binding Mechanism and Selectivity:

• Selectivity arises from the requirement to form a specific POI-PROTAC-E3 ternary complex, adding a layer of discrimination beyond warhead binding ^[21].
• Both the POI ligand and E3 ligase ligand can be optimized to favor degradation of a single isoform or paralogue, even when ligand-binding sites are highly conserved.
• This mechanism enables conversion of non-selective inhibitors into highly selective degraders.

Examples of Improved Selectivity and Reduced Toxicity:

• MZ1 selectively degrades BRD4 over BRD2/BRD3 using the non-selective BET inhibitor JQ1 as the warhead ^[8].
• A PROTAC derived from a pan-HDAC inhibitor selectively degraded HDAC6 ^[13].
• DT2216 selectively degrades BCL-xL without causing thrombocytopenia by exploiting E3 ligases minimally expressed in platelets, unlike traditional inhibitors such as Navitoclax ^[10].
• SD-36 selectively degrades STAT3, sparing closely related STAT1 and STAT4.
  1. 3. Overcoming Drug Resistance

Drug resistance, often due to protein over expression, compensatory pathways, or point mutations, limits the long-term efficacy of conventional inhibitors. PROTACs offer multiple mechanisms to overcome resistance.

Mechanisms for Circumventing Resistance:

1. Complete Protein Removal: Degrading the entire POI prevents resistance caused by target upregulation or non-enzymatic (scaffolding/regulatory) functions ^[12].
2. Mutation Tolerance: PROTACs only require transient binding to initiate degradation, making them less sensitive to point mutations that impair inhibitor occupancy ^[33].
3. Use of Non-Inhibitory Ligands: Even loss-of-function ligands or agonists can trigger degradation ^[12].
4. Lower Drug Pressure: Catalytic activity and low dosing reduce evolutionary selection pressure for resistant mutations ^[25].

Examples Demonstrating Resistance Overcome:

• BTK Mutants: Degraders such as NX-2127 effectively degrade BTK variants, including the clinically important C481S mutation resistant to ibrutinib.
• Hormone Receptors: AR degraders (ARV-110, ARCC-4) reduce AR levels even in mutation-driven resistant cancers, including cases where antagonists convert to agonists ^[15].
• BCR-ABL Mutants: Dasatinib-based PROTACs such as SIAIS056 degrade multiple clinically relevant imatinib and dasatinib-resistant BCR-ABL1 mutants ^[12].
• Viral Resistance: PROTACs have been developed against drug-resistant Hepatitis C Virus (HCV) strains, effectively degrading viral proteins despite mutational escape.

Molecular Components and Rational Design Strategies

Figure 4. Chemical Architecture of a PROTAC Design Showing: Warhead (Target Binder), Linker and E3 Ligase (Ligand).

1. 1. Expanding the E3 Ligase Toolbox

The design of PROTACs centers on three components: a POI-binding warhead, an E3 ligase ligand, and a linker that optimally orients both proteins to form a productive ternary complex. CRBN and VHL dominate current PROTAC design due to well characterized ligands and favorable drug-like properties, although expanding the E3 toolbox (e.g., DCAF15, KEAP1, RNF114, RNF43) is a key priority for improving tissue selectivity and overcoming resistance ^{[1, 31]}.

Warhead affinity need not be high as long as it supports ternary complex formation; even non-inhibitory ligands can be repurposed for degradation. Linkers remain the most empirical design element, influencing orientation, cooperativity, permeability, and PK. PEG and alkyl linkers are common, while rigidified linkers can improve oral exposure. Computational tools including docking, MD, and AI-based linker generators now accelerate rational design by predicting geometries and cooperativity determinants.

1. 2. Advanced PROTAC Modalities

Next-generation PROTAC modalities aim to improve tissue selectivity, reduce systemic toxicity, and provide external control over degradation activity. Prodrug or “pro-PROTAC” ^[4] strategies mask active moieties with cleavable groups, enabling activation only under disease-specific conditions, such as hypoxia, high ROS, or tumor-associated enzymes. Light-controlled systems include photocaged PROTACs, which become active upon UV/visible irradiation, and photo switchable degraders that toggle between active and inactive conformations.

Other innovations include radiation-activated RT-PROTACs for spatially restricted degradation during radiotherapy ^[26], CLIPTACs, which assemble intracellularly from two smaller fragments to bypass permeability limitations; and conjugate-based formats such as antibody-PROTAC conjugates and aptamer-PROTACs that provide cell-type-specific delivery. These modalities significantly expand the therapeutic window and allow greater control over degrader activity in vivo.

1. 3. PROTAC Warhead Design and POI Ligands

The warhead determines POI recognition and contributes to degradation potency.

Key Principles of Warhead Selection:

• High affinity is not essential and may even be counterproductive by stabilizing nonproductive binary complexes.
• Warheads need not bind active sites any binding site enabling ternary complex formation is sufficient.
• Warheads are interchangeable, allowing rapid expansion across disease targets (>40 proteins reported).
• Ligands often originate from known inhibitors or structure-guided design.

Figure 5. E3 ligase ligands used in PROTAC Design.

Computational Support:

• Traditionally based on synthesis and SAR screening.
• Newer approaches include DNA encoded libraries (DELs) for high-throughput warhead discovery ^[1].
  4. Linker Design Strategies: Geometry and “Linkerology”

The linker influences ternary complex formation, degradation efficiency, and physicochemical properties.

Common Linker Types:

• No universal rules; design is largely empirical.
• PEG (54%) and alkyl chains (31%) dominate due to tunable length, flexibility, and solubility benefits ^[25].
• Rigid linkers (alkynes, piperazine, piperidine) improve permeability and oral PK (e.g., ARV-110, ARV-471) ^[1].

Figure 6. Structure of the linkers used in PROTAC Design.

Geometric and Functional Considerations:

• Length: Too short leads to steric clash; too long leads to weak PPIs, both can reduce degradation.
• Orientation: Must position POI and E3 ligase to facilitate productive PPIs and present accessible lysine residues for ubiquitination.
• Attachment Site: Should be at solvent-exposed positions that preserve warhead and E3 ligand affinity.

Computational Acceleration:

• Tools like docking, MD simulations, PPI modelling, and conformational sampling guide rational linker design.
• AI/ML platforms (PROTAC-RL) use large datasets (e.g., PROTAC-DB, 6,000+ molecules) to generate optimized linker architectures with desired PK profiles ^[14].

Therapeutic Applications across Major Diseases

1. 1. PROTACs in Cancer (Oncology)

PROTACs are transforming cancer therapy by selectively degrading oncogenic proteins, overcoming drug resistance, and expanding targeting possibilities beyond traditional inhibition.

Table 2. Selected PROTACs in Cancer Therapy and Their Clinical Status.

(Abbreviations: PROTAC-Proteolysis-Targeting Chimera, ERα-Estrogen Receptor alpha, AR -Androgen Receptor, BTK-Bruton's Tyrosine Kinase, BCL-xL-B-cell lymphoma-extra large, BRD9-Bromodomain-containing protein 9, BCL6-B-cell lymphoma 6, CRBN-Cereblon, VHL-Von Hippel-Lindau, IV-Intravenous, mCRPC-Metastatic Castration-Resistant Prostate Cancer, NHL-Non-Hodgkin Lymphoma, NDA-New Drug Application, HER2-Human Epidermal Growth Factor Receptor 2).

1. Targeting “Undruggable” Oncoproteins

• SMIs cannot target ~80% of human proteins due to the absence of ligandable pockets; PROTACs bypass this by requiring only transient binding to form a ternary complex ^[1].
• Transcription factors: MYC, STAT3 (e.g., KT-333 in Phase I trials) ^[22].

• Mutant oncoproteins: KRAS^G12C (LC-2), BRAF^V600E (P4B) ^[6].
• Scaffolding proteins: FAK degraders demonstrate potent degradation where inhibitors fail.

2. Overcoming Drug Resistance

PROTACs eliminate the entire target protein, addressing resistance arising from mutations or over expression.

• AR degraders: ARV-110 active against AR mutations and splice variants (e.g., AR-V7) ^[12].
• BTK mutants: NX-2127 degrades resistant BTK variants such as C481S ^[8].
• BCR-ABL mutants: Dasatinib-based PROTACs degrade multiple TKI-resistant BCR-ABL1 mutants.

3. Key Clinical Targets and Advancements

Over 50 PROTACs are in clinical evaluation, primarily in oncology.

Hormone receptors:

• • ARV-110 (mCRPC) shows strong PSA reduction in mutation-positive patients.
  • ARV-766: Next-generation AR degrader.
  • ARV-471 (ER degrader): Leading Phase III candidate with robust ER suppression.

Kinase targets:

3. • BRD4: dBET6 and ARV-771 degrade BET proteins more effectively than inhibitors.
   • CDKs: CDK4/6 degraders such as BTX-9341 in Phase I trials^[32].
   • EGFR mutants: CFT8919 for EGFR-L858R in NSCLC trials^[30].
   • BCL-XL: DT2216 selectively degrades BCL-XL with reduced platelet toxicity.
4. Tumor-Specific PROTAC Strategies

• Bioresponsive PROTACs: Hypoxia, ROS, and radiation-activated systems enable tumor restricted degradation.
• Ligand-directed delivery: Antibody PROTAC conjugates (e.g., trastuzumab PROTAC), aptamer PROTACs (HER2targeted), folate-guided PROTACs ^[33].
• Nano-PROTACs: Nanoparticle carriers improve tumor accumulation, PK properties, and solubility.
  2. PROTACs in Viral Diseases

PROTACs offer a novel antiviral strategy by degrading essential viral proteins and reducing the emergence of resistant viral strains.

Table 3. Selected PROTACs in Viral Diseases.

(Abbreviations: PROTAC-Proteolysis-Targeting Chimera, HCV-Hepatitis C virus, NA-Neuraminidase, HA-Hemagglutinin, M^Pro-Main Protease (SARS?CoV?2), CDK9-Cyclin-Dependent Kinase 9, PGES?2-Prostaglandin E Synthase-2).

• 1. 1. Hepatitis Viruses
• HCV: Telaprevir-based PROTAC DGY-08-097 (PROTAC 70) degrades NS3/4A, effective even against drug-resistant NS3 variants ^[1].
• HAV/HBV: RG7834-based PROTACs degrade host/viral proteins critical for replication ^[19].
  1. 2. HIV/AIDS

PROTACs target essential proteins for viral replication and immune evasion.

• HIV-1 integrase: Peptide PROTACs degrade integrase, suppressing replication.
• Nef: Nef-targeting PROTACs restore immune receptor expression and block viral replication.
• Vif: PROTAC L15 degrades Vif and demonstrates antiviral activity ^[19].
• Delivery: Exosome-mediated systems show potential for targeting latent HIV reservoirs.
  1. 3. Coronaviruses (e.g., SARS-CoV-2)

• Mpro degraders: Compounds such as HP211206 and MPD2 degrade SARS-CoV-2 Mpro, including resistant variants ^[24].
• Indomethacin-based PROTACs: Enhance antiviral potency via PGES-2 degradation and show broad coronavirus activity.
• Targets include Mpro, PLpro, and RdRP ^[37].
  1. 4. Influenza and Other Viruses:

PROTACs offer novel antiviral strategies for influenza, human cytomegalovirus (HCMV), and other viral infections.

Influenza:

• • Oseltamivir-PROTACs degrade neuraminidase, effective against resistant strains.
  • HA degraders (oleanolic acid-based) show broad IAV activity.
  • Host-directed strategies (e.g., GSPT1 degradation) inhibit multiple viruses including IAV and SARS-CoV-2.
  • PROTAC-engineered live attenuated influenza virus demonstrates vaccine potential.

HCMV: THAL-SNS-032 shows enhanced anti-HCMV and dual antiviral effects ^[38].

1. 3. Overcoming and Predicting Drug Resistance

Overcoming Resistance to Traditional SMIs

PROTACs address resistance that limits small-molecule inhibitors by degrading targets rather than merely inhibiting them.

• Complete Protein Elimination:  Removes enzymatic and non-enzymatic (scaffolding/ regulatory) functions, counteracting resistance from gain/loss of function mutations and target overexpression.
• Mutation Tolerance : Event-driven action requires only transient binding; even lower-affinity ligands can drive degradation.

Key Examples:

• • AR: ARV-110 / ARCC-4 overcome CRPC resistance from AR overexpression, LBD mutations (e.g., F876L, T877A) and splice variants (e.g., AR-V7).
  • BTK: BTK degraders eliminate ibrutinib-resistant mutants such as C481S.
  • BCR-ABL: SIAIS056 degrades multiple imatinib/dasatinib resistant BCR-ABL1 mutants (e.g., T315I) ^[12].
  • EGFR: PROTACs target multi-mutant EGFR (e.g., Del19/T790M/C797S) in resistant NSCLC.
  • Viruses: HCV NS3/4A PROTACs retain efficacy against resistant viral variants ^[2].

Resistance to PROTACs and Mitigation

PROTACs can themselves encounter distinct resistance mechanisms:

Observed Mechanisms:

• • Loss/mutation/down regulation of E3 ligases (e.g., CRBN, VHL) or associated components (CUL2, ELOB, RBX1, UBE2M).
  • Upregulated DUBs (e.g., USP15) reversing ubiquitination.
  • Increased efflux pumps (MDR1) lowering intracellular PROTAC levels ^[9].
  • Pathway rewiring e.g., ER down regulation and HER/MAPK activation with ARV-471.
  • Hook effect at high concentrations (excess binary complexes reducing ternary complex formation) ^[18].

Mitigation Strategies:

• Expanding and switching E3 ligases (e.g., from VHL to CRBN) for the same POI.
• Genomic/proteomic profiling to identify resistance drivers and biomarkers.
• Combination therapies, e.g., with efflux inhibitors or chemo (BRD4 degraders restoring doxorubicin sensitivity) ^[14].
• Computational design/ML to optimize ternary complex stability and minimize resistance risk.
  4. Neurodegenerative Diseases (NDDs)

Neurodegenerative diseases arise from toxic protein buildup. PROTACs can selectively degrade tau, α-synuclein, LRRK2, and mutant huntingtin, offering a promising approach for treating Alzheimer’s, Parkinson’s, and Huntington’s diseases

Table 4. PROTACs Targeting Neurodegenerative Diseases.

PROTACs are promising for NDDs by clearing aggregation-prone or dysregulated proteins often inaccessible to SMIs.

• Alzheimer’s disease (AD):

Tau degraders: Peptide PROTACs (e.g., TH006, Keap1-based constructs) and small molecules (e.g., C004019, QC-01-175) lower tau levels and improve neuronal function in models^[38].

GSK-3β degraders (e.g., PG-21): Reduce tau phosphorylation and Aβ production, protecting neurons.

• Parkinson’s disease (PD) / Huntington’s disease (HD):

α-Synuclein PROTACs (e.g., PROTAC 66): Decrease α-syn levels, mitigating toxicity in PD models.

LRRK2 and mHTT: Degraders such as XL01126 (BBB-penetrant LRRK2 PROTAC) show potential for PD and HD.

1. 5. Metabolic and Cardiovascular Diseases

Table 5. Key PROTAC Targets in Metabolic and Cardiovascular Diseases.

• HMG-CoA reductase (HMGCR):
  Statin-based PROTACs (e.g., P22A, VHL-lovastatin conjugates) degrade HMGCR, potentially avoiding statin-induced compensatory upregulation and achieving stronger cholesterol lowering in vivo ^[38].

Other targets:

PTP1B: Degradation may improve insulin sensitivity in diabetes/obesity.

Nuclear receptors and transcription factors (e.g., LXR-β, SREBP): Degraders modulate lipid and cholesterol homeostasis and show benefit in atherosclerosis models ^[18].

6. Applications in Immune and Inflammatory Disorders

PROTACs allow precise modulation of complex immune and inflammatory pathways that are difficult to fully control with classical inhibitors.

A. IRAK4 and Clinical Proof-of-Concept

• IRAK4 integrates TLR and IL-1R signalling and has both kinase and scaffolding functions ^[36].
• KT-474 (SAR444656): First IRAK4 degrader in Phase II trials for atopic dermatitis (AD) and hidradenitis suppurativa (HS); shows deep IRAK4 degradation, broad cytokine suppression, and clinical symptom improvement.
• KT-413: Dual degrader of IRAK4 and IMiD substrates (Ikaros/Aiolos) for MYD88-mutant tumors ^[8].
• Other molecules (e.g., PROTAC47) achieve >90% IRAK4 degradation in cells.

B. Other Inflammatory Targets

Transcription factors & signaling regulators:

• • NF-κB axis: Degraders targeting proteasome subunits (e.g., PSMD13) or upstream regulators block NF-κB activation.
  • STATs: STAT3 and STAT6 degrader KT-621 show anti-inflammatory and anti-allergic potential.

Kinases and enzymes:

• • BTK degraders provide anti-inflammatory effects via BCR/NF-κB signalling inhibition ^[38].
  • RIPK2 PROTACs rapidly and completely degrade RIPK2, relevant for IBD and other autoimmune diseases ^[19].
  • H-PGDS PROTACs (e.g., PROTAC52): Reduce PGD2, with promise in asthma and allergic inflammation.

Immune checkpoints (PD-L1) :
Small-molecule and antibody-based PROTACs induce PD-L1 degradation, enhancing antitumor immunity.

A. Epigenetic and Other Immune Targets

• HDAC degraders (e.g., HDAC3, HDAC6) modulate inflammatory responses and bone/joint pathology.
• TNF-α and receptors and CD147 have been targeted with PROTACs in models of autoimmune and inflammatory disease.

B. Skin Diseases and Immuno-Inflammation

PROTACs (particularly peptide PROTACs) are being explored for eczema, dermatitis, psoriasis, and acne, owing to precise targeting and potential topical delivery.

KT-474 provides the leading clinical example for inflammatory skin disease ^[18].

C. Synergy with Immunotherapy in Cancer

PROTACs can reshape the tumor microenvironment (TME) by degrading immunosuppressive proteins (e.g., NAMPT in myeloid cells) and enhancing responses to checkpoint inhibitors.

Table 6. Key PROTAC Targets and Therapeutic Applications in Inflammatory, Metabolic, and Dermatological Disorders.

Expanding Scope: Metabolic Disorders and Dermatology

1. Metabolic Disorders

Building on the HMGCR and PTP1B examples:

PROTACs are being developed for diabetes, obesity, and dyslipidemia by degrading:

1. • HMGCR (P22A and related degraders) to improve lipid control.
   • SREBP and nuclear receptors (e.g., LXR-β) to reprogram metabolic gene expression.
   • PTP1B to enhance insulin signalling.
2. Dermatology

Dermatology is a rapidly emerging area, especially for topical PROTACs with reduced systemic exposure.

1. Androgen-Driven Conditions (Alopecia, Acne)
   • GT20029: First topical AR-targeting PROTAC in clinical trials for androgenetic alopecia and acne; Phase I showed good safety, and Phase II in male AGA has met its primary endpoint.
2. Pigmentation, Anti-Ageing, and Inflammation
   • Pigmentation: Tyrosinase-targeting PROTACs (e.g., Rhein-based degraders) reduce melanin production.
   • Anti-ageing: Peptide PROTACs target proteins involved in collagen loss and matrix degradation, showing anti-wrinkle/skin-barrier benefits in models.
   • Inflammation: Topical and peptide PROTACs degrading inflammatory mediators (e.g., IRAK4, NF-κB/STAT-related targets) are being explored for eczema, dermatitis, and acne ^[18].
3. Dermal Delivery Strategies
   • Skin-penetrating peptides (SPPs) and other delivery enhancers are key to achieving sufficient skin and follicular penetration for peptide and large-molecule PROTACs.

Challenges, Clinical Status, and Future Directions of PROTACs

1. Physicochemical Properties and Bioavailability

PROTACs typically occupy beyond the Rule-of-Five (bRo5) space, creating major developability challenges.

Key Issues

• • High MW (≈700-1,300 Da), high PSA and HBD countlead to low solubility, poor permeability, and limited oral bioavailability ^[38].
  • Many clinical PROTACs (e.g., ARV-110) require relatively high doses due to poor PK.

Improvement Strategies

• • Molecular optimisation: Reduce MW, HBDs, rotatable bonds; use rigidified and H-bondmasked linkers to lower PSA and improve permeability.
  • E3 choice: CRBN-based PROTACs often show more favourable oral profiles than VHL-based analogues.
  • Formulation approaches: NanoDDSs and amorphous solid dispersions (ASDs) increase solubility and exposure (e.g., ARCC-4) ^[7].

2. Pharmacological-Specific Challenges

• On-target toxicity: Complete protein removal in normal tissues can cause more severe, irreversible effects than partial inhibition.
• Off-target degradation (neosubstrates): Especially with early CRBN ligands, unintended substrates can be recruited and degraded.
• Drug resistance: Often driven by genetic alterations in E3 ligase machinery (e.g., CRBN, CUL2) rather than POI mutations.
• Limited E3 repertoire: Heavy reliance on CRBN/VHL restricts tissue- and context-specific targeting ^[8].
• PK/PD complexity:Event-driven pharmacology requires new metrics (DC50, Dmax) rather than traditional occupancy-based parameters.
  5. Clinical Status and Future Directions:

PROTAC technology has successfully transitioned from academia to the pharmaceutical industry, marking a paradigm shift in drug discovery.

Current landscape

• • >50 PROTACs in clinical development, largely for oncology.
  • Leading examples: ARV-110 (AR, prostate cancer), ARV-471 / vepdegestrant (ER, breast cancer; advanced to Phase III and NDA stage), with additional candidates such as KT-333 (STAT3) and multiple BTK degraders ^[13].

Future priorities

• • Expanding E3 ligase space beyond CRBN/VHL to increase target diversity and tissue selectivity.
  • Advanced delivery: Lipid nanoparticles, antibody-PROTAC conjugates, and prodrug strategies for improved oral or targeted delivery.
  • CNS targeting: Design of brain-penetrant PROTACs via MW/PSA reduction, transporter hijacking, and nanocarriers.
  • Next-generation designs: Reversible PROTACs, multi-target degraders, and AI/ML-guided design to optimize ternary complex formation and PK/PD ^[18].

The Hook Effect

• Concept: At high concentrations, PROTACs favor non-productive binary complexes (PROTAC–POI or PROTAC–E3) over the ternary complex, leading to reduced degradation at higher doses ^[1].
• Relevance: Intrinsic to bifunctional design; often used as a mechanistic signature of true PROTAC activity.
• Mitigation
  • Enhance positive cooperativity and formation of stabilizing neocontacts in the ternary complex.
  • Carefully map concentration–response curves to define a non-hook therapeutic window.
  • Structural strategies (e.g., macrocyclization) and nanodelivery/split-mix nano-PROTACs to modulate local exposure ^[26].

Covalent and Reversible PROTACs

Non-covalent PROTACs: Standard format; maintain full catalytic, event-driven degradation and recyclability.

Irreversible covalent PROTACs (IC):

• POI-covalent: Often sacrifice catalytic turnover and may impair ubiquitin transfer; some fail to induce degradation.
• E3-covalent: Can retain catalytic degradation of multiple POI molecules.

Reversible covalent (RC) PROTACs:

• • Use reversible covalent motifs (e.g., cyanoacrylamides for BTK).
  • Combine strong, selective engagement with preserved catalytic cycling.
  • Can improve intracellular accumulation and target residence time.

Advanced Molecular Strategies for Controllability (Prodrugs)

“Smart” or pro-PROTACs aim to introduce spatiotemporal control and reduce systemic toxicity.

Light-controlled PROTACs

• Photocaged PROTACs: Inactive until UV/visible light removes a photolabile group on the POI or E3 ligand site.
• Photoswitchable PROTACs (PHOTACs): Reversibly toggled between active/inactive states via isomerization (e.g., azobenzene).
• RT-PROTACs: X-ray–triggered release for deep-tissue, radiotherapy-linked degradation ^[38].

Tumor microenvironment-responsive systems

• Hypoxia-activated PROTACs (HAP-TACs): Use bioreductive triggers (e.g., indolequinone) to unmask active PROTACs in hypoxic tumors.
• ROS-activated PROTACs: Triggered by elevated reactive oxygen species.
• Dual-gated (DAO-PROTACs): Require two independent stimuli (e.g., hypoxia + cathepsin L), enhancing disease specificity ^[2].

Intracellular assembly (CLIPTACs)

Split the PROTAC into two smaller, permeable fragments that undergo bioorthogonal click chemistry in cells, bypassing poor permeability associated with fully assembled, high-MW PROTACs.

Emerging Modalities and Conjugate Technologies

The development of clinical-grade PROTACs has driven the evolution of peptide-based degraders, biomacromolecule PROTAC conjugates, and nano-PROTACs, all aimed at improving targetability, PK, and safety.

Peptide-Based PROTACs: Design, Advantages, and Scope

Peptide-based PROTACs were the first PROTACs (e.g., Protac-1 recruiting SCF^β-TRCP to degrade MetAP-2) and use peptidic ligands that mimic natural protein–protein interaction motifs.

Key features and advantages

• Use peptide POI ligands, E3-binding peptides, linkers, and often cell-penetrating sequences.
• Offer high selectivity, biocompatibility, and low toxicity, with good suitability for “undruggable” PPI targets (e.g., transcription factors).
• Allow multitarget and modular design by concatenating different peptide segments.

Therapeutic examples

• Oncology: Peptide PROTACs against p300 and STAT3; cyclic peptide PROTACs reducing PD-L1 levels ^[19].
• Neurodegeneration: KEAP1-recruiting peptide PROTACs that degrade tau in Alzheimer’s models.
• Hematologic/skin disease and membrane proteins are promising areas due to peptide compatibility.

Limitations and solutions

• Main issues: high MW and poor permeability, historically requiring microinjection.
• New approaches: tumor-penetrating peptides (e.g., iRGD) and nanodelivery systems to improve uptake.

Biomacromolecule-PROTAC Conjugates (AOCs/APCs)

Antibody-PROTAC Conjugates (AOCs/DACs)

AOCs (DACs) couple PROTACs to monoclonal antibodies, paralleling ADCs.

• Mechanism: Antibody binds a cell-surface antigen causes endocytosis leading to intracellular release of the PROTAC for target degradation^[23].
• Advantages: High cell/tissue specificity, reduced off-target exposure, and antibody-like PK (extended half-life).

Examples:

• Trastuzumab–PROTAC constructs degrading BRD4 selectively in HER2? cells.
• ROR1-directed DACs.
• AbTACs recruiting RNF43 to degrade PD-L1 at the cell surface.

Challenges: Very large, complex conjugates with difficult synthesis, formulation, and potential immunogenicity.

AptamerPROTAC Conjugates (APCs)

APCs use nucleic acid aptamers as targeting ligands.

Mechanism: Aptamer binds a specific receptor (e.g., HER2, nucleolin) and mediates receptor-driven uptake of the PROTAC ^[37].

Advantages: High specificity, low immunogenicity, good tissue penetration; enable tumor-selective degradation.

Examples:

• • HER2-targeting APCs for selective degradation in HER2? tumors.
  • AS1411-PROTAC constructs enhancing nucleolin/BET degradation.
  • Aptamer-PROTAC ZL216, showing good serum stability, solubility, and potent nucleolin degradation.

Nano-Drug Delivery Systems (Nano-PROTACs)

NanoDDS-based PROTACs tackle the core liabilities of PROTACs are high MW, polarity, low solubility, and poor bioavailabilityand help localize activity.

General advantages

• Improve solubility, stability, circulation time, and cellular uptake.
• Enable passive (EPR) and active targeting, reducing systemic off-target effects and helping modulate the hook effect via controlled local exposure.

Key strategies

1. Physical encapsulation: Encapsulation in liposomes or polymeric nanoparticles to enhance stability and enable co-delivery with other drugs.
2. Chemical conjugation: Covalent linkage of PROTACs to nanocarriers with stimuli-responsive linkers (pH, enzymes, redox, radiation) for TME-triggered release.
3. Carrier-free self-assembly: PROTACs co-assemble with other hydrophobic/π–π stacking molecules (e.g., photosensitizers), achieving very high drug loading and avoiding carrier toxicity.
4. “Split-and-mix” / CLIPTAC-like platforms: Deliver two smaller ligands (POI and E3 ligands) separately and assemble the active PROTAC in situ, improving permeability and reducing hook-effect–driven self-inhibition ^[26].

Representative nanoplatforms

• Polymeric nanoparticles (PNPs): Stimuli-responsive systems (e.g., PSRNs) delivering CDK4/6 PROTACs that release cargo in acidic, cathepsin B-rich tumor sites.
• Lipid-based nanoparticles (LBNPs): Widely used to enhance PROTAC exposure and tumor accumulation; also explored for delivering bioPROTACs.
• Inorganic nanoparticles (INPs): Gold or silica NPs for theranostics; e.g., UCNP–MSN systems enabling NIR-triggered release of photocaged BRD4 PROTACs.
• Exosome-based carriers: Natural vesicles with excellent biocompatibility and barrier penetration, promising for precise PROTAC delivery (including to hard-to-reach sites).

Clinical Translation, Technology, and Future Directions

PROTACs have rapidly evolved from a conceptual degradation technology into a clinically validated therapeutic modality. Their successespecially in oncologyhas been enabled by improvements in molecular design, computational modeling, and diversification of targeted protein degradation (TPD) systems beyond the proteasome.

Clinical Landscape and Regulatory Challenges

Clinical Status

Since the first PROTAC entered clinical trials in 2019, the pipeline has grown to 30–50 candidates. Most clinically advanced degraders target hormone-driven cancers.

• ARV-471 (ER degrader): Completed Phase III, submitted the first-ever PROTAC NDA to the US FDA.
• ARV-110 (AR degrader): In Phase II/III for mCRPC ^[8].
• Additional clinical degraders target STAT3 (KT-333), BTK, IRAK4, BRD9 (FHD-609), and BCL-XL (DT2216).

Regulatory and Clinical Challenges

• Physicochemical limitations: Large MW (700-1300 Da)^[18], high polarity, poor permeability and oral bioavailability (e.g., ARV-110 ~23% in rats).
• Safety risks: Complete protein removal increases risk of irreversible toxicity and off-target/neosubstrate degradation.
• Regulatory uncertainty: New evaluation paradigms are required, including degradation biomarkers, and quantitative degradation assays.

Computational and AI-Assisted PROTAC Design

Designing optimal linkers and ternary complexes remains labor-intensive. Computational tools now drive PROTAC design by predicting linker geometry, ternary complex formation, and degradation efficiency.

Figure 7. Computer aided drug design in the development of PROTAC.

Key tools and approaches:

Ternary complex modeling: Using PRosettaC, PROTAC-Model, RosettaDock, RDKit.

AI/ML integration:

• AlphaFold for high-fidelity 3D protein structures and modeling when crystal structures are unavailable.
• DeepPROTACs for degradation prediction.
• AIMLinker, ShapeLinker for generative linker design.

These tools significantly accelerate rational design and reduce trial-and-error synthesis ^[14].

PROTAC Databases and Resources

Bioinformatic resources support rational PROTAC design by providing access to chemical structures, target information, linkers, and biological activity, enabling systematic optimization and prediction of protein degradation efficiency ^[42].

Table 7. Key Databases and Resources for PROTAC Design and Research.