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Implications of frequent hitter E3 ligases in targeted protein degradation screens

Nature Chemical Biology volume 21, pages 474–481 (2025)Cite this article

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Abstract

Targeted protein degradation (TPD) offers a promising approach for chemical probe and drug discovery that uses small molecules or biologics to direct proteins to the cellular machinery for destruction. Among the >600 human E3 ligases, CRBN and VHL have served as workhorses for ubiquitin–proteasome system-dependent TPD. Identification of additional E3 ligases capable of supporting TPD would unlock the full potential of this mechanism for both research and pharmaceutical applications. This perspective discusses recent strategies to expand the scope of TPD and the surprising convergence of these diverse screening efforts on a handful of E3 ligases, specifically DCAF16, DCAF11 and FBXO22. We speculate that a combination of properties, including superficial ligandability, potential for promiscuous substrate interactions and high occupancy in Cullin–RING complexes, may position these E3 ligases as ‘low-hanging fruit’ in TPD screens. We also discuss complementary approaches that might further expand the E3 ligase landscape supporting TPD.

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Fig. 1: TPD mediated by DCAF16.
Fig. 2: TPD mediated by DCAF11 and FBXO22.
Fig. 3: Representative approaches for identifying TPD-competent E3 ligases.

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References

  1. Huang, X. & Dixit, V. M. Drugging the undruggables: exploring the ubiquitin system for drug development. Cell Res. 26, 484–498 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. De Nardo, D. et al. Interleukin-1 receptor-associated kinase 4 (IRAK4) plays a dual role in myddosome formation and Toll-like receptor signaling. J. Biol. Chem. 293, 15195–15207 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  3. Yu, F., Cai, M., Shao, L. & Zhang, J. Targeting protein kinases degradation by PROTACs. Front. Chem. 9, 679120 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Wells, J. A. & Kumru, K. Extracellular targeted protein degradation: an emerging modality for drug discovery. Nat. Rev. Drug Discov. 23, 126–140 (2024).

    Article  PubMed  Google Scholar 

  5. Schapira, M., Calabrese, M. F., Bullock, A. N. & Crews, C. M. Targeted protein degradation: expanding the toolbox. Nat. Rev. Drug Discov. 18, 949–963 (2019).

    Article  CAS  PubMed  Google Scholar 

  6. Banik, S. M. et al. Lysosome-targeting chimaeras for degradation of extracellular proteins. Nature 584, 291–297 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Ji, C. H. et al. The AUTOTAC chemical biology platform for targeted protein degradation via the autophagy–lysosome system. Nat. Commun. 13, 904 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Paudel, R. R., Lu, D., Roy Chowdhury, S., Monroy, E. Y. & Wang, J. Targeted protein degradation via lysosomes. Biochemistry 62, 564–579 (2023).

    Article  CAS  PubMed  Google Scholar 

  9. Schreiber, S. L. The rise of molecular glues. Cell 184, 3–9 (2021).

    Article  CAS  PubMed  Google Scholar 

  10. Kronke, J. et al. Lenalidomide causes selective degradation of IKZF1 and IKZF3 in multiple myeloma cells. Science 343, 301–305 (2014).

    Article  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  12. Kozicka, Z. & Thoma, N. H. Haven’t got a glue: protein surface variation for the design of molecular glue degraders. Cell Chem. Biol. 28, 1032–1047 (2021).

    Article  CAS  PubMed  Google Scholar 

  13. Domostegui, A., Nieto-Barrado, L., Perez-Lopez, C. & Mayor-Ruiz, C. Chasing molecular glue degraders: screening approaches. Chem. Soc. Rev. 51, 5498–5517 (2022).

    Article  CAS  PubMed  Google Scholar 

  14. Wang, B., Cao, S. & Zheng, N. Emerging strategies for prospective discovery of molecular glue degraders. Curr. Opin. Struct. Biol. 86, 102811 (2024).

    Article  CAS  PubMed  Google Scholar 

  15. Nalawansha, D. A. & Crews, C. M. PROTACs: an emerging therapeutic modality in precision medicine. Cell Chem. Biol. 27, 998–1014 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  18. Hanzl, A. & Winter, G. E. Targeted protein degradation: current and future challenges. Curr. Opin. Chem. Biol. 56, 35–41 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Bulatov, E. & Ciulli, A. Targeting Cullin–RING E3 ubiquitin ligases for drug discovery: structure, assembly and small-molecule modulation. Biochem. J. 467, 365–386 (2015).

    Article  CAS  PubMed  Google Scholar 

  20. Reichermeier, K. M. et al. PIKES analysis reveals response to degraders and key regulatory mechanisms of the CRL4 network. Mol. Cell 77, 1092–1106 (2020).

    Article  CAS  PubMed  Google Scholar 

  21. Harper, J. W. & Schulman, B. A. Cullin–RING ubiquitin ligase regulatory circuits: a quarter century beyond the F-box hypothesis. Annu. Rev. Biochem. 90, 403–429 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Schapira, M., Tyers, M., Torrent, M. & Arrowsmith, C. H. WD40 repeat domain proteins: a novel target class? Nat. Rev. Drug Discov. 16, 773–786 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. 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  PubMed  Google Scholar 

  24. Zhang, X., Thielert, M., Li, H. & Cravatt, B. F. SPIN4 is a principal endogenous substrate of the E3 ubiquitin ligase DCAF16. Biochemistry 60, 637–642 (2021).

    Article  CAS  PubMed  Google Scholar 

  25. Zhang, X., Crowley, V. M., Wucherpfennig, T. G., Dix, M. M. & Cravatt, B. F. Electrophilic PROTACs that degrade nuclear proteins by engaging DCAF16. Nat. Chem. Biol. 15, 737–746 (2019). This study demonstrated DCAF16 as a TPD-competent E3 ligase through a phenotypic screen measuring FKBP12 degradation induced by heterobifunctional compounds containing an FKBP12 ligand coupled to electrophilic fragments.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Shergalis, A. G. et al. CRISPR screen reveals BRD2/4 molecular glue-like degrader via recruitment of DCAF16. ACS Chem. Biol. 18, 331–339 (2023).

    Article  CAS  PubMed  Google Scholar 

  27. Li, Y. D. et al. Template-assisted covalent modification underlies activity of covalent molecular glues. Nat. Chem. Biol. 20, 1640–1649 (2024). This study found that the vinyl sulfonamide JQ1 derivative, when bound to BRD4-BD2, recruits DCAF16 and facilitates modification of DCAF16 at C58 by the ligand, leading to BRD4 degradation.

  28. Blake, R. A. GNE-0011, a novel monovalent BRD4 degrader. Cancer Res. 79, 4452 (2019).

  29. Hassan, M. M. et al. Exploration of the tunability of BRD4 degradation by DCAF16 trans-labelling covalent glues. Eur. J. Med. Chem. 279, 116904 (2024).

    Article  CAS  PubMed  Google Scholar 

  30. Lim, M. et al. DCAF16-based covalent handle for the rational design of monovalent degraders. ACS Cent. Sci. 10, 1318–1331 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Han, T. et al. Anticancer sulfonamides target splicing by inducing RBM39 degradation via recruitment to DCAF15. Science 356, 6336 (2017).

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

    Article  CAS  PubMed  Google Scholar 

  33. Hsia, O. et al. Targeted protein degradation via intramolecular bivalent glues. Nature 627, 204–211 (2024). This study demonstrated that the heterobifunctional compound JQ1-E7820 simultaneously engages both bromodomains of BRD4, inducing a conformational change that strengthens a weak interaction between BRD4 and DCAF16, ultimately leading to BRD4 degradation.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Zhang, X. et al. DCAF11 supports targeted protein degradation by electrophilic proteolysis-targeting chimeras. J. Am. Chem. Soc. 143, 5141–5149 (2021). This study used cell-based screens with a focused library of FKBP12-directed bifunctional electrophilic compounds across a panel of human cancer cell lines to identify DCAF11 as a TPD-competent E3 ligase.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Lo, J. Y., Spatola, B. N. & Curran, S. P. WDR23 regulates NRF2 independently of KEAP1. PLoS Genet. 13, e1006762 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  36. Djakbarova, U., Marzluff, W. F. & Koseoglu, M. M. DDB1 and CUL4 associated factor 11 (DCAF11) mediates degradation of stem-loop binding protein at the end of S phase. Cell Cycle 15, 1986–1996 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Xue, G. et al. Discovery of a drug-like, natural product-inspired DCAF11 ligand chemotype. Nat. Commun. 14, 7908 (2023). This study demonstrated that heterobifunctional compounds containing an alkenyl oxindole moiety induce protein degradation by covalently targeting DCAF11.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Wang, Y. et al. Alkenyl oxindole is a novel PROTAC moiety that recruits the CRL4DCAF11 E3 ubiquitin ligase complex for targeted protein degradation. PLoS Biol. 22, e3002550 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Li, Z. et al. Allele-selective lowering of mutant HTT protein by HTT–LC3 linker compounds. Nature 575, 203–209 (2019).

    Article  CAS  PubMed  Google Scholar 

  40. Tin, G. et al. Discovery of a DCAF11-dependent cyanoacrylamide-containing covalent degrader of BET-proteins. Bioorg. Med. Chem. Lett. 107, 129779 (2024).

    Article  CAS  PubMed  Google Scholar 

  41. Sarott, R. C. et al. Chemical specification of E3 ubiquitin ligase engagement by cysteine-reactive chemistry. J. Am. Chem. Soc. 145, 21937–21944 (2023).

    Article  CAS  PubMed  Google Scholar 

  42. Parker, G. et al. Discovery of novel small molecules that recruit DCAF11 for selective degradation of BRD4. Eur. J. Cancer 174, S81 (2022).

    Article  Google Scholar 

  43. Jin, J. et al. Systematic analysis and nomenclature of mammalian F-box proteins. Genes Dev. 18, 2573–2580 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. 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  PubMed  PubMed Central  Google Scholar 

  45. Nie, D. Y. et al. Recruitment of FBXO22 for targeted degradation of NSD2. Nat. Chem. Biol. 20, 1597–1607 (2024). This study demonstrated that an alkylamine-containing degrader targeting NSD2 operates by converting the alkylamine to an aldehyde, which modifies C326 of FBXO22 and triggers NSD2 degradation.

  46. Kagiou, C. et al. Alkylamine-tethered molecules recruit FBXO22 for targeted protein degradation. Nat. Commun. 15, 5409 (2024). This study demonstrated that attaching an alkylamine moiety to an FKBP12 ligand results in the conversion of the alkylamine to an aldehyde in culture media. The aldehyde moiety modifies C326 of FBXO22, thereby inducing FKBP12 degradation.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Basu, A. A. et al. A CRISPR activation screen identifies FBXO22 supporting targeted protein degradation. Nat. Chem. Biol. 20, 1608–1616 (2024). This study reported a CRISPR transcriptional activation screen that identified FBXO22, which, upon induced expression, enhances the activity of a hypoactive electrophilic PROTAC degrader of FKBP12.

  48. Branon, T. C. et al. Efficient proximity labeling in living cells and organisms with TurboID. Nat. Biotechnol. 36, 880–887 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Bai, C. et al. SKP1 connects cell cycle regulators to the ubiquitin proteolysis machinery through a novel motif, the F-box. Cell 86, 263–274 (1996).

    Article  CAS  PubMed  Google Scholar 

  50. Geiger, T., Wehner, A., Schaab, C., Cox, J. & Mann, M. Comparative proteomic analysis of eleven common cell lines reveals ubiquitous but varying expression of most proteins. Mol. Cell Proteomics 11, M111.014050 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  51. Belcher, B. P., Ward, C. C. & Nomura, D. K. Ligandability of E3 ligases for targeted protein degradation applications. Biochemistry 62, 588–600 (2023).

    Article  CAS  PubMed  Google Scholar 

  52. Gosavi, P. M. et al. Profiling the landscape of drug resistance mutations in neosubstrates to molecular glue degraders. ACS Cent. Sci. 8, 417–429 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Xie, X. et al. KBTBD4 cancer hotspot mutations drive neomorphic degradation of HDAC1/2 corepressor complexes. Preprint at bioRxiv https://doi.org/10.1101/2024.05.14.593970 (2024).

  54. Hanzl, A. et al. Functional E3 ligase hotspots and resistance mechanisms to small-molecule degraders. Nat. Chem. Biol. 19, 323–333 (2023).

    Article  CAS  PubMed  Google Scholar 

  55. McCarthy, W. J., van der Zouwen, A. J., Bush, J. T. & Rittinger, K. Covalent fragment-based drug discovery for target tractability. Curr. Opin. Struct. Biol. 86, 102809 (2024).

    Article  CAS  PubMed  Google Scholar 

  56. Gehringer, M. & Laufer, S. A. Emerging and re-emerging warheads for targeted covalent inhibitors: applications in medicinal chemistry and chemical biology. J. Med. Chem. 62, 5673–5724 (2019).

    Article  CAS  PubMed  Google Scholar 

  57. Zheng, S. & Crews, C. M. Electrophilic screening platforms for identifying novel covalent ligands for E3 ligases. Biochemistry 60, 2367–2370 (2021).

    Article  CAS  PubMed  Google Scholar 

  58. Yamamoto, J., Ito, T., Yamaguchi, Y. & Handa, H. Discovery of CRBN as a target of thalidomide: a breakthrough for progress in the development of protein degraders. Chem. Soc. Rev. 51, 6234–6250 (2022).

    Article  CAS  PubMed  Google Scholar 

  59. Lv, L. et al. Discovery of a molecular glue promoting CDK12–DDB1 interaction to trigger cyclin K degradation. eLife 9, e59994 (2020).

  60. Slabicki, M. et al. The CDK inhibitor CR8 acts as a molecular glue degrader that depletes cyclin K. Nature 585, 293–297 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Mayor-Ruiz, C. et al. Rational discovery of molecular glue degraders via scalable chemical profiling. Nat. Chem. Biol. 16, 1199–1207 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Spradlin, J. N. et al. Harnessing the anti-cancer natural product nimbolide for targeted protein degradation. Nat. Chem. Biol. 15, 747–755 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Toriki, E. S. et al. Rational chemical design of molecular glue degraders. ACS Cent. Sci. 9, 915–926 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Liu, Y. et al. Expanding PROTACtable genome universe of E3 ligases. Nat. Commun. 14, 6509 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Schreiber, S. L. A chemical biology view of bioactive small molecules and a binder-based approach to connect biology to precision medicines. Isr. J. Chem. 59, 52–59 (2019).

    Article  CAS  PubMed  Google Scholar 

  66. Soares, P. et al. Group-based optimization of potent and cell-active inhibitors of the von Hippel–Lindau (VHL) E3 ubiquitin ligase: structure–activity relationships leading to the chemical probe (2S,4R)-1-((S)-2-(1-cyanocyclopropanecarboxamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (VH298). J. Med. Chem. 61, 599–618 (2018).

    Article  CAS  PubMed  Google Scholar 

  67. Buckley, D. L. et al. Small-molecule inhibitors of the interaction between the E3 ligase VHL and HIF1α. Angew. Chem. Int. Ed. Engl. 51, 11463–11467 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Buckley, D. L. et al. Targeting the von Hippel–Lindau E3 ubiquitin ligase using small molecules to disrupt the VHL/HIF-1α interaction. J. Am. Chem. Soc. 134, 4465–4468 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Hickey, C. M. et al. Co-opting the E3 ligase KLHDC2 for targeted protein degradation by small molecules. Nat. Struct. Mol. Biol. 31, 311–322 (2024).

    Article  CAS  PubMed  Google Scholar 

  70. Ramachandran, S. et al. Structure-based design of a phosphotyrosine-masked covalent ligand targeting the E3 ligase SOCS2. Nat. Commun. 14, 6345 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Niphakis, M. J. & Cravatt, B. F. Ligand discovery by activity-based protein profiling. Cell Chem. Biol. 31, 1636–1651 (2024).

    Article  CAS  PubMed  Google Scholar 

  72. Tao, Y. et al. Targeted protein degradation by electrophilic PROTACs that stereoselectively and site-specifically engage DCAF1. J. Am. Chem. Soc. 144, 18688–18699 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Schroder, M. et al. DCAF1-based PROTACs with activity against clinically validated targets overcoming intrinsic- and acquired-degrader resistance. Nat. Commun. 15, 275 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Vulpetti, A. et al. Discovery of new binders for DCAF1, an emerging ligase target in the targeted protein degradation field. ACS Med. Chem. Lett. 14, 949–954 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Gironda-Martinez, A., Donckele, E. J., Samain, F. & Neri, D. DNA-encoded chemical libraries: a comprehensive review with succesful stories and future challenges. ACS Pharm. Transl. Sci. 4, 1265–1279 (2021).

    Article  CAS  Google Scholar 

  76. Li, A. S. M. et al. Discovery of nanomolar DCAF1 small molecule ligands. J. Med. Chem. 66, 5041–5060 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Ward, C. C. et al. Covalent ligand screening uncovers a RNF4 E3 ligase recruiter for targeted protein degradation applications. ACS Chem. Biol. 14, 2430–2440 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Henning, N. J. et al. Discovery of a covalent FEM1B recruiter for targeted protein degradation applications. J. Am. Chem. Soc. 144, 701–708 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We gratefully acknowledge the support of the National Institutes of Health (R00 CA248715 to X.Z. and R35 CA231991 to B.F.C.).

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  1. Department of Chemistry, Northwestern University, Evanston, IL, USA

    Xiaoyu Zhang

  2. Vividion Therapeutics, San Diego, CA, USA

    Gabriel M. Simon

  3. Department of Chemistry, The Scripps Research Institute, La Jolla, CA, USA

    Benjamin F. Cravatt

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  1. Xiaoyu Zhang
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  3. Benjamin F. Cravatt
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X.Z. and B.F.C. conceived the topic and wrote the manuscript. G.M.S. contributed to discussions and edited the manuscript.

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Correspondence to Xiaoyu Zhang or Benjamin F. Cravatt.

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B.F.C. is a founder and scientific advisor to Vividion Therapeutics and Magnet Therapeutics. X.Z. and G.M.S. declare no relevant competing interests.

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Zhang, X., Simon, G.M. & Cravatt, B.F. Implications of frequent hitter E3 ligases in targeted protein degradation screens. Nat Chem Biol 21, 474–481 (2025). https://doi.org/10.1038/s41589-024-01821-z

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