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Cited by 433Protein Degradation and InhibitorsUbiquitin and proteasome pathwaysCancer-related Molecular Pathways

The CDK inhibitor CR8 acts as a molecular glue degrader that depletes cyclin K

Open access
Słabicki, Mikołaj, Kozicka, Zuzanna, Petzold, Georg, Li, Yen-Der, Manojkumar, Manisha, Bunker, Richard D., Donovan, Katherine A., Sievers, Quinlan L. +16 more·2020·Nature
DOI 10.1038/s41586-020-2374-xPMC PMC7486275
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Abstract

Molecular glue compounds induce protein-protein interactions that, in the context of a ubiquitin ligase, lead to protein degradation 1. Unlike traditional enzyme inhibitors, these molecular glue degraders act substoichiometrically to catalyse the rapid depletion of previously inaccessible targets 2. They are clinically effective and highly sought-after, but have thus far only been discovered serendipitously. Here, through systematically mining databases for correlations between the cytotoxicity of 4,518 clinical and preclinical small molecules and the expression levels of E3 ligase components across hundreds of human cancer cell lines 3-5, we identify CR8-a cyclin-dependent kinase (CDK) inhibitor 6-as a compound that acts as a molecular glue degrader. The CDK-bound form of CR8 has a solvent-exposed pyridyl moiety that induces the formation of a complex between CDK12-cyclin K and the CUL4 adaptor protein DDB1, bypassing the requirement for a substrate receptor and presenting cyclin K for ubiquitination and degradation. Our studies demonstrate that chemical alteration of surface-exposed moieties can confer gain-of-function glue properties to an inhibitor, and we propose this as a broader strategy through which target-binding molecules could be converted into molecular glues.

AI snapshot

Written from the paper’s full text.

TL;DR

Systematic mining of drug-sensitivity and E3-ligase expression data across cancer cell lines identified CR8 as a molecular glue degrader. CR8-bound CDK12 recruits DDB1 to ubiquitinate and deplete cyclin K, with a surface-exposed pyridyl group conferring the glue activity.

Key findings

  • A correlation screen of 4,518 clinical/preclinical compounds across 578 cancer cell lines and 499 E3 ligase components linked R-CR8 toxicity to DDB1 expression; DDB1-targeting sgRNAs conferred resistance. Proteome-wide mass spectrometry of >8,000 proteins found cyclin K as the only consistently decreased protein after R-CR8, and degradation was rescued by MLN7243, MLN4924, and MG132.
  • CRISPR screens implicated DDB1, CUL4B, RBX1, NEDD8, and NAE1/UBA3, but no canonical DCAF substrate receptor. In vitro, CR8 induced CDK12-cycK binding to DDB1 with an affinity range of 100–500 nM and strengthened the complex 500- to 1000-fold by ITC; the CUL4A-RBX1-DDB1 core alone supported cyclin K ubiquitination.
  • A 3.5 Å crystal structure showed CR8 bridging CDK12 and DDB1, with CDK12 acting as a drug-induced substrate receptor. CDK13 was recruited less efficiently than CDK12, while CDK9 was not; the 2-pyridyl/phenylpyridine moiety was required, as roscovitine and DRF053 bound more weakly and failed to degrade the reporter, and flavopiridol supported in vitro ubiquitination but not cellular degradation.
  • DDB1 depletion reduced CR8 cytotoxicity about ten-fold but did not affect other CDK inhibitors; CRBN overexpression decreased cyclin K degradation and altered CR8 sensitivity.

Methods

  • Bioinformatic mining of PRISM viability data for 4,518 compounds across 578 cell lines, correlated with CCLE expression and copy-number data for 499 E3 ligase components, followed by CRISPR-based validation of drug–E3 pairs.
  • Cellular work used HEK293T and other Cas9-expressing lines, a cycKeGFP/mCherry stability reporter, genome-wide and E3-focused CRISPR screens, DCAF arrayed screens, quantitative proteomics, qPCR, immunoblots, and drug-sensitivity assays.
  • Biochemical and structural work used recombinant CDK12-cycK and DDB1 proteins, co-immunoprecipitation, in vitro ubiquitination, TR-FRET, ITC, BRET, and crystallization with structure determination at 3.5 Å.

Limitations

  • The paper does not state a dedicated limitations section. Evident from the text: no canonical DCAF was identified despite multiple screens, and CDK12 autoubiquitination is proposed but not demonstrated.
  • Evident: structural density for the CDK12 C-terminal region was only tentatively assigned because of multiple conformations, and cellular degradation did not always track with in vitro ubiquitination results.
  • Evident: the genome-wide CR8 resistance screen used one DMSO and one CR8 condition, and the proteomics experiment had a single CR8 replicate, limiting statistical robustness.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1038/s41586-020-2374-x

Cites

52 of 52 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

23 of 38 cited sources checked · 15 supported · 0 flagged · accuracy not yet measured

  • Findingcitation

    THZ531, a bulky covalent CDK12/13 inhibitor predicted to clash with DDB1, locks the CDK12 C-terminal extension in a conformation incompatible with DDB1 recruitment.

    “THZ53124, a bulky covalent CDK12/13 inhibitor predicted to clash with DDB1 (Extended Data Fig. 6d–f), locks the CDK12 C-terminal extension in a conformation incompatible with DDB1 recruitment (Extended Data Fig. 6d).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [24] Zhang, T. et al. Covalent targeting of remote cysteine residues to develop CDK12 and CDK13 inhibitors. Nat. Chem. Biol. 12, 876–884 (2016). source
  • Premisecitation

    This behavior resembles CRL4CRBN and thalidomide analogues, where an apparent affinity threshold must be crossed to drive drug-induced target degradation.

    “This behaviour is reminiscent of CRL4CRBN and thalidomide analogues11,26, where an apparent affinity threshold must be overcome to drive drug-induced target degradation.”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [11] Petzold, G., Fischer, E. S. & Thomä, N. H. Structural basis of lenalidomide-induced CK1α degradation by the CRL4CRBN ubiquitin ligase. Nature 532, 127–130 (2016). source
    • cites [26] Sievers, Q. L. et al. Defining the human C2H2 zinc finger degrome targeted by thalidomide analogs through CRBN. Science 362, eaat0572 (2018). source
  • Findingown data

    Mining correlations between cytotoxicity of 4,518 clinical and preclinical small molecules and E3 ligase expression across hundreds of cancer cell lines identified CR8, a CDK inhibitor, as a molecular glue degrader.

    “Through systematic mining of databases for correlations between the cytotoxicity of 4,518 clinical and pre-clinical small molecules and E3 ligase expression levels across hundreds of human cancer cell lines,3–5 we identified CR8, a cyclin-dependent kinase (CDK) inhibitor,6 as a compound that acts as a molecular glue degrader.”
    Quote in the paperSubstantial claim3 of 4 sources checked
    • cites [3] Yu, C. et al. High-throughput identification of genotype-specific cancer vulnerabilities in mixtures of barcoded tumor cell lines. Nat. Biotechnol. 34, 419–423 (2016). source

      The Abstract (page unavailable) describes correlating compound sensitivity with gene expression across cancer cell lines, supporting the database-mining approach attributed to this source. But this paper reports screening 8,400 compounds in 102 lines, not 4,518 compounds across hundreds of lines, and the cited passages do not identify E3 ligase expression as the correlate. The sentence presents CR8's identification as the citing authors’ own finding, not this source’s. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [4] Corsello, S. M. et al. Discovering the anticancer potential of non-oncology drugs by systematic viability profiling. Nat. Cancer 1, 235–248 (2020). source

      The Abstract (page unavailable) describes a resource measuring the growth-inhibitory activity of 4,518 drugs across 578 human cancer cell lines, and Results, ‘Predictive models of killing activity’ (page unavailable), reports that mRNA expression predicted drug response. The cited passages do not establish the more specific correlation with E3 ligase expression. Citations 3–5 attach to the database-mining clause; the sentence presents CR8’s identification as the citing authors’ own finding, not this source’s. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [5] Ghandi, M. et al. Next-generation characterization of the Cancer Cell Line Encyclopedia. Nature 569, 503–508 (2019). source
    • cites [6] Bettayeb, K. et al. CR8, a potent and selective, roscovitine-derived inhibitor of cyclin-dependent kinases. Oncogene 27, 5797–5807 (2008). source
  • Findingown data

    CDK12, a known CR8 target whose activity requires cycK, is a crucial component for CR8-induced destabilization of cycKeGFP.

    “In addition, we identified cyclin-dependent kinase 12 (CDK12), which is a known target of CR819 and whose activity depends on the interaction with cycK20, as a crucial component for CR8-induced cycKeGFP destabilisation (Fig. 1g, Extended Data Fig. 2h–k).”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [19] Bösken, C. A. et al. The structure and substrate specificity of human Cdk12/Cyclin K. Nat. Commun. 5, 3505 (2014). source

      Citation 20 is attached to the claim that CDK12 activity depends on its interaction with cyclin K, not to the CR8-target or destabilization claims. The Results sections on the CDK12/cyclin K structure and CTD kinase activity describe their interaction and an active complex, but the passages do not establish that CDK12 activity requires cyclin K binding. (LLM, conf=0.78)

      Experimental — accuracy not yet measured
    • cites [20] Cheng, S.-W. G. et al. Interaction of cyclin-dependent kinase 12/CrkRS with cyclin K1 is required for the phosphorylation of the C-terminal domain of RNA polymerase II. Mol. Cell. Biol. 32, 4691–4704 (2012). source
  • Findingown data

    The N- and C-lobes of CDK12 lie close to DDB1 residues in BPA loop 111-114, BPC helix 2 986-990, and C-terminal loop 1078-1081, which are otherwise involved in DCAF binding.

    “The N- and C-lobes of CDK12 are proximal to DDB1 residues located in a loop of the BPA domain (amino acid (aa) 111–114), BPC-helix 2 (aa 986–990), and a loop in the C-terminal domain (aa 1078–1081) that are otherwise involved in DCAF binding (Extended Data Fig. 5).”
    Quote in the paperSubstantial claim
  • Classified as probably not a substantial claim. 17
  • May combine two claims. 38

  • Classified as probably not a substantial claim.

    Premisecitation

    Molecular glue compounds cause protein-protein interactions that, when a ubiquitin ligase is involved, result in protein degradation.

    “Molecular glue compounds induce protein-protein interactions that, in the context of a ubiquitin ligase, lead to protein degradation.1”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [1] Stanton, B. Z., Chory, E. J. & Crabtree, G. R. Chemically induced proximity in biology and medicine. Science 359, eaao5902 (2018). source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Aryl sulphonamides degrade RBM39 by engaging DCAF15, the substrate receptor of the CRL4DCAF15 E3 ligase.

    “Similarly, aryl sulphonamides degrade the essential RNA-binding protein RBM39 by engaging DCAF15, the substrate receptor of the CRL4DCAF15 E3 ubiquitin ligase13–15.”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites [13] Han, T. et al. Anticancer sulfonamides target splicing by inducing RBM39 degradation via recruitment to DCAF15. Science 356, eaal3755 (2017). source
    • cites [14] Uehara, T. et al. Selective degradation of splicing factor CAPERα by anticancer sulfonamides. Nat. Chem. Biol. 13, 675–680 (2017). source
    • cites [15] Faust, T. B. et al. Structural complementarity facilitates E7820-mediated degradation of RBM39 by DCAF15. Nat. Chem. Biol. 16, 7–14 (2020). source

  • May combine two claims.

    Interpretationcitation

    The nucleotide-bound conformation of CDK12 appears incompatible with approaching DDB1, while its C-terminal extension can adopt multiple conformations.

    “suggesting that although the nucleotide-bound conformation of CDK12 seems incompatible with approaching DDB1 (Extended Data Fig. 6b), its C-terminal extension is free to adopt multiple conformations21.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [21] Dixon-Clarke, S. E., Elkins, J. M., Cheng, S. W. G., Morin, G. B. & Bullock, A. N. Structures of the CDK12/CycK complex with AMP-PNP reveal a flexible C-terminal kinase extension important for ATP binding. Sci. Rep. 5, 17122 (2015). source

  • Classified as probably not a substantial claim.

    Premisecitation

    By changing ubiquitin ligase selectivity, these compounds redirect the ligase to carry out repeated rounds of target ubiquitination catalytically.

    “By reprogramming ubiquitin ligase selectivity, these molecules divert the ligase to drive multiple rounds of target ubiquitination in a catalytic manner16.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [16] Bondeson, D. P. et al. Catalytic in vivo protein knockdown by small-molecule PROTACs. Nat. Chem. Biol. 11, 611–617 (2015). source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Thalidomide analogues bring zinc-finger transcription factors and other targets to CRBN, the substrate receptor of the CRL4CRBN E3 ligase.

    “Thalidomide analogues recruit zinc-finger transcription factors and other targets to CRBN8–11, the substrate receptor of the cullin-RING E3 ubiquitin ligase CUL4A/B-RBX1-DDB1-CRBN (CRL4CRBN)12.”
    Quote in the paperNot a substantial claim3 of 5 sources checked
    • cites [8] Lu, G. et al. The myeloma drug lenalidomide promotes the cereblon-dependent destruction of Ikaros proteins. Science 343, 305–309 (2014). source

      The paper’s indexed Abstract reports that lenalidomide promotes binding of the zinc-finger transcription factors IKZF1 and IKZF3 to cereblon, and identifies cereblon as a substrate-recognition component of a cullin-dependent ubiquitin ligase. It does not establish recruitment of other targets; it says additional cereblon substrates might exist. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [9] Krönke, J. et al. Lenalidomide causes selective degradation of IKZF1 and IKZF3 in multiple myeloma cells. Science 343, 301–305 (2014). source

      The paper reports that lenalidomide increases binding of the zinc-finger transcription factors IKZF1 and IKZF3 to CRBN, which it identifies as a CRL4 substrate receptor (Abstract; page unavailable). It does not establish recruitment of other targets: it says alternative substrates may mediate other effects. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [10] Matyskiela, M. E. et al. A novel cereblon modulator recruits GSPT1 to the CRL4CRBN ubiquitin ligase. Nature 535, 252–257 (2016). source
    • cites [11] Petzold, G., Fischer, E. S. & Thomä, N. H. Structural basis of lenalidomide-induced CK1α degradation by the CRL4CRBN ubiquitin ligase. Nature 532, 127–130 (2016). source
    • cites [12] Fischer, E. S. et al. Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide. Nature 512, 49–53 (2014). source

  • Classified as probably not a substantial claim.

    Premisecitation

    CR8 is a broad CDK inhibitor reported to bind CDK1, CDK2, CDK3, CDK5, CDK7, CDK9, and CDK12.

    “CR8 is a pleiotropic CDK inhibitor reported to bind CDK1/2/3/5/7/9/126,19”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [6] Bettayeb, K. et al. CR8, a potent and selective, roscovitine-derived inhibitor of cyclin-dependent kinases. Oncogene 27, 5797–5807 (2008). source
    • cites [19] Bösken, C. A. et al. The structure and substrate specificity of human Cdk12/Cyclin K. Nat. Commun. 5, 3505 (2014). source

      The Results section, “Inhibition of Cdk12 compared with Cdk9” (page not available), reports that CR8 inhibits Cdk12 and is a potent inhibitor of Cdk9. Our copy does not establish CR8 binding to either kinase or address its activity against CDK1, CDK2, CDK3, CDK5, or CDK7. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Molecular glue degraders, unlike classical enzyme inhibitors, work catalytically at sub-stoichiometric amounts to rapidly remove targets that were previously hard to reach.

    “Unlike traditional enzyme inhibitors, such molecular glue degraders act sub-stoichiometrically to catalyse rapid depletion of previously inaccessible targets.2”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [2] Chopra, R., Sadok, A. & Collins, I. A critical evaluation of the approaches to targeted protein degradation for drug discovery. Drug Discov. Today. Technol. 31, 5–13 (2019). source

      The section “E3 ligase receptors as templates for targeted degradation” (page unavailable) reports that IMiD compounds induce degradation of transcription factors and other proteins. But the paper’s explicit discussion of catalytic turnover, low target occupancy compared with classical inhibitors, and depletion within minutes to hours appears in the PROTACs section and concerns heterobifunctional degraders. It does not establish those properties, or that the targets were previously inaccessible, for molecular glue degraders. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Known cullin-RING ligases use specific substrate receptors, and DDB1 binds more than 20 DCAFs to bring them to the CUL4-RBX1 core.

    “Thus far, all known cullin-RING ligases engage their substrates through specific substrate receptors, and DDB1 serves as an adaptor protein able to bind over 20 such receptors (also known as DDB1-CUL4-associated-factors, DCAFs)17,18 to recruit them to the CUL4-RBX1 ligase core.”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [17] Angers, S. et al. Molecular architecture and assembly of the DDB1-CUL4A ubiquitin ligase machinery. Nature 443, 590–593 (2006). source
    • cites [18] Lee, J. & Zhou, P. DCAFs, the missing link of the CUL4-DDB1 ubiquitin ligase. Mol. Cell 26, 775–780 (2007). source

  • May combine two claims.

    Findingcitation

    THZ531 lowered the TR-FRET signal and reduced in vitro cycK ubiquitination below DMSO control levels.

    “Consistently, THZ531 further decreased the TR-FRET signal and diminished cycK ubiquitination in vitro below DMSO control levels (Fig. 3d, Extended Data Fig. 6c, f)24.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [24] Zhang, T. et al. Covalent targeting of remote cysteine residues to develop CDK12 and CDK13 inhibitors. Nat. Chem. Biol. 12, 876–884 (2016). source

      The ‘THZ531 covalently inhibits CDK12 and 13’ section reports kinase inhibition, and the ‘In vitro kinase assays’ section describes phosphorylation measurements, but neither reports a TR-FRET signal or cyclin K ubiquitination. Searches for TR-FRET and cyclin K ubiquitination found no passage addressing the claimed reductions relative to DMSO. Page numbers were not available in our copy. (LLM, conf=0.75)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    The density for the CDK12 C-terminal region was only tentatively assigned, likely because of multiple conformations, but the C-terminal tail clearly engages DDB1 and adopts a conformation different from isolated CDK12-cycK structures.

    “The density for this region could only be tentatively assigned, likely due to the presence of multiple conformations, but the CDK12 C-terminal tail clearly engages with DDB1 and assumes a conformation different from those seen in isolated CDK12-cycK structures (Extended Data Fig. 6a, b, d)19,21.”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [19] Bösken, C. A. et al. The structure and substrate specificity of human Cdk12/Cyclin K. Nat. Commun. 5, 3505 (2014). source
      Experimental — accuracy not yet measured
    • cites [21] Dixon-Clarke, S. E., Elkins, J. M., Cheng, S. W. G., Morin, G. B. & Bullock, A. N. Structures of the CDK12/CycK complex with AMP-PNP reveal a flexible C-terminal kinase extension important for ATP binding. Sci. Rep. 5, 17122 (2015). source

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Drug-induced interactions involving a ubiquitin ligase can cause protein degradation, an emerging way to inactivate therapeutic targets that conventional pharmacology cannot address.

    “In the context of a ubiquitin ligase, drug-induced interactions can lead to protein degradation, which is an emerging strategy for the inactivation of therapeutic targets intractable by conventional pharmacological means2.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [2] Chopra, R., Sadok, A. & Collins, I. A critical evaluation of the approaches to targeted protein degradation for drug discovery. Drug Discov. Today. Technol. 31, 5–13 (2019). source

  • Classified as probably not a substantial claim.

    Premisecitation

    Molecular glues are small-molecule drugs that induce or stabilize protein-protein interactions.

    “Molecular glues are a class of small molecule drugs that induce or stabilise protein-protein interactions1.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [1] Stanton, B. Z., Chory, E. J. & Crabtree, G. R. Chemically induced proximity in biology and medicine. Science 359, eaao5902 (2018). source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductown data

    MOLPROBITY analysis shows that 93.9% of residues in the final model are in favorable Ramachandran regions, with 0.6% outliers.

    “Analysis with MOLPROBITY48. indicates that 93.9% of the residues in final model are in favourable regions of the Ramachandran plot, with 0.6% outliers.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [48] Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010). source

  • May combine two claims.

    Findingown data

    The TR-FRET affinity rank order correlated with in vitro cycK ubiquitination, with DRF053 and roscovitine producing less processive ubiquitination.

    “The affinity rank-order observed in our TR-FRET assay correlated with the degree of cycK ubiquitination in vitro, in which DRF053 and roscovitine showed less processive ubiquitination (Fig. 3d).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    DCAF15 expression correlated with indisulam and tasisulam toxicity, matching its known role in degrading RBM39 through CRL4DCAF15.

    “DCAF15 gene expression correlated with indisulam and tasisulam toxicity, consistent with its known function as a degrader of the essential protein RBM39 by the CRL4DCAF15 E3 ubiquitin ligase, thus demonstrating the potential of the approach (Extended Data Fig. 1b, c).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Flavopiridol produced moderate cycK ubiquitination in vitro but did not degrade cycK in cells.

    “While flavopiridol gave rise to moderate cycK ubiquitination in vitro (Fig. 3d), it failed to degrade cycK in cells (Fig. 3e).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Inhibiting the E1 ubiquitin-activating enzyme, cullin neddylation, or the proteasome rescued compound-induced cycK degradation.

    “compound-induced cycK degradation could be rescued by inhibition of the E1 ubiquitin-activating enzyme (MLN7243), cullin neddylation (MLN4924) and the proteasome (MG132) (Fig. 1d).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Roscovitine, the parent compound that lacks the 2-pyridyl but keeps the proximal phenyl ring, also facilitated complex formation, with three-fold lower apparent affinity.

    “Roscovitine23, the parent compound of CR8 that lacks the 2-pyridyl substituent but retains the phenyl ring proximal to Arg928 (Fig. 3a, c), also facilitated complex formation, albeit with a three-fold lower apparent affinity (Extended Data Fig. 8a).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [23] Meijer, L. et al. Biochemical and cellular effects of roscovitine, a potent and selective inhibitor of the cyclin-dependent kinases cdc2, cdk2 and cdk5. Eur. J. Biochem. 243, 527–536 (1997). source

  • May combine two claims.

    Findingown data

    Mutating DDB1 residues Ile909, Arg928, or Arg947 each reduced CR8-induced recruitment of CDK12.

    “Mutation of DDB1 residues Ile909, Arg928, and Arg947 each diminished drug-induced recruitment of the kinase (Extended Data Fig. 5f), highlighting the contribution of the phenylpyridine moiety to complex formation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Mutating or truncating the CDK12 C-terminal extension abolished basal CDK12-DDB1 binding, while CR8 could still promote complex formation to varying degrees.

    “Mutations in, or truncation of, the CDK12 C-terminal extension (Extended Data Fig. 5c) abolished basal binding between CDK12 and DDB1, whereas complex formation could still be facilitated by CR8 to a varying extent (Extended Data Fig. 7h, i).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In screens against 29 DCAF or DCAF-like genes across four cell lines, sgRNAs against CUL4-RBX1-DDB1 components caused CR8 resistance, but no DCAF substrate receptor was found.

    “While sgRNAs targeting the previously identified components of the CUL4-RBX1-DDB1 complex consistently caused resistance to CR8, a DCAF substrate receptor could not be identified (Extended Data Fig. 3).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The main sequence difference between CDK9 and CDK12/13 is the C-terminal extension, which in the structure contacts DDB1 BPA and BPC.

    “The key difference between CDK9 and CDK12/13 primary sequence lies in the C-terminal extension (Extended Data Fig. 7a, b), which in our structure nestles up against DDB1 BPA and BPC propellers (Fig. 2d, Extended Data Fig. 5i).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    CR8 occupies the CDK12 ATP pocket and makes discrete contacts with DDB1 BPC residues through its hydrophobic phenylpyridine ring system, burying about 150 Å2.

    “CR8 occupies the ATP binding pocket of CDK12 and forms discrete contacts with residues in the BPC domain of DDB1 (~150 Å2) through its hydrophobic phenylpyridine ring system (Fig. 3a, b).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In vitro, CDK12-cycK did not markedly enrich DDB1 without CR8, but equimolar CR8 produced stoichiometric complex formation.

    “The kinase domain of CDK12 (CDK12713−1052) bound to cycK1−267 did not markedly enrich DDB1 over the bead binding control in the absence of CR8, whereas equimolar amounts of the compound led to stoichiometric complex formation (Fig. 2a).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    CDK13, which is closely related to CDK12, but not the more divergent CDK9, was recruited to DDB1 by CR8, though with lower affinity.

    “The closely related CDK13 (90.8 % sequence identity), but not the more divergent CDK9 (45.5 % sequence identity) (Extended Data Fig. 7a–c), was recruited to DDB1 in the presence of CR8, albeit with a lower binding affinity (Extended Data Fig. 7d–f).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    CR8 binds the CDK12 active site and bridges the CDK12-DDB1 interface, while cycK binds CDK12 on the opposite side and does not contact DDB1.

    “CR8 binds the active site of CDK12 and bridges the CDK12-DDB1 interface, while cycK binds CDK12 on the opposite site and does not contact DDB1.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    A weak CDK12-cycK-DDB1 interaction was detectable without compound in vitro, and CR8 strengthened complex formation 500- to 1000-fold by ITC.

    “While weak CDK12-cycK-DDB1 interaction was still detectable in the absence of the compound in vitro, CR8 strengthened complex formation 500- to 1000-fold as estimated by isothermal titration calorimetry (ITC) (Extended Data Fig. 4f–k).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    DDB1 is involved in CR8-mediated cycK degradation but not in cycK degradation that occurs without the compound.

    “validated the involvement of DDB1 in CR8-mediated cycK degradation (Fig. 1g, Extended Data Fig. 2h), but not in compound-independent cycK degradation (Extended Data Fig. 2i).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Neither DRF053 nor roscovitine caused degradation of the cycKeGFP reporter in cells.

    “As neither DRF053 nor roscovitine induced degradation of the cycKeGFP reporter in cells (Fig. 3e)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    sgRNAs targeting DDB1 cause only partial depletion of DDB1, with about half of DDB1 alleles modified and DDB1 protein levels reduced by approximately 50%.

    “sgRNAs targeting DDB1 provide partial depletion of DDB1 (50% DDB1 alleles modified, reducing DDB1 levels by roughly 50%)”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    MLN4924 had only minor effects on viability but decreased CR8 sensitivity, showing that CRL neddylation significantly contributes to CR8 toxicity.

    “Global inhibition of CRL activity by MLN4924 had only minor effects on cell viability (Extended Data Fig. 9a), but resulted in decreased sensitivity to CR8 (Extended Data Fig. 9b), showing that CRL neddylation significantly contributes to CR8 toxicity.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    CRBN overexpression affected CR8 sensitivity and reduced cycK degradation, presumably by lowering the free DDB1 pool.

    “Overexpression of the substrate receptor CRBN also affected sensitivity to CR8 and decreased cycK degradation (Extended Data Fig. 9c–g), presumably by reducing the free pool of DDB1.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Findingown data

    The DDB1ΔBPB-R-CR8-CDK12-cycK complex crystallized in space group P3121, with three complexes in the asymmetric unit.

    “The DDB1ΔBPB–R-CR8-CDK12713−1052-cycK1−267 complex formed crystals belonging space group P3121, with three complexes in the crystallographic asymmetric unit (ASU).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    DDB1-compound interactions are highly plastic, and diverse surface-exposed groups in CR8, DRF053, roscovitine, and flavopiridol can promote CDK12-cycK recruitment.

    “Our results thus show that the DDB1-compound interactions display significant plasticity and that structurally diverse surface-exposed moieties in CR8, DRF053, roscovitine and flavopiridol can facilitate CDK12-cycK recruitment.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    When CR8 is bound to CDK, its exposed pyridyl group makes CDK12-cyclin K associate with DDB1, the CUL4 adaptor, without a substrate receptor, and this presents cyclin K for ubiquitination and degradation.

    “A solvent-exposed pyridyl moiety of CR8, in its CDK-bound form, induces CDK12-cyclin K complex formation with DDB1, the CUL4 adaptor protein, bypassing the requirement for a substrate receptor and presenting cyclin K (cycK) for ubiquitination and degradation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    CR8-bound CDK12-cycK is recruited to the CUL4-RBX1-DDB1 core through DDB1, and CR8 tightens the complex enough to drive cycK degradation without a canonical DCAF.

    “Thus, our data indicate that CR8-engaged CDK12-cycK is recruited to the CUL4-RBX1-DDB1 ligase core through DDB1, and the compound tightens the complex sufficiently to drive CR8-induced cycK degradation in the absence of a canonical DCAF substrate receptor.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    CDK12 acts as a glue-induced substrate receptor and positions cycK where CRL4 substrates normally sit.

    “Taken together, our data demonstrates that CDK12 assumes the role of a glue-induced substrate receptor and places cycK in a position typically occupied by CRL4 substrates (Fig. 2f).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    CR8 induces specific protein-protein interactions between CDK12/13 and DDB1, and the C-terminal extension contributes to binding but is not required for drug-dependent kinase recruitment.

    “Hence, our data show that the pan-selective CDK inhibitor CR8 induces specific protein-protein interactions between CDK12/13 and DDB1 and suggest that the C-terminal extension, while contributing to binding, is not essential for drug-dependent kinase recruitment.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Three technical replicates were performed for each experiment.

    “Three technical replicates were carried out per experiment.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Single-residue mutations designed to increase surface hydrophobicity produce ordered protein assemblies.

    “Accordingly, single residue mutations designed to increase surface hydrophobicity give rise to ordered protein assemblies33.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [33] Garcia-Seisdedos, H., Empereur-Mot, C., Elad, N. & Levy, E. D. Proteins evolve on the edge of supramolecular self-assembly. Nature 548, 244–247 (2017). source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    The sgRNA guide sequences were confirmed by Sanger sequencing.

    “guide sequence confirmed by Sanger sequencing.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    CR8 bypasses the need for a canonical DCAF and instead hijacks the essential adaptor DDB1.

    “CR8 thus bypasses the requirement for a canonical DCAF and instead hijacks the essential adaptor protein DDB1.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The CRL4-dependent gain-of-function glue degrader activity of CR8 strongly contributes to its cellular potency and adds an ortholog-specific layer of CDK inactivation through cycK degradation.

    “Together, the data demonstrate that the CRL4-dependent gain-of-function glue degrader activity of CR8 strongly contributes to its cellular potency and provides an additional layer of ortholog-specific CDK inactivation through cycK degradation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    Bound compounds such as enzyme inhibitors can in principle mimic such amino acid changes and strongly alter the protein interaction landscape, suggesting compound-induced protein-protein interactions may be more common than previously recognized.

    “Bound compounds, such as enzyme inhibitors, can in principle mimic such amino acid changes with dramatic effects on the protein interaction landscape, suggesting that compound-induced protein-protein interactions may be more common than previously recognised.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    CDK12 is not a constitutive E3 ligase component but acts as a drug-induced substrate receptor that links DDB1 to the ubiquitination target.

    “CDK12 is not a constitutive E3 ligase component, but rather serves as a drug-induced substrate receptor, linking DDB1 to the ubiquitination target.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Between one and three replicates were performed for each titration.

    “Between one and three replicates were performed per titration.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Previously reported molecular glue degraders use a ligandable pocket on the ligase to recruit targets, whereas CR8 binds the CDK12 active site and does not need an independent ligand site on DDB1.

    “While previously reported molecular glue degraders engage a ligandable pocket on the ligase to recruit target proteins, CR8 instead binds the active site of CDK12 and does not rely on an independent ligand binding site on DDB1 (Extended Data Fig. 4h).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning

    The partial depletion of DDB1 by sgRNAs suggests that the surviving cells are selected for heterozygous or hypomorphic clones.

    “which suggests selection towards heterozygous or hypomorphic clones.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Surface-exposed single-residue mutations can promote higher-order protein complexes, as the hemoglobin Glu-to-Val mutation induces polymerization in sickle cell anemia.

    “Surface-exposed single residue mutations have been shown to promote the formation of higher-order protein complexes, as the haemoglobin Glu to Val mutation, for example, induces polymerization in sickle cell anaemia32.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [32] Dykes, G. W., Crepeau, R. H. & Edelstein, S. J. Three-dimensional reconstruction of the 14-filament fibers of hemoglobin S. J. Mol. Biol. 130, 451–472 (1979). source
49 of 49

  • The paper relies on this without support.

    Molecular glue degraders have so far been found only by chance, and few strategies exist for finding or designing them.

    “They are clinically effective and highly sought-after, but have thus far only been discovered serendipitously.”
    “Although highly sought-after, molecular glue degraders have only been found serendipitously, and there are currently limited strategies available for identifying or designing such compounds.”

  • The paper relies on this without support.

    Before this work, molecular glue degraders were known to act only through substrate-recruiting modules of E3 ligases.

    “Molecular glue degraders have thus far only been shown to engage substrate-recruiting E3 ligase modules.”

  • The authors state this as an assumption.

    Biotinylated DDB1 binds Alexa488-labeled cycK-CDK12 with equimolar stoichiometry.

    “assuming equimolar binding of biotinDDB1 to Alexa488cycK-CDK12 using the equations described previously8.”

  • The authors state this as an assumption.

    The compound binding follows a single-site model.

    “assuming a single binding site using Prism 7 (GraphPad).”
4 of 4

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • CR8 (225) acts as a molecular glue within cells, triggering Cyclin K's ubiquitination and proteasomal degradation; unlike conventional molecular glues, CR8 bypasses classical E3 ubiquitin ligase substrate receptors like CRBN and DCAF15, binding directly to CDK12, and the CDK12-DDB1 interaction results in Cyclin K's ubiquitination and degradation.

    “CR8 (225), a small molecule, acts as a molecular glue within cells, triggering Cyclin K’s ubiquitination and proteasomal degradation (Table 4). Unlike conventional molecular glues, CR8 bypasses classical E3 ubiquitin ligase substrate receptors like CRBN and DCAF15, binding directly to CDK12. This CDK12-DDB1 interaction results in Cyclin K’s ubiquitination and degradation, offering a unique means”

    From Recent advances in targeting the “undruggable” proteins: from drug discovery to clinical trials (2023) · cites this paper as [530]

  • A panel of CDK12 inhibitors act as MGs to enable a neo-morphic interaction between CDK12 and DDB1, inducing CRL4-catalyzed Cyclin K ubiquitination and degradation.

    “a panel of CDK12 inhibitors have been shown to act as MGs to enable a neo-morphic interaction between CDK12 and DDB1, inducing CRL4-catalyzed Cyclin K ubiquitination and degradation27–29.”

    From Defining molecular glues with a dual-nanobody cannabidiol sensor (2022) · cites this paper as [29]

  • Weak interactions between MG protein partners are echoed by low μM affinities reported for DCAF15-RBM39, DDB1-CDK12-Cyclin K, and JAZ1-COI1.

    “Moreover, their weak interactions are echoed by the low μM affinities recently reported for the DCAF15-RBM39 and DDB1-CDK12-Cyclin K interacting partners22,29 as well as the bipartite degron of JAZ1 that docks to the F-box protein COI1 via an α-helix next to the binding pocket of jasmonate-isoleucine, another MG plant hormone45.”

    From Defining molecular glues with a dual-nanobody cannabidiol sensor (2022) · cites this paper as [29]

  • The CDK12 inhibitor CR8 recruits CDK12 to DDB1 and leads to the degradation of CCNK.

    “Slabicki et al. used a correlation of DDB1 mRNA level with drug-sensitivity across hundreds of cancer cell lines as a strategy to discover that the CDK12 inhibitor CR8 recruits CDK12 to DDB1 and leads to the degradation of CCNK.”

    From Discovery of a molecular glue promoting CDK12-DDB1 interaction to trigger cyclin K degradation (2020) · cites this paper as Słabicki et al., 2020

  • Two studies identified CDK12 inhibitors that induce CDK12-DDB1 interactions to trigger CCNK degradation.

    “While we are preparing our work for publication, two studies identified CDK12 inhibitors that induce CDK12-DDB1 interactions to trigger CCNK degradation (Mayor-Ruiz et al., 2020; Słabicki et al., 2020).”

    From Discovery of a molecular glue promoting CDK12-DDB1 interaction to trigger cyclin K degradation (2020) · cites this paper as Słabicki et al., 2020

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Guide count differs between cloning and validation methods

    Minor

    The cloning method describes 170 oligo pairs targeting 95 E3 ligases, while the subsequent validation method says it used 192 sgRNAs targeting those 95 ligases. The text does not account for the additional 22 guides.

1 of 1

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Related

Authors
Słabicki, M., Kozicka, Z., Petzold, G., Li, Y.-D., Manojkumar, M., Bunker, R. D., Donovan, K. A., Sievers, Q. L., Koeppel, J., Suchyta, D., Sperling, A. S., Fink, E. C., Gasser, J. A., Wang, L. R., Corsello, S. M., Sellar, R. S., Jan, M., Gillingham, D., Scholl, C., Fröhling, S., Golub, T. R., Fischer, E. S., Thomä, N. H., & Ebert, B. L.
Affiliations
  • University of Basel (CH)
  • Friedrich Miescher Institute (CH)
  • Broad Institute (US)
  • Harvard University (US)
  • German Cancer Research Center (DE)
Corresponding author
  • Thomä, Nicolas H.
  • Ebert, Benjamin L.
ORCID
Venue
Nature
Publisher
Springer Science and Business Media LLC
Published
2020-06-03
Volume
585
Issue
7824
Pages
293-297
ISSN
  • 0028-0836 (ISSN-L)
  • 1476-4687
DOI
10.1038/s41586-020-2374-x
PMC
PMC7486275
OpenAlex
W3028901078
Cited by
433
References
57
Citations by year
2026: 51 · 2025: 96 · 2024: 77 · 2023: 69 · 2022: 60 · 2021: 60 · 2020: 19
FWCI
17.43
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
  • Digital Access to Scholarship at Harvard (DASH) (Harvard University) (acceptedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DORA PSI (Paul Scherrer Institute) (submittedVersion)
  • edoc (University of Basel) (submittedVersion)
  • UCL Discovery (University College London) (submittedVersion)
Funders
  • Howard Hughes Medical Institute
  • European Hematology Association (TRTH160)
  • Cancer Research UK (29036)
  • National Institutes of Health (KL2TR002542)
  • National Heart, Lung, and Blood Institute (R01HL082945)
  • National Cancer Institute (R35CA253125, P01CA108631, K08CA230220, P50CA206963, R01CA214608, P01CA066996, R01CA218278)
  • National Institute of General Medical Sciences (T32GM007753)
  • National Center for Advancing Translational Sciences
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
  • Health Sciences › Medicine › Oncology › Cancer-related Molecular Pathways
Keywords
CR8, molecular glues, cyclin K, CDK12, DDB1, ubiquitination, protein degradation, cyclin-dependent kinase inhibitors, cancer cell lines
MeSH terms
DNA-Binding Proteins, Humans, Models, Molecular, Protein Binding, Purines, Pyridines, Cyclins, Cyclin-Dependent Kinases, Cell Line, Tumor, Proteasome Endopeptidase Complex, Small Molecule Libraries, Ubiquitination, Proteolysis

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read under definition version 3 (extract-v3) on 2026-09-28.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-09-28.

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