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

In the paper

“Molecular glue compounds induce protein-protein interactions that, in the context of a ubiquitin ligase, lead to protein degradation.1”

What the sources say

• Supported

  The Protein degradation section describes auxin inducing an interaction with an E3 ubiquitin ligase complex, followed by ubiquitination and rapid proteasomal degradation. The Degrading or inactivating pathogenic proteins section likewise describes compounds recruiting ubiquitin ligases to target proteins and causing their degradation. These passages support the mechanism attributed to the source. (LLM, conf=0.87)

  cites [1] Stanton, B. Z., Chory, E. J. & Crabtree, G. R. Chemically induced proximity in biology and medicine. Science 359, eaao5902 (2018). source

In the paper

“Similarly, aryl sulphonamides degrade the essential RNA-binding protein RBM39 by engaging DCAF15, the substrate receptor of the CRL4DCAF15 E3 ubiquitin ligase13–15.”

What the sources say

• ?Not checked — no full text of the sourcecites [13] Han, T. et al. Anticancer sulfonamides target splicing by inducing RBM39 degradation via recruitment to DCAF15. Science 356, eaal3755 (2017). source
• ?Not checked — no full text of the sourcecites [14] Uehara, T. et al. Selective degradation of splicing factor CAPERα by anticancer sulfonamides. Nat. Chem. Biol. 13, 675–680 (2017). source
• Supported

  The Discussion states that aryl-sulfonamides promote RBM39 degradation by CRL4DCAF15. The Results also show that aryl-sulfonamides bind DCAF15 and recruit RBM39 for degradation. These passages support the attributed mechanism. (LLM, conf=0.93)

  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

In the paper

“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.”

What the sources say

• Supported

  Citation 21 is attached to the claim that CDK12’s C-terminal extension can adopt multiple conformations, not to the preceding DDB1 comparison. The Results section, “Conformational plasticity of the C-terminal kinase extension,” describes two distinct conformations, and the Discussion says the region may explore multiple conformations in solution. Page numbers were not provided. (LLM, conf=0.93)

  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

In the paper

“By reprogramming ubiquitin ligase selectivity, these molecules divert the ligase to drive multiple rounds of target ubiquitination in a catalytic manner16.”

What the sources say

• Supported

  The Results section ('PROTACs mediate catalytic ubiquitination') says PROTACs redefine the VHL–E2 ligase complex’s activity toward a new substrate, allowing one PROTAC molecule to induce ubiquitination of multiple RIPK2 molecules. The same section reports more modified RIPK2 molecules than PROTAC molecules in its in vitro reactions, supporting catalytic, repeated action. (LLM, conf=0.94)

  cites [16] Bondeson, D. P. et al. Catalytic in vivo protein knockdown by small-molecule PROTACs. Nat. Chem. Biol. 11, 611–617 (2015). source

In the paper

“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.”

What the sources say

• Partly supported

  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)

  cites [8] Lu, G. et al. The myeloma drug lenalidomide promotes the cereblon-dependent destruction of Ikaros proteins. Science 343, 305–309 (2014). sourceExperimental — accuracy not yet measured
• Partly supported

  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)

  cites [9] Krönke, J. et al. Lenalidomide causes selective degradation of IKZF1 and IKZF3 in multiple myeloma cells. Science 343, 301–305 (2014). sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [10] Matyskiela, M. E. et al. A novel cereblon modulator recruits GSPT1 to the CRL4CRBN ubiquitin ligase. Nature 535, 252–257 (2016). source
• ?Not checked — no full text of the sourcecites [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
• Supported

  Citation 12 is attached to the identification of CRBN as the substrate receptor of the CRL4CRBN ligase, not to the preceding recruitment claim, which cites references 8–11. The paper’s Abstract identifies CRBN as that substrate receptor, and its ‘CRBN functions as a DCAF for the CRL4CRBN ligase’ section explains its role in the CUL4 complex (page unavailable). (LLM, conf=0.93)

  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

In the paper

“CR8 is a pleiotropic CDK inhibitor reported to bind CDK1/2/3/5/7/9/126,19”

What the sources say

• ?Not checked — no full text of the sourcecites [6] Bettayeb, K. et al. CR8, a potent and selective, roscovitine-derived inhibitor of cyclin-dependent kinases. Oncogene 27, 5797–5807 (2008). source
• Partly supported

  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)

  cites [19] Bösken, C. A. et al. The structure and substrate specificity of human Cdk12/Cyclin K. Nat. Commun. 5, 3505 (2014). sourceExperimental — accuracy not yet measured

In the paper

“Unlike traditional enzyme inhibitors, such molecular glue degraders act sub-stoichiometrically to catalyse rapid depletion of previously inaccessible targets.2”

What the sources say

• Partly supported

  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)

  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). sourceExperimental — accuracy not yet measured

In the paper

“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.”

What the sources say

• ?Not checked — no full text of the sourcecites [17] Angers, S. et al. Molecular architecture and assembly of the DDB1-CUL4A ubiquitin ligase machinery. Nature 443, 590–593 (2006). source
• Supported

  The CUL4-DDB1 Ubiquitin Ligase section describes cullin-RING ligases using substrate receptors and identifies DDB1 as the CUL4 adaptor. The paper also reports identification of 60 novel DCAFs—more than the claimed 20—and its Conclusion describes DCAFs as the means by which CUL4-DDB1 recruits substrates. No page numbers are available in our copy. (LLM, conf=0.87)

  cites [18] Lee, J. & Zhou, P. DCAFs, the missing link of the CUL4-DDB1 ubiquitin ligase. Mol. Cell 26, 775–780 (2007). source

In the paper

“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.”

What the sources say

• ?Could not verify

  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)

  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). sourceExperimental — accuracy not yet measured

In the paper

“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.”

What the sources say

• Supported

  The Results section, “A kinase C-terminal helix extends to the bound ATP” (page unavailable), describes the C-terminal tail’s conformation in an isolated Cdk12/CycK crystal structure. In the citing sentence, this paper supports the reference to the isolated structure used for comparison; the DDB1 engagement and tentative density are observations about the citing paper’s structure, not findings attributed to this source. (LLM, conf=0.90)

  cites [19] Bösken, C. A. et al. The structure and substrate specificity of human Cdk12/Cyclin K. Nat. Commun. 5, 3505 (2014). sourceExperimental — accuracy not yet measured
• Supported

  The Results section, “Conformational plasticity of the C-terminal kinase extension” (page unavailable), describes distinct C-terminal conformations in isolated CDK12/CycK complexes. The citation serves as a reference for those isolated structures; the DDB1 engagement and tentative density assignment are observations of the citing study, not findings attributed to this source. (LLM, conf=0.88)

  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

In the paper

“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.”

What the sources say

• Supported

  The Proteolysis targeting chimeras (PROTACs) section explains that small molecules bring a target protein together with an E3 ubiquitin ligase, leading to ubiquitination and degradation. The Introduction describes targeted degradation as a way to address proteins that have been difficult to drug, including transcription factors. (LLM, conf=0.92)

  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

In the paper

“Molecular glues are a class of small molecule drugs that induce or stabilise protein-protein interactions1.”

What the sources say

• Supported

  The “Tool kits to explore proximity in biology” section (page unavailable) describes small molecules that bring proteins together, including the immunosuppressant rapamycin, which forms a complex with FKBP and a TOR-kinase domain. This supports the cited functional description of molecular glues, although the passage does not use that term. (LLM, conf=0.86)

  cites [1] Stanton, B. Z., Chory, E. J. & Crabtree, G. R. Chemically induced proximity in biology and medicine. Science 359, eaao5902 (2018). source

In the paper

“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.”

What the sources say

• Supported

  The citation identifies MolProbity as the analysis tool, rather than attributing the citing paper’s final-model percentages to Chen et al. In ‘Validation analyses > Torsion-angle combinations,’ the source says MolProbity evaluates Ramachandran conformations and reports percentages of favored residues and outliers. (LLM, conf=0.93)

  cites [48] Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010). source

In the paper

“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).”

In the paper

“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).”

In the paper

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

In the paper

“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).”

In the paper

“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).”

What the sources say

• ?Not checked — no full text of the sourcecites [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

In the paper

“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.”

In the paper

“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).”

In the paper

“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).”

In the paper

“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).”

In the paper

“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).”

In the paper

“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).”

In the paper

“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).”

In the paper

“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.”

In the paper

“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).”

In the paper

“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).”

In the paper

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

In the paper

“sgRNAs targeting DDB1 provide partial depletion of DDB1 (50% DDB1 alleles modified, reducing DDB1 levels by roughly 50%)”

In the paper

“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.”

In the paper

“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.”

In the paper

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

In the paper

“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.”

In the paper

“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.”

In the paper

“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.”

In the paper

“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).”

In the paper

“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.”

In the paper

“Three technical replicates were carried out per experiment.”

In the paper

“Accordingly, single residue mutations designed to increase surface hydrophobicity give rise to ordered protein assemblies33.”

What the sources say

• ?Not checked — no full text of the sourcecites [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

In the paper

“guide sequence confirmed by Sanger sequencing.”

In the paper

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

In the paper

“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.”

In the paper

“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.”

In the paper

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

In the paper

“Between one and three replicates were performed per titration.”

In the paper

“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).”

In the paper

“which suggests selection towards heterozygous or hypomorphic clones.”

In the paper

“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.”

What the sources say

• ?Not checked — no full text of the sourcecites [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