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Cited by 153Protein Degradation and InhibitorsUbiquitin and proteasome pathwaysChemical Synthesis and Analysis

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Systematic exploration of different E3 ubiquitin ligases: an approach towards potent and selective CDK6 degraders

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Steinebach, Christian, Ng, Yuen Lam Dora, Sosič, Izidor, Lee, Chih-Shia, Chen, Sirui, Lindner, Stefanie, Vu, Lan Phuong, Bricelj, Aleša, Haschemi, Reza, Monschke, Marius, Steinwarz, Elisabeth, Wagner, Karl G., Bendas, Gerd, Luo, Ji, Gütschow, Michael, Krönke, Jan·2020·Chemical Science
DOI 10.1039/d0sc00167hPMC PMC7552917
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Abstract

Cyclin-dependent kinase 6 (CDK6) is an important regulator of the cell cycle. Together with CDK4, it phosphorylates and inactivates retinoblastoma (Rb) protein.

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Figures

Fig. 1 Chemical structures of the CDK4/6 inhibitors palbociclib (1) and ribociclib (2), selected E3 ligase ligands, i.e. the CRBN ligand pomalidomide (3), the VHL inhibitor VH298 (4), an IAP ligand (5), and the MDM2 antagonist idasanutlin (6), as well as published CDK6 selective PROTACs YKL-06-102 (7), and BSJ-03-123 (8).
Fig. 1 Chemical structures of the CDK4/6 inhibitors palbociclib (1) and ribociclib (2), selected E3 ligase ligands, i.e. the CRBN ligand pomalidomide (3), the VHL inhibitor VH298 (4), an IAP ligand (5), and the MDM2 antagonist idasanutlin (6), as well as published CDK6 selective PROTACs YKL-06-102 (7), and BSJ-03-123 (8).CC BY · PubMed Central
Scheme 1 Synthesis of the CRBN-addressing CDK4/6 degrader 11. Reagents and conditions: (a) Z-6-aminohexanoic acid, DCC, DMAP, tBuOH, CH2Cl2, rt, 16 h; (b) Pd/C, H2, EtOAc, 16 h, rt; (c) DIPEA, DMSO, 90 °C, 24 h; (d) TFA, CH2Cl2, 40 °C, 2 h; (e) HATU, DMSO, rt, 16 h.
Scheme 1 Synthesis of the CRBN-addressing CDK4/6 degrader 11. Reagents and conditions: (a) Z-6-aminohexanoic acid, DCC, DMAP, tBuOH, CH2Cl2, rt, 16 h; (b) Pd/C, H2, EtOAc, 16 h, rt; (c) DIPEA, DMSO, 90 °C, 24 h; (d) TFA, CH2Cl2, 40 °C, 2 h; (e) HATU, DMSO, rt, 16 h.CC BY · PubMed Central
Scheme 2 Synthesis of the VHL-addressing CDK4/6 degrader 34 with a different connection of the linkers. Reagents and conditions: (a) 2-[2-(2-chloroethoxy)ethoxy]ethanol, MsCl, Et3N, CH2Cl2, rt, 16 h; (b) (i) Cs2CO3, DMF, rt, 16 h; (ii) 60 °C, 3 h; (c) NaI, acetone, 60 °C, 48 h; (d) DIPEA, DMSO/DMF, 80 °C, 24 h.
Scheme 2 Synthesis of the VHL-addressing CDK4/6 degrader 34 with a different connection of the linkers. Reagents and conditions: (a) 2-[2-(2-chloroethoxy)ethoxy]ethanol, MsCl, Et3N, CH2Cl2, rt, 16 h; (b) (i) Cs2CO3, DMF, rt, 16 h; (ii) 60 °C, 3 h; (c) NaI, acetone, 60 °C, 48 h; (d) DIPEA, DMSO/DMF, 80 °C, 24 h.CC BY · PubMed Central
Fig. 2 CDK6 degradation by CRBN-based degrader 11 is conserved in murine cell lines. (A) Human cell lines MM.1S and murine cell lines 32D and Ba/F3 were treated with vehicle, pomalidomide (POM), CDK6-selective degrader BSJ-03-123 (8), negative control 21, or PROTAC 11 at 0.1 μM for 16 h; (B) CDK6-degradation is induced in a dose-dependent manner in murine cells. Murine cell lines 32D and Ba/F3 were treated with PROTAC 11 at indicated concentrations for 16 h.
Fig. 2 CDK6 degradation by CRBN-based degrader 11 is conserved in murine cell lines. (A) Human cell lines MM.1S and murine cell lines 32D and Ba/F3 were treated with vehicle, pomalidomide (POM), CDK6-selective degrader BSJ-03-123 (8), negative control 21, or PROTAC 11 at 0.1 μM for 16 h; (B) CDK6-degradation is induced in a dose-dependent manner in murine cells. Murine cell lines 32D and Ba/F3 were treated with PROTAC 11 at indicated concentrations for 16 h.CC BY · PubMed Central
Fig. 3 VHL-based PROTACs induce strong and selective CDK6 degradation. MM.1S cells were treated with 0.1 μM VH298 (4), the degrader BSJ-03-123 (8), negative control 32, or PROTACs 28–31 and improved PROTACs 33–34 for 16 h.
Fig. 3 VHL-based PROTACs induce strong and selective CDK6 degradation. MM.1S cells were treated with 0.1 μM VH298 (4), the degrader BSJ-03-123 (8), negative control 32, or PROTACs 28–31 and improved PROTACs 33–34 for 16 h.CC BY · PubMed Central
Fig. 4 VHL-based PROTAC 34 of the ‘phenoxy’ subseries (A) induces CDK6-selective degradation in a dose-dependent manner (B). MM.1S cells were treated with 1 μM of the VHL ligand VH298, BSJ-03-123 (8), negative control 32, or PROTAC 34 at the indicated concentrations for 16 h. (C) Quantification of (B) and calculation of the DC50-value. The value for 1 μM was excluded due to a slight hook effect. Mechanistic characterization of 34; the activity is CRL2VHL- and proteasome-dependent (D). Co-treatment of 34 and VH298 (100-fold excess) abrogated CDK4/6 degradation. MG132 and MLN4924 prevented proteasomal degradation of CDK4/6. MM.1S cells were treated with MG132, MLN4924 or VH298 in the presence or absence of CDK4/6d 34 (0.1 μM). (E) Persisting effects of PROTAC 34 on CDK4/6 degradation after drug washout. MM.1S cells were treated with PROTACs at 0.1 μM for 16 h before washout with PBS (=0 h), then kept in plain media until indicated time points.
Fig. 4 VHL-based PROTAC 34 of the ‘phenoxy’ subseries (A) induces CDK6-selective degradation in a dose-dependent manner (B). MM.1S cells were treated with 1 μM of the VHL ligand VH298, BSJ-03-123 (8), negative control 32, or PROTAC 34 at the indicated concentrations for 16 h. (C) Quantification of (B) and calculation of the DC50-value. The value for 1 μM was excluded due to a slight hook effect. Mechanistic characterization of 34; the activity is CRL2VHL- and proteasome-dependent (D). Co-treatment of 34 and VH298 (100-fold excess) abrogated CDK4/6 degradation. MG132 and MLN4924 prevented proteasomal degradation of CDK4/6. MM.1S cells were treated with MG132, MLN4924 or VH298 in the presence or absence of CDK4/6d 34 (0.1 μM). (E) Persisting effects of PROTAC 34 on CDK4/6 degradation after drug washout. MM.1S cells were treated with PROTACs at 0.1 μM for 16 h before washout with PBS (=0 h), then kept in plain media until indicated time points.CC BY · PubMed Central
Fig. 5 (A) Quantification of long-term treatment experiments (see Fig. S9, ESI†) and (B) drug stability data at pH 1 and pH 7.4. Acetonitrile solutions of the PROTACs were mixed with two different buffers and incubated for 24 hours at 37 °C. Subsequently, aliquots were analysed by LC/MS and normalized to acetonitrile solutions.
Fig. 5 (A) Quantification of long-term treatment experiments (see Fig. S9, ESI†) and (B) drug stability data at pH 1 and pH 7.4. Acetonitrile solutions of the PROTACs were mixed with two different buffers and incubated for 24 hours at 37 °C. Subsequently, aliquots were analysed by LC/MS and normalized to acetonitrile solutions.CC BY · PubMed Central
Fig. 6 (A) CDK6d 27 and 34 show more pronounced effects on cell viability in the human HEL cells compared to palbociclib (palbo). For cell viability, cells were treated with palbo, 27, or 34 for 96 h. (B) CDK6 degradation after treatment with PROTACs 27 and 34 for 16 h was confirmed by western blotting.
Fig. 6 (A) CDK6d 27 and 34 show more pronounced effects on cell viability in the human HEL cells compared to palbociclib (palbo). For cell viability, cells were treated with palbo, 27, or 34 for 96 h. (B) CDK6 degradation after treatment with PROTACs 27 and 34 for 16 h was confirmed by western blotting.CC BY · PubMed Central
Fig. 7 (A) IAP-based degrader 35 induces CDK4/6 degradation in a dose-dependent manner. MM.1S cells were treated with the IAP ligand 5, the CDK6-selective degrader BSJ-03-123, negative control 36, or degrader 35 at the indicated concentrations for 16 h. In addition to the target proteins CDK4/6, the expression levels of relevant members of the inhibitor of apoptosis (IAP) protein family were blotted; (B) IAP-based PROTAC 35 is more as potent as palbociclib (palbo) and ligand 5 on decreasing cell viability in the multiple myeloma cell line MM.1S and the acute lymphoblastic leukemia cell line Nalm-6. Cells were treated with palbo, 5, or 35 for 96 h at 0.1 μM or 1 μM. All results were normalized to non-treated conditions and data represent mean ± SD of biological triplicates.
Fig. 7 (A) IAP-based degrader 35 induces CDK4/6 degradation in a dose-dependent manner. MM.1S cells were treated with the IAP ligand 5, the CDK6-selective degrader BSJ-03-123, negative control 36, or degrader 35 at the indicated concentrations for 16 h. In addition to the target proteins CDK4/6, the expression levels of relevant members of the inhibitor of apoptosis (IAP) protein family were blotted; (B) IAP-based PROTAC 35 is more as potent as palbociclib (palbo) and ligand 5 on decreasing cell viability in the multiple myeloma cell line MM.1S and the acute lymphoblastic leukemia cell line Nalm-6. Cells were treated with palbo, 5, or 35 for 96 h at 0.1 μM or 1 μM. All results were normalized to non-treated conditions and data represent mean ± SD of biological triplicates.CC BY · PubMed Central
Fig. 8 (A) Selectivity profile of CDK4/6d hijacking four different E3 ligases. (B) Radar plot of molecular descriptors of highly active and less active CDK6 degrading PROTACs (see Tables S1–S4, ESI†). Average values for the recently published analysis of more than 400 degraders (‘Maple set’) are given.
Fig. 8 (A) Selectivity profile of CDK4/6d hijacking four different E3 ligases. (B) Radar plot of molecular descriptors of highly active and less active CDK6 degrading PROTACs (see Tables S1–S4, ESI†). Average values for the recently published analysis of more than 400 degraders (‘Maple set’) are given.CC BY · PubMed Central

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  1. Schumacher, 1965, Br. J. Pharmacol., 25, 324

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Metadata

Authors
Steinebach, C., Ng, Y. L. D., Sosič, I., Lee, C.-S., Chen, S., Lindner, S., Vu, L. P., Bricelj, A., Haschemi, R., Monschke, M., Steinwarz, E., Wagner, K. G., Bendas, G., Luo, J., Gütschow, M., & Krönke, J.
Affiliations
  • University of Bonn (DE)
  • Universität Ulm (DE)
  • University of Ljubljana (SI)
  • National Cancer Institute (US)
Corresponding author
  • Gütschow, Michael
  • Krönke, Jan
ORCID
Venue
Chemical Science
Publisher
Royal Society of Chemistry (RSC)
Published
2020
Volume
11
Issue
13
Pages
3474-3486
ISSN
  • 2041-6520 (ISSN-L)
  • 2041-6539
DOI
10.1039/d0sc00167h
PMC
PMC7552917
OpenAlex
W3009753365
Cited by
148
References
77
Citations by year
2026: 20 · 2025: 24 · 2024: 27 · 2023: 20 · 2022: 27 · 2021: 22 · 2020: 8
FWCI
6.56
Type
journal article
Language
en
Open access
Licence
Locations
  • Chemical Science (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
  • Deutsche Forschungsgemeinschaft (FOR2372, Kr-3886/2-1, SFB1074)
  • Javna Agencija za Raziskovalno Dejavnost RS (P1-0208)
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, CDK4/6, E3 ubiquitin ligase, VHL, cIAP1, targeted protein degradation, tumor cell proliferation, leukemia, multiple myeloma, breast cancer

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