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Cited by 260Protein Degradation and InhibitorsUbiquitin and proteasome pathwaysChemical Synthesis and Analysis

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Homo-PROTACs: bivalent small-molecule dimerizers of the VHL E3 ubiquitin ligase to induce self-degradation

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Maniaci, Chiara, Hughes, Scott J., Testa, Andrea, Chen, Wenzhang, Lamont, Douglas J., Rocha, Sonia, Alessi, Dario R., Romeo, Roberto, Ciulli, Alessio·2017·Nature Communications
DOI 10.1038/s41467-017-00954-1PMC PMC5635026
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Abstract

E3 ubiquitin ligases are key enzymes within the ubiquitin proteasome system which catalyze the ubiquitination of proteins, targeting them for proteasomal degradation. E3 ligases are gaining importance as targets to small molecules, both for direct inhibition and to be hijacked to induce the degradation of non-native neo-substrates using bivalent compounds known as PROTACs (for ‘proteolysis-targeting chimeras’). We describe Homo-PROTACs as an approach to dimerize an E3 ligase to trigger its suicide-type chemical knockdown inside cells. We provide proof-of-concept of Homo-PROTACs using diverse molecules composed of two instances of a ligand for the von Hippel-Lindau (VHL) E3 ligase. The most active compound, CM11, dimerizes VHL with high avidity in vitro and induces potent, rapid and proteasome-dependent self-degradation of VHL in different cell lines, in a highly isoform-selective fashion and without triggering a hypoxic response. This approach offers a novel chemical probe for selective VHL knockdown, and demonstrates the potential for a new modality of chemical intervention on E3 ligases.

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Crossref · 10.1038/s41467-017-00954-1

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Figures

Fig. 1 Structure-guided design of Homo-PROTACs to induce VHL dimerization. a Crystal structures of VHL in complex with VH298 (PDB code 5LLI)19. VHL is shown as a pink surface and the bound ligand as sticks representation with purple carbons, nitrogen atoms in blue, oxygen in red and sulphur in dark yellow. b Chemical structure of VHL inhibitors VH032 and VH298. c–e General chemical structures and design of Homo-PROTACs compounds. Linkage sites at the acetyl and phenyl groups are indicated in red
Fig. 1 Structure-guided design of Homo-PROTACs to induce VHL dimerization. a Crystal structures of VHL in complex with VH298 (PDB code 5LLI)19. VHL is shown as a pink surface and the bound ligand as sticks representation with purple carbons, nitrogen atoms in blue, oxygen in red and sulphur in dark yellow. b Chemical structure of VHL inhibitors VH032 and VH298. c–e General chemical structures and design of Homo-PROTACs compounds. Linkage sites at the acetyl and phenyl groups are indicated in redCC BY · PubMed Central
Fig. 2 Synthesis of symmetric homo-PROTAC compounds derivatized from the terminal acetyl group. The shown route yielded symmetric trans-trans CM09, CM10 and CM11 and negative control symmetric cis-cis compound CMP98
Fig. 2 Synthesis of symmetric homo-PROTAC compounds derivatized from the terminal acetyl group. The shown route yielded symmetric trans-trans CM09, CM10 and CM11 and negative control symmetric cis-cis compound CMP98CC BY · PubMed Central
Fig. 3 Synthesis of negative control homo-PROTAC compound derivatized from the terminal acetyl group. Compound CMP99 has cis-trans configuration
Fig. 3 Synthesis of negative control homo-PROTAC compound derivatized from the terminal acetyl group. Compound CMP99 has cis-trans configurationCC BY · PubMed Central
Fig. 4 Homo-PROTACs CM09, CM10 and CM11 induce selective knockdown of the long isoform of VHL. a HeLa cells were treated with 0.1% DMSO, VH032 (150 µM) and 1 µM of the indicated compounds for 10 h. Abundance of individual proteins was analyzed by western blotting using corresponding specific antibodies accordingly after SDS-PAGE. The data demonstrated is a representative example of experiments performed in biological duplicates. b Different cells lines were treated with siRNA targeting VHL proteins or negative control siRNA (for 48 h), as well as with CM11 (1 µM) or 0.1% v/v DMSO for 10 h. The bands observed to selectively disappear in the presence of CM09–11 are closely migrating isoforms of pVHL30 (see refs 52, 53). The data demonstrated is a representative example of experiments performed as four biological replicates
Fig. 4 Homo-PROTACs CM09, CM10 and CM11 induce selective knockdown of the long isoform of VHL. a HeLa cells were treated with 0.1% DMSO, VH032 (150 µM) and 1 µM of the indicated compounds for 10 h. Abundance of individual proteins was analyzed by western blotting using corresponding specific antibodies accordingly after SDS-PAGE. The data demonstrated is a representative example of experiments performed in biological duplicates. b Different cells lines were treated with siRNA targeting VHL proteins or negative control siRNA (for 48 h), as well as with CM11 (1 µM) or 0.1% v/v DMSO for 10 h. The bands observed to selectively disappear in the presence of CM09–11 are closely migrating isoforms of pVHL30 (see refs 52, 53). The data demonstrated is a representative example of experiments performed as four biological replicatesCC BY · PubMed Central
Fig. 5 CM11 induces pVHL30 depletion in a concentration and time-dependent fashion. (left) Dose-response profile of HeLa cells treated with increasing concentration of CM11 for 4 or 24 h. (right) Time-course immunoblots of lysates from HeLa cells treated 1 µM of CM11 up to 48 h. Control treatments of 0.1% DMSO, CoCl2 (100 µM), IOX2 (150 µM), and VH032 (250 µM or 1 µM) are included. All data demonstrated is a representative example of experiments performed in biological duplicates
Fig. 5 CM11 induces pVHL30 depletion in a concentration and time-dependent fashion. (left) Dose-response profile of HeLa cells treated with increasing concentration of CM11 for 4 or 24 h. (right) Time-course immunoblots of lysates from HeLa cells treated 1 µM of CM11 up to 48 h. Control treatments of 0.1% DMSO, CoCl2 (100 µM), IOX2 (150 µM), and VH032 (250 µM or 1 µM) are included. All data demonstrated is a representative example of experiments performed in biological duplicatesCC BY · PubMed Central
Fig. 6 CM11 activity is CRL2VHL and proteasome-dependent. HeLa cells treated with CM11 in the absence or presence of proteasome inhibitor MG132, neddylation inhibitor MLN4924, VHL inhibitor VH032 or PHD2 inhibitor IOX4 as negative control. The data demonstrated here is representative of one biological replicate
Fig. 6 CM11 activity is CRL2VHL and proteasome-dependent. HeLa cells treated with CM11 in the absence or presence of proteasome inhibitor MG132, neddylation inhibitor MLN4924, VHL inhibitor VH032 or PHD2 inhibitor IOX4 as negative control. The data demonstrated here is representative of one biological replicateCC BY · PubMed Central
Fig. 7 Biophysical studies of Homo-PROTACs binding to VHL. a Superposition of the integrated ITC heat curves of CM11 (blue), CMP99 (black) or CMP98 (green) titrations against VCB. b SEC assay of complex formation after incubation of CM11 (red), CMP98 (pale green), CMP99 (purple), VH032 (dotted purple) or DMSO (black) with VCB. c AlphaLISA: intensity values titrating CM09, CM10, CM11 and CMP98 against VCB. Each point is mean ( ± SEM) intensity of four technical replicates
Fig. 7 Biophysical studies of Homo-PROTACs binding to VHL. a Superposition of the integrated ITC heat curves of CM11 (blue), CMP99 (black) or CMP98 (green) titrations against VCB. b SEC assay of complex formation after incubation of CM11 (red), CMP98 (pale green), CMP99 (purple), VH032 (dotted purple) or DMSO (black) with VCB. c AlphaLISA: intensity values titrating CM09, CM10, CM11 and CMP98 against VCB. Each point is mean ( ± SEM) intensity of four technical replicatesCC BY · PubMed Central
Fig. 8 Proposed model for the mechanism of action of Homo-PROTAC CM11
Fig. 8 Proposed model for the mechanism of action of Homo-PROTAC CM11CC BY · PubMed Central

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  1. Uehara, T. et al. Selective degradation of splicing factor CAPERα by anticancer sulfonamides. Nat. Chem. Biol. 13, 675–680 (2017). · DOI · Paper page

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Authors
Maniaci, C., Hughes, S. J., Testa, A., Chen, W., Lamont, D. J., Rocha, S., Alessi, D. R., Romeo, R., & Ciulli, A.
Affiliations
  • University of Dundee (GB)
  • University of Messina (IT)
Corresponding author
Ciulli, Alessio
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2017-10-10
Volume
8
Issue
1
Pages
830-830
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-017-00954-1
PMC
PMC5635026
OpenAlex
W2761240402
Cited by
245
References
75
Citations by year
2026: 16 · 2025: 20 · 2024: 21 · 2023: 36 · 2022: 37 · 2021: 27 · 2020: 37 · 2019: 37 · 2018: 12 · 2017: 1
FWCI
9.94
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • Discovery Research Portal (University of Dundee) (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Newcastle University ePrints (Newcastle Univesity) (cc-by)
Funders
  • Wellcome Trust (100476, 100476/Z/12/Z)
  • Cancer Research UK (C99667/A12918, 12918)
  • Ministero dell’Istruzione, dell’Università e della Ricerca
  • Medical Research Council (MC_UU_12016, MC_UU_12016/2)
  • FP7 Ideas: European Research Council (311460)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Degradation and Inhibitors
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ubiquitin and proteasome pathways
  • Physical Sciences › Chemistry › Organic Chemistry › Chemical Synthesis and Analysis
Keywords
PROTACs, VHL E3 ubiquitin ligase, ligand-induced dimerization, proteasomal degradation, isoform selectivity, ubiquitin-proteasome system, chemical probes
MeSH terms
Humans, Ligands, Dimerization, Ubiquitin, Ubiquitin-Protein Ligases, Proteasome Endopeptidase Complex, Von Hippel-Lindau Tumor Suppressor Protein, Small Molecule Libraries, Proteolysis

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