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Cited by 429Protein Degradation and InhibitorsChemical Synthesis and AnalysisUbiquitin and proteasome pathways

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Current strategies for the design of PROTAC linkers: a critical review

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Troup, Robert I., Fallan, Charlene, Baud, Matthias G. J.·2020·Exploration of Targeted Anti-tumor Therapy
DOI 10.37349/etat.2020.00018PMC PMC9400730
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Abstract

PROteolysis TArgeting Chimeras (PROTACs) are heterobifunctional molecules consisting of two ligands; an “anchor” to bind to an E3 ubiquitin ligase and a “warhead” to bind to a protein of interest, connected by a chemical linker. Targeted protein degradation by PROTACs has emerged as a new modality for the knock down of a range of proteins, with the first agents now reaching clinical evaluation. It has become increasingly clear that the length and composition of the linker play critical roles on the physicochemical properties and bioactivity of PROTACs. While linker design has historically received limited attention, the PROTAC field is evolving rapidly and currently undergoing an important shift from synthetically tractable alkyl and polyethylene glycol to more sophisticated functional linkers. This promises to unlock a wealth of novel PROTAC agents with enhanced bioactivity for therapeutic intervention. Here, the authors provide a timely overview of the diverse linker classes in the published literature, along with their underlying design principles and overall influence on the properties and bioactivity of the associated PROTACs. Finally, the authors provide a critical analysis of current strategies for PROTAC assembly. The authors highlight important limitations associated with the traditional “trial and error” approach around linker design and selection, and suggest potential future avenues to further inform rational linker design and accelerate the identification of optimised PROTACs. In particular, the authors believe that advances in computational and structural methods will play an essential role to gain a better understanding of the structure and dynamics of PROTAC ternary complexes, and will be essential to address the current gaps in knowledge associated with PROTAC design.

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Figures

Figure 1. A. General structure of a PROTAC. The E3 ligase targeting “anchor” (blue) is connected to the specific POI targeting warhead (green) via a variable linker; B. mechanism of PROTAC-mediated target degradation via RING-type E3 ligases. (i) Ub transfer from E1 to E2 by trans-thioesterification is followed by complex formation with an E3 ligase; (ii) the PROTAC binds to both the E3 ligase and POI to form a TC, where the E3 ligase is shown as an assembly of scaffolding proteins (Sc), adapter proteins (Ad), and a SBD. This brings the E2 ligase into proximity to the POI; (iii) this leads to the transfer of multiple Ub units to surface exposed lysine residues; (iv) the resulting polyubiquitin chain is recognised by the proteasome, leading to the proteolytic degradation of the POI; and (v) the PROTAC is released and can catalyse the transfer of Ub to additional POIs
Figure 1. A. General structure of a PROTAC. The E3 ligase targeting “anchor” (blue) is connected to the specific POI targeting warhead (green) via a variable linker; B. mechanism of PROTAC-mediated target degradation via RING-type E3 ligases. (i) Ub transfer from E1 to E2 by trans-thioesterification is followed by complex formation with an E3 ligase; (ii) the PROTAC binds to both the E3 ligase and POI to form a TC, where the E3 ligase is shown as an assembly of scaffolding proteins (Sc), adapter proteins (Ad), and a SBD. This brings the E2 ligase into proximity to the POI; (iii) this leads to the transfer of multiple Ub units to surface exposed lysine residues; (iv) the resulting polyubiquitin chain is recognised by the proteasome, leading to the proteolytic degradation of the POI; and (v) the PROTAC is released and can catalyse the transfer of Ub to additional POIsCC BY · PubMed Central
Figure 2. Structures of BRD4 degraders MZ1 (1), ARV-825 (2), and dBET1 (3). The anchors of (1) and (2–3) targeting the Von Hippel-Lindau tumour suppressor protein (VHL) and cereblon (CRBN) respectively are highlighted in blue; the common JQ1 based warhead is highlighted in green, and the linkers in black
Figure 2. Structures of BRD4 degraders MZ1 (1), ARV-825 (2), and dBET1 (3). The anchors of (1) and (2–3) targeting the Von Hippel-Lindau tumour suppressor protein (VHL) and cereblon (CRBN) respectively are highlighted in blue; the common JQ1 based warhead is highlighted in green, and the linkers in blackCC BY · PubMed Central
Figure 3. Early PROTAC development: the first published PROTAC degrader (4), which conjugates the angiogenesis inhibitor ovalicin to the IκBα phosphopeptide (denoted IκBα); structures of second generation PROTACs targeting the ER (5) and AR (6) receptors; the first all small-molecule PROTAC incorporating the MDM2 ligand nutlin-3 (7). In all PROTACs, the anchor is coloured blue and the warhead green
Figure 3. Early PROTAC development: the first published PROTAC degrader (4), which conjugates the angiogenesis inhibitor ovalicin to the IκBα phosphopeptide (denoted IκBα); structures of second generation PROTACs targeting the ER (5) and AR (6) receptors; the first all small-molecule PROTAC incorporating the MDM2 ligand nutlin-3 (7). In all PROTACs, the anchor is coloured blue and the warhead greenCC BY · PubMed Central
Figure 4. Commonly used anchor ligands. Structure of the high-affinity ligand VH032 (8) commonly used to recruit VHL. Structures of thalidomide (9) and its analogues pomalidomide (10) and lenalidomide (11), which recruit CRBN. Methylbestatin (12) and a higher-affinity derivative of LCL161 (13) are most commonly used to target cIAP. Nutlin-3 (14) has been used to target MDM2
Figure 4. Commonly used anchor ligands. Structure of the high-affinity ligand VH032 (8) commonly used to recruit VHL. Structures of thalidomide (9) and its analogues pomalidomide (10) and lenalidomide (11), which recruit CRBN. Methylbestatin (12) and a higher-affinity derivative of LCL161 (13) are most commonly used to target cIAP. Nutlin-3 (14) has been used to target MDM2CC BY · PubMed Central
Figure 5. Structure of the BTK covalent inhibitor ibrutinib (15) and its PROTAC counterpart P131 (16), a potent degrader of WT and C481S mutant BTK. Promiscuous kinase inhibitor foretinib (17) is incorporated into PROTAC degraders of MAPK kinases, which differ by linker composition and site of conjugation to the VHL ligand. SJFα (18) is selective for p38α and SJFδ (19) for p38δ. In all PROTACs, the anchor is coloured blue and the warhead green
Figure 5. Structure of the BTK covalent inhibitor ibrutinib (15) and its PROTAC counterpart P131 (16), a potent degrader of WT and C481S mutant BTK. Promiscuous kinase inhibitor foretinib (17) is incorporated into PROTAC degraders of MAPK kinases, which differ by linker composition and site of conjugation to the VHL ligand. SJFα (18) is selective for p38α and SJFδ (19) for p38δ. In all PROTACs, the anchor is coloured blue and the warhead greenCC BY · PubMed Central
Figure 6. Effect of PROTAC linker length and conjugation site. A. In a representative PROTAC synthesis by Cyrus et al. [65], 20 was reacted with DSG to install an amide-linked alkyl linker. The product 21 was then reacted with the E3-binding pentapeptide sequence to displace the second succinimidyl moiety and obtain PROTAC 22; B. commercially available linker building blocks used by Cyrus et al. [65], include DSG (23), DSS (24), and Fmoc-protected acid 25. The structure of oestradiol (26) is shown with arrows pointing to potential sites of linker conjugation; C. extension of the PEG linker in EGFR and HER2 degrader 27 by one unit abolished HER2 activity to afford selective EGFR PROTAC 28
Figure 6. Effect of PROTAC linker length and conjugation site. A. In a representative PROTAC synthesis by Cyrus et al. [65], 20 was reacted with DSG to install an amide-linked alkyl linker. The product 21 was then reacted with the E3-binding pentapeptide sequence to displace the second succinimidyl moiety and obtain PROTAC 22; B. commercially available linker building blocks used by Cyrus et al. [65], include DSG (23), DSS (24), and Fmoc-protected acid 25. The structure of oestradiol (26) is shown with arrows pointing to potential sites of linker conjugation; C. extension of the PEG linker in EGFR and HER2 degrader 27 by one unit abolished HER2 activity to afford selective EGFR PROTAC 28CC BY · PubMed Central
Figure 7. Structures of representative PROTACs with PEG/alkyl linkers. BTK degraders (29) where the linkers are alkyl/ether chains of various combinations between 3 and 19 atoms. MDM2-targeting PROTAC with direct conjugation of the warhead to the anchor (30), although this did not function as a degrader. TBK1 PROTACs with alkyl/ether linkers between 7 and 29 atoms in length (31). In all PROTACs, the anchor is coloured blue and the warhead green
Figure 7. Structures of representative PROTACs with PEG/alkyl linkers. BTK degraders (29) where the linkers are alkyl/ether chains of various combinations between 3 and 19 atoms. MDM2-targeting PROTAC with direct conjugation of the warhead to the anchor (30), although this did not function as a degrader. TBK1 PROTACs with alkyl/ether linkers between 7 and 29 atoms in length (31). In all PROTACs, the anchor is coloured blue and the warhead greenCC BY · PubMed Central
Figure 8. Key methods to assemble PROTAC libraries using alkyl and ether linkers. A. Nucleophilic aromatic substitution of 32 with linkers carrying an array of functionalities (34–38) was used to build a toolbox of compounds for CRBN PROTAC development (33); B. representative example synthesis of linkers with varying ether combinations; commercially available 39 was sequentially alkylated with 40 and 42. The chloride handle in 43 was subsequently converted to a Cbz-protected amine (45) after further manipulations; C. synthesis of a PROTAC library (47) by alkylation of lenalidomide (11) with various alkyl bromides/iodides (46)
Figure 8. Key methods to assemble PROTAC libraries using alkyl and ether linkers. A. Nucleophilic aromatic substitution of 32 with linkers carrying an array of functionalities (34–38) was used to build a toolbox of compounds for CRBN PROTAC development (33); B. representative example synthesis of linkers with varying ether combinations; commercially available 39 was sequentially alkylated with 40 and 42. The chloride handle in 43 was subsequently converted to a Cbz-protected amine (45) after further manipulations; C. synthesis of a PROTAC library (47) by alkylation of lenalidomide (11) with various alkyl bromides/iodides (46)CC BY · PubMed Central
Figure 9. PROTACs with rigid linkers. Replacement of the amine connecting group to thalidomide in 48 with a rigid alkyne led to 49, which exhibited enhanced cell growth inhibition in 2/3 tested cell lines. Modifications to the anchor of 50 afforded 51, and further changes to the linker and anchor provided 52, which retained high degradation potency with a > 200 reduction in MW vs. 50
Figure 9. PROTACs with rigid linkers. Replacement of the amine connecting group to thalidomide in 48 with a rigid alkyne led to 49, which exhibited enhanced cell growth inhibition in 2/3 tested cell lines. Modifications to the anchor of 50 afforded 51, and further changes to the linker and anchor provided 52, which retained high degradation potency with a > 200 reduction in MW vs. 50CC BY · PubMed Central
Figure 10. PROTACs with aromatic linkers. The benzyl linker in 53 provided conformational restriction and a pi-stacking interaction with Y98 in VHL. PROTACs 55-57 incorporating a disubstituted phenyl did not display AR degradation, in contrast to parent PROTAC 54
Figure 10. PROTACs with aromatic linkers. The benzyl linker in 53 provided conformational restriction and a pi-stacking interaction with Y98 in VHL. PROTACs 55-57 incorporating a disubstituted phenyl did not display AR degradation, in contrast to parent PROTAC 54CC BY · PubMed Central
Figure 11. Use of triazoles in library synthesis. A. Azide intermediate 59 was reacted with alkynes bearing warheads for CRBN or VHL to afford two series of triazole-containing PROTACs 60 and 61 with variable linker lengths; B. amino azide intermediate 62 was conjugated to various anchors and reacted with alkyne derivatives of niraparib (63) or olaparib (64) to screen different warhead/anchor combinations. This identified potent MDM2-recruiting PROTAC 65
Figure 11. Use of triazoles in library synthesis. A. Azide intermediate 59 was reacted with alkynes bearing warheads for CRBN or VHL to afford two series of triazole-containing PROTACs 60 and 61 with variable linker lengths; B. amino azide intermediate 62 was conjugated to various anchors and reacted with alkyne derivatives of niraparib (63) or olaparib (64) to screen different warhead/anchor combinations. This identified potent MDM2-recruiting PROTAC 65CC BY · PubMed Central
Figure 12. Use of triazoles to exploit intermolecular interactions. The nitrogen atoms in the triazole formed hydrogen bonds with R97 in Sirt2 in the crystal structure of 65. Following the click reaction with azide 69, triazole-containing PROTAC 70 retained these interactions in docking analysis of the TC
Figure 12. Use of triazoles to exploit intermolecular interactions. The nitrogen atoms in the triazole formed hydrogen bonds with R97 in Sirt2 in the crystal structure of 65. Following the click reaction with azide 69, triazole-containing PROTAC 70 retained these interactions in docking analysis of the TCCC BY · PubMed Central
Figure 13. In-situ formation of CLIPTACs 74 and 75 by the click reaction between tetrazine 71 and TCO 72 or 73
Figure 13. In-situ formation of CLIPTACs 74 and 75 by the click reaction between tetrazine 71 and TCO 72 or 73CC BY · PubMed Central
Figure 14. Structures of potent BET degrader ARV-771 (76) and its inactive epimer ARV-766 (77)
Figure 14. Structures of potent BET degrader ARV-771 (76) and its inactive epimer ARV-766 (77)CC BY · PubMed Central
Figure 15. A. Structures of photocleavable groups DMNB and diethylamino coumarin; B. irradiation of DMNB-protected PROTAC 78 at 365 nm releases potent BET degrader dBET1 (3)
Figure 15. A. Structures of photocleavable groups DMNB and diethylamino coumarin; B. irradiation of DMNB-protected PROTAC 78 at 365 nm releases potent BET degrader dBET1 (3)CC BY · PubMed Central
Figure 16. PROTACs with photoswitchable linkers. PROTACs 79 and 80 are active degraders in their trans configuration, whereas 81 is active in its cis configuration. The wavelengths required for conversion between the trans and cis forms are given above and below the arrows
Figure 16. PROTACs with photoswitchable linkers. PROTACs 79 and 80 are active degraders in their trans configuration, whereas 81 is active in its cis configuration. The wavelengths required for conversion between the trans and cis forms are given above and below the arrowsCC BY · PubMed Central
Figure 17. PROTACs with linkers optimised to improve physical properties. Replacement of the PEG linker in 82 with piperazine and pyrimidine moieties (alongside other changes) greatly reduced lipophilicity and metabolic clearance of 83. Chessum et al. [126], developed pirin-targeting probe 84 in only three focused design iterations (through 85 and 86) by seeking to optimise physical properties instead of potency
Figure 17. PROTACs with linkers optimised to improve physical properties. Replacement of the PEG linker in 82 with piperazine and pyrimidine moieties (alongside other changes) greatly reduced lipophilicity and metabolic clearance of 83. Chessum et al. [126], developed pirin-targeting probe 84 in only three focused design iterations (through 85 and 86) by seeking to optimise physical properties instead of potencyCC BY · PubMed Central
Figure 18. Use of co-crystal structures to identify linker conjugation sites. Arrows indicate sites on VH298 (87) that are solvent exposed. This was used to inform the design of three combinations of analogous VH032 (8) in homo-PROTACs 88-90. The morpholine nitrogen in SGK3 inhibitor 91 was shown to be solvent-exposed, and was used for linker conjugation in PROTAC 92
Figure 18. Use of co-crystal structures to identify linker conjugation sites. Arrows indicate sites on VH298 (87) that are solvent exposed. This was used to inform the design of three combinations of analogous VH032 (8) in homo-PROTACs 88-90. The morpholine nitrogen in SGK3 inhibitor 91 was shown to be solvent-exposed, and was used for linker conjugation in PROTAC 92CC BY · PubMed Central
Figure 19. Rational design of PROTACs from TC crystal structures. The crystal structure of MZ1 (1) suggested that the tert-butyl moiety was a better site for linker conjugation, which was used to product AT1 (93). Macrocyclisation of 1 to retain its binding conformation in the TC crystal structure afforded 94
Figure 19. Rational design of PROTACs from TC crystal structures. The crystal structure of MZ1 (1) suggested that the tert-butyl moiety was a better site for linker conjugation, which was used to product AT1 (93). Macrocyclisation of 1 to retain its binding conformation in the TC crystal structure afforded 94CC BY · PubMed Central
Figure 20. Use of co-crystal structure to guide changes to linker composition. The crystal structure of 95 in complex with SMARCA2BD identified the piperazine ring as suitable for conjugation. The PEG linkage in 96 was replaced with a benzyl to improve hydrophobic interactions and exploit a potential pi-stack to Y98 in VHL (97). Extension by one atom to obtain the same length as 96 afforded 53
Figure 20. Use of co-crystal structure to guide changes to linker composition. The crystal structure of 95 in complex with SMARCA2BD identified the piperazine ring as suitable for conjugation. The PEG linkage in 96 was replaced with a benzyl to improve hydrophobic interactions and exploit a potential pi-stack to Y98 in VHL (97). Extension by one atom to obtain the same length as 96 afforded 53CC BY · PubMed Central
Figure 21. Use of computational docking to inform rational linker design. A. Docking pose of wogonin (97) was used to identify suitable conjugation vectors whilst retaining key binding interactions (site indicated by arrow), which was achieved in PROTAC 98; B. experimental SAR studies combined with computational docking was used to probe plausible TC ensembles formed by HDAC6 degraders 99 and 100, and suggested that each degrader employed a different set of amino acids to form distinct productive TCs. Similarly, modelling of the TC using a combination of docking and molecular dynamics was used to explain the orthogonal selectivities displayed by 101/102 towards MCL-1/BLC-2
Figure 21. Use of computational docking to inform rational linker design. A. Docking pose of wogonin (97) was used to identify suitable conjugation vectors whilst retaining key binding interactions (site indicated by arrow), which was achieved in PROTAC 98; B. experimental SAR studies combined with computational docking was used to probe plausible TC ensembles formed by HDAC6 degraders 99 and 100, and suggested that each degrader employed a different set of amino acids to form distinct productive TCs. Similarly, modelling of the TC using a combination of docking and molecular dynamics was used to explain the orthogonal selectivities displayed by 101/102 towards MCL-1/BLC-2CC BY · PubMed Central
Figure 22. Rational PROTAC design using crystal structures and computational methods. The crystal structures of TC bound 103 and 104, alongside protein-protein docking, revealed potential binding orientations in the TC. Docking was used to identify the shortest distance that could bridge BRD4 and CRBN in the TC, and was used to design 105 and 106
Figure 22. Rational PROTAC design using crystal structures and computational methods. The crystal structures of TC bound 103 and 104, alongside protein-protein docking, revealed potential binding orientations in the TC. Docking was used to identify the shortest distance that could bridge BRD4 and CRBN in the TC, and was used to design 105 and 106CC BY · PubMed Central
Figure 23. The generative model DeLinker was used to generate novel PROTAC linkers 107-109 to attain the same objective as 97 in improving the interactions of 96 in the TC
Figure 23. The generative model DeLinker was used to generate novel PROTAC linkers 107-109 to attain the same objective as 97 in improving the interactions of 96 in the TCCC BY · PubMed Central

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Authors
Troup, R. I., Fallan, C., & Baud, M. G. J.
Affiliations
  • University of Southampton (GB)
  • AstraZeneca (United Kingdom) (GB)
Corresponding author
  • Fallan, Charlene
  • Baud, Matthias G. J.
ORCID
Venue
Exploration of Targeted Anti-tumor Therapy
Publisher
Open Exploration Publishing
Published
2020-10-11
Volume
1
Issue
5
Pages
273-312
ISSN
2692-3114 (ISSN-L)
DOI
10.37349/etat.2020.00018
PMC
PMC9400730
OpenAlex
W3093395956
Cited by
424
References
183
Citations by year
2026: 91 · 2025: 102 · 2024: 96 · 2023: 66 · 2022: 48 · 2021: 19 · 2020: 1
FWCI
3.28
Type
journal article
Language
en
Open access
Licence
Locations
  • Exploration of Targeted Anti-tumor Therapy (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • ePrints Soton (University of Southampton) (acceptedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • AstraZeneca
  • Engineering and Physical Sciences Research Council (EP/R513325/1)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Degradation and Inhibitors
  • Physical Sciences › Chemistry › Organic Chemistry › Chemical Synthesis and Analysis
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ubiquitin and proteasome pathways
Keywords
PROTACs, linker design, targeted protein degradation, E3 ubiquitin ligase, heterobifunctional molecules, ternary complex structure, rational drug design, polyethylene glycol linker, alkyl linkers

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