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Electrophilic PROTACs that degrade nuclear proteins by engaging DCAF16

Nature Chemical Biology volume 15, pages 737–746 (2019)Cite this article

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Abstract

Ligand-dependent protein degradation has emerged as a compelling strategy to pharmacologically control the protein content of cells. So far, however, only a limited number of E3 ligases have been found to support this process. Here, we use a chemical proteomic strategy that leverages broadly reactive, cysteine-directed electrophilic fragments coupled to selective ligands for intracellular proteins (for example, SLF for FKBP12, JQ1 for BRD4) to screen for heterobifunctional degrader compounds (or proteolysis targeting chimeras, PROTACs) that operate by covalent adduction of E3 ligases. This approach identified DCAF16—a poorly characterized substrate recognition component of CUL4-DDB1 E3 ubiquitin ligases—as a target of electrophilic PROTACs that promote the nuclear-restricted degradation of proteins. We find that only a modest fraction (~10–40%) of DCAF16 needs to be modified to support protein degradation, pointing to the potential for electrophilic PROTACs to induce neosubstrate degradation without substantially perturbing the function of the participating E3 ligase.

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Fig. 1: Discovery of an electrophilic PROTAC that promotes the loss of nuclear FKBP12.
Fig. 2: KB02-SLF promotes the loss of nuclear FKBP12.
Fig. 3: KB02-SLF promotes proteasomal degradation of FKBP12 via the action of CRLs.
Fig. 4: DCAF16 mediates KB02-SLF-induced degradation of FKBP12.
Fig. 5: Evaluation of DCAF16 cysteines involved in KB02-SLF-induced degradation of FKBP12.
Fig. 6: KB02-JQ1 degrades BRD4 by sub-stoichiometric modification of DCAF16.

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All data generated or analyzed during this study are included in this published article (and its Supplementary Information files) or are available from the corresponding author on reasonable request.

References

  1. Cromm, P. M. & Crews, C. M. Targeted protein degradation: from chemical biology to drug discovery. Cell Chem. Biol. 24, 1181–1190 (2017).

    Article  CAS  Google Scholar 

  2. Lebraud, H. & Heightman, T. D. Protein degradation: a validated therapeutic strategy with exciting prospects. Essays Biochem. 61, 517–527 (2017).

    Article  Google Scholar 

  3. Bondeson, D. P. & Crews, C. M. Targeted protein degradation by small molecules. Annu. Rev. Pharmacol. Toxicol. 57, 107–123 (2017).

    Article  CAS  Google Scholar 

  4. Collins, I., Wang, H., Caldwell, J. J. & Chopra, R. Chemical approaches to targeted protein degradation through modulation of the ubiquitin-proteasome pathway. Biochem. J. 474, 1127–1147 (2017).

    Article  CAS  Google Scholar 

  5. Hughes, S. J. & Ciulli, A. Molecular recognition of ternary complexes: a new dimension in the structure-guided design of chemical degraders. Essays Biochem. 61, 505–516 (2017).

    Article  Google Scholar 

  6. Lu, G. et al. The myeloma drug lenalidomide promotes the cereblon-dependent destruction of Ikaros proteins. Science 343, 305–309 (2014).

    Article  CAS  Google Scholar 

  7. Ito, T. & Handa, H. Cereblon and its downstream substrates as molecular targets of immunomodulatory drugs. Int. J. Hematol. 104, 293–299 (2016).

    Article  Google Scholar 

  8. Lopez-Girona, A. et al. Cereblon is a direct protein target for immunomodulatory and antiproliferative activities of lenalidomide and pomalidomide. Leukemia 26, 2326–2335 (2012).

    Article  CAS  Google Scholar 

  9. Ito, T. et al. Identification of a primary target of thalidomide teratogenicity. Science 327, 1345–1350 (2010).

    Article  CAS  Google Scholar 

  10. Matyskiela, M. E. et al. A novel cereblon modulator recruits GSPT1 to the CRL4CRBN ubiquitin ligase. Nature 535, 252–257 (2016).

    Article  CAS  Google Scholar 

  11. Uehara, T. et al. Selective degradation of splicing factor CAPERalpha by anticancer sulfonamides. Nat. Chem. Biol. 13, 675–680 (2017).

    Article  CAS  Google Scholar 

  12. Han, T. et al. Anticancer sulfonamides target splicing by inducing RBM39 degradation via recruitment to DCAF15. Science 356, eaal3755 (2017).

  13. Lu, J. et al. Hijacking the E3 Ubiquitin ligase cereblon to efficiently target BRD4. Chem. Biol. 22, 755–763 (2015).

    Article  CAS  Google Scholar 

  14. Sakamoto, K. M. et al. PROTACs: chimeric molecules that target proteins to the Skp1-Cullin-F box complex for ubiquitination and degradation. Proc. Natl Acad. Sci. USA 98, 8554–8559 (2001).

    Article  CAS  Google Scholar 

  15. Winter, G. E. et al. Drug development. Phthalimide conjugation as a strategy for in vivo target protein degradation. Science 348, 1376–1381 (2015).

    Article  CAS  Google Scholar 

  16. Gadd, M. S. et al. Structural basis of PROTAC cooperative recognition for selective protein degradation. Nat. Chem. Biol. 13, 514–521 (2017).

    Article  CAS  Google Scholar 

  17. Qin, C. et al. Discovery of QCA570 as an exceptionally potent and efficacious proteolysis targeting chimera (PROTAC) degrader of the bromodomain and extra-terminal (bet) proteins capable of inducing complete and durable tumor regression. J. Med. Chem. 61, 6685–6704 (2018).

    Article  CAS  Google Scholar 

  18. Zhou, B. et al. Discovery of a small-molecule degrader of bromodomain and extra-terminal (BET) proteins with picomolar cellular potencies and capable of achieving tumor regression. J. Med. Chem. 61, 462–481 (2018).

    Article  CAS  Google Scholar 

  19. Zorba, A. et al. Delineating the role of cooperativity in the design of potent PROTACs for BTK. Proc. Natl Acad. Sci. USA 115, E7285–E7292 (2018).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  21. Lai, A. C. & Crews, C. M. Induced protein degradation: an emerging drug discovery paradigm. Nat. Rev. Drug Discov. 16, 101–114 (2017).

    Article  CAS  Google Scholar 

  22. Okuhira, K. et al. Specific degradation of CRABP-II via cIAP1-mediated ubiquitylation induced by hybrid molecules that crosslink cIAP1 and the target protein. FEBS Lett. 585, 1147–1152 (2011).

    Article  CAS  Google Scholar 

  23. Schneekloth, J. S. et al. Chemical genetic control of protein levels: selective in vivo targeted degradation. J. Am. Chem. Soc. 126, 3748–3754 (2004).

    Article  CAS  Google Scholar 

  24. Schneekloth, A. R., Pucheault, M., Tae, H. S. & Crews, C. M. Targeted intracellular protein degradation induced by a small molecule: en route to chemical proteomics. Bioorg. Med. Chem. Lett. 18, 5904–5908 (2008).

    Article  CAS  Google Scholar 

  25. Bondeson, D. P. et al. Lessons in PROTAC design from selective degradation with a promiscuous warhead. Cell Chem. Biol. 25, 78–87 e75 (2018).

    Article  CAS  Google Scholar 

  26. Huang, H. T. et al. A chemoproteomic approach to query the degradable kinome using a multi-kinase degrader. Cell Chem. Biol. 25, 88–99 e86 (2018).

    Article  CAS  Google Scholar 

  27. Bar-Peled, L. et al. Chemical proteomics identifies druggable vulnerabilities in a genetically defined cancer. Cell 171, 696–709 e623 (2017).

    Article  CAS  Google Scholar 

  28. Backus, K. M. et al. Proteome-wide covalent ligand discovery in native biological systems. Nature 534, 570–574 (2016).

    Article  CAS  Google Scholar 

  29. Wang, C., Weerapana, E., Blewett, M. M. & Cravatt, B. F. A chemoproteomic platform to quantitatively map targets of lipid-derived electrophiles. Nat. Methods 11, 79–85 (2014).

    Article  Google Scholar 

  30. Amara, J. F. et al. A versatile synthetic dimerizer for the regulation of protein-protein interactions. Proc. Natl Acad. Sci. USA 94, 10618–10623 (1997).

    Article  CAS  Google Scholar 

  31. Nabet, B. et al. The dTAG system for immediate and target-specific protein degradation. Nat. Chem. Biol. 14, 431–441 (2018).

    Article  CAS  Google Scholar 

  32. Soucy, T. A. et al. An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer. Nature 458, 732–736 (2009).

    Article  CAS  Google Scholar 

  33. Kawakami, T. et al. NEDD8 recruits E2-ubiquitin to SCF E3 ligase. EMBO J. 20, 4003–4012 (2001).

    Article  CAS  Google Scholar 

  34. Sakata, E. et al. Direct interactions between NEDD8 and ubiquitin E2 conjugating enzymes upregulate cullin-based E3 ligase activity. Nat. Struct. Mol. Biol. 14, 167–168 (2007).

    Article  CAS  Google Scholar 

  35. Deshaies, R. J., Emberley, E. D. & Saha, A. Control of cullin-ring ubiquitin ligase activity by nedd8. Subcell. Biochem. 54, 41–56 (2010).

    Article  CAS  Google Scholar 

  36. Weerapana, E. et al. Quantitative reactivity profiling predicts functional cysteines in proteomes. Nature 468, 790–795 (2010).

    Article  CAS  Google Scholar 

  37. Jin, J., Arias, E. E., Chen, J., Harper, J. W. & Walter, J. C. A family of diverse Cul4-Ddb1-interacting proteins includes Cdt2, which is required for S phase destruction of the replication factor Cdt1. Mol. Cell 23, 709–721 (2006).

    Article  CAS  Google Scholar 

  38. Sansam, C. L. et al. DTL/CDT2 is essential for both CDT1 regulation and the early G2/M checkpoint. Genes Dev. 20, 3117–3129 (2006).

    Article  CAS  Google Scholar 

  39. Higa, L. A. et al. L2DTL/CDT2 interacts with the CUL4/DDB1 complex and PCNA and regulates CDT1 proteolysis in response to DNA damage. Cell Cycle 5, 1675–1680 (2006).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  41. Guerrero-Santoro, J. et al. The cullin 4B-based UV-damaged DNA-binding protein ligase binds to UV-damaged chromatin and ubiquitinates histone H2A. Cancer Res. 68, 5014–5022 (2008).

    Article  CAS  Google Scholar 

  42. Kim, S., Kim, D., Cho, S. W., Kim, J. & Kim, J. S. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 24, 1012–1019 (2014).

    Article  CAS  Google Scholar 

  43. Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

    Article  CAS  Google Scholar 

  44. Wu, Y., Li, Q. & Chen, X. Z. Detecting protein-protein interactions by Far western blotting. Nat. Protoc. 2, 3278–3284 (2007).

    Article  CAS  Google Scholar 

  45. Filippakopoulos, P. et al. Selective inhibition of BET bromodomains. Nature 468, 1067–1073 (2010).

    Article  CAS  Google Scholar 

  46. Raina, K. et al. PROTAC-induced BET protein degradation as a therapy for castration-resistant prostate cancer. Proc. Natl Acad. Sci. USA 113, 7124–7129 (2016).

    Article  CAS  Google Scholar 

  47. Delmore, J. E. et al. BET bromodomain inhibition as a therapeutic strategy to target c-Myc. Cell 146, 904–917 (2011).

    Article  CAS  Google Scholar 

  48. Tanaka, M. et al. Design and characterization of bivalent BET inhibitors. Nat. Chem. Biol. 12, 1089–1096 (2016).

    Article  CAS  Google Scholar 

  49. Fan, J. et al. Tetrameric acetyl-CoA acetyltransferase 1 is important for tumor growth. Mol. Cell 64, 859–874 (2016).

    Article  CAS  Google Scholar 

  50. Blewett, M. M. et al. Chemical proteomic map of dimethyl fumarate-sensitive cysteines in primary human T cells. Sci. Signal 9, rs10 (2016).

    Article  Google Scholar 

  51. Weerapana, E., Speers, A. E. & Cravatt, B. F. Tandem orthogonal proteolysis-activity-based protein profiling (TOP-ABPP)—a general method for mapping sites of probe modification in proteomes. Nat. Protoc. 2, 1414–1425 (2007).

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported by the NIH (grant nos. CA087660 (B.F.C.), CA231991 (B.F.C.), CA211526 (M.M.D.), CA212467 (V.M.C.)) and the Damon-Runyon Cancer Research Foundation (grant no.DRG-2341-18 (X.Z.)).

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Authors and Affiliations

  1. The Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA, USA

    Xiaoyu Zhang, Vincent M. Crowley, Thomas G. Wucherpfennig, Melissa M. Dix & Benjamin F. Cravatt

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  1. Xiaoyu Zhang
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  2. Vincent M. Crowley
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Contributions

X.Z. and B.F.C. conceived the research and wrote the paper. X.Z. developed methods, performed experiments and analyzed data. X.Z. and M.M.D. analyzed chemical proteomic data. X.Z. designed and synthesized KB02-SLF, KB02-PEG0-SLF, KB02-PEG4-SLF, KB03-SLF, KB05-SLF and C-KB02-SLF. V.M.C. designed and synthesized KB02-JQ1. T.G.W. designed and synthesized lenalidomide-SLF. V.M.C. characterized all the compounds.

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Correspondence to Xiaoyu Zhang or Benjamin F. Cravatt.

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Competing interests

B.F.C. is a founder and scientific advisor to Vividion Therapeutics, a biotechnology company interested in developing small-molecule therapeutics.

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Supplementary information

Supplementary Information

Supplementary Figures 1–14

Reporting Summary

Supplementary Note

Synthetic procedures

Supplementary Dataset 1

isoTOP-ABPP data for HEK293T cells treated with KB02-SLF (10 µM, 2 h) or DMSO and summary of the total number of E3 ligases (and the cysteines in these proteins) that have been identified in isoTOP-ABPP experiments reported in three different publications 1–3 and this study.

Supplementary Dataset 2

Complete proteomic data for light and heavy amino acid-labeled HEK293T cells stably expressing FLAG-FKBP12_NLS that were treated with DMSO or KB02-SLF (2 or 10 µM), respectively, for 2 h in the presence of MG132 (10 µM), lysed, subject to anti-FLAG immunoprecipitation and the affinity-enriched proteins combined, digested with trypsin and analyzed by LC–MS/MS.

Supplementary Dataset 3

Complete proteomic data for comparison of DCAF16+/+ and DCAF16−/− clones by anti-FLAG affinity enrichment coupled to mass spectrometry-based proteomics of KB02-SLFtreated HEK293 cells stably expressing FLAG-FKBP12_NLS.

Supplementary Dataset 4

Competitive ABPP data for HEK293T cells treated with KB02-SLF (2 µM, 1.5 h), KB02-JQ1 (20 µM, 1.5 h) or DMSO (1.5 h).

Supplementary Dataset 5

Complete proteomics data for fold-change in protein abundance between heavy- and light-isotopically labeled HEK293T cells treated with KB02-JQ1 (20 µM, heavy)/DMSO (light), KB02-SLF (2 µM, heavy)/DMSO (light) or DMSO (heavy)/DMSO (light) for 24 h.

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Zhang, X., Crowley, V.M., Wucherpfennig, T.G. et al. Electrophilic PROTACs that degrade nuclear proteins by engaging DCAF16. Nat Chem Biol 15, 737–746 (2019). https://doi.org/10.1038/s41589-019-0279-5

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