Lenalidomide derivatives and proteolysis-targeting chimeras for controlling neosubstrate degradation

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

Statistical analysis and reproducibility

The data are presented as mean ± standard deviation (SD). Significant

changes were analysed using Student’s t tests or one-way analysis of

variance (ANOVA), followed by Tukey’s tests using Microsoft Excel

(version 16.66) or GraphPad Prism (version 9) (GraphPad, Inc.). Each

value determined from the dose–response curve was calculated using

GraphPad Prism (version 9). All experiments were repeated more than

twice, and the number of replications is described in the figure

legends.

17.

18.

19.

20.

Reporting summary

Further information on research design is available in the Nature

Portfolio Reporting Summary linked to this article.

Nature Communications | (2023)14:4683

21.

Bartlett, J. B., Dredge, K. & Dalgleish, A. G. The evolution of thalidomide and its IMiD derivatives as anticancer agents. Nat. Rev.

Cancer 4, 314–322 (2004).

Palumbo, A. & Anderson, K. Multiple myeloma. N. Engl. J. Med. 364,

1046–1060 (2011).

List, A. et al. Lenalidomide in the myelodysplastic syndrome with

chromosome 5q deletion. N. Engl. J. Med. 355, 1456–1465 (2006).

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

Krönke, J. et al. Lenalidomide causes selective degradation of IKZF1

and IKZF3 in multiple myeloma cells. Science 343, 301–305 (2014).

Lu, G. et al. The myeloma drug lenalidomide promotes the

cereblon-dependent destruction of Ikaros proteins. Science 343,

305–309 (2014).

Krönke, J. et al. Lenalidomide induces ubiquitination and degradation of CK1α in del(5q) MDS. Nature 523, 183–188 (2015).

Matyskiela, M. E. et al. SALL4 mediates teratogenicity as a

thalidomide-dependent cereblon substrate. Nat. Chem. Biol. 14,

981–987 (2018).

Donovan, K. A. et al. Thalidomide promotes degradation of SALL4, a

transcription factor implicated in Duane Radial syndrome. eLife 7,

e38430 (2018).

Yamanaka, S. et al. Thalidomide and its metabolite

5-hydroxythalidomide induce teratogenicity via the cereblon neosubstrate PLZF. EMBO J. 40, e105375 (2021).

Lai, A. C. & Crews, C. M. Induced protein degradation: an emerging

drug discovery paradigm. Nat. Rev. Drug Discov. 16, 101–114 (2017).

Schneider, M. et al. The PROTACtable genome. Nat. Rev. Drug

Discov. 20, 789–797 (2021).

Surka, C. et al. CC-90009, a novel cereblon E3 ligase modulator,

targets acute myeloid leukemia blasts and leukemia stem cells.

Blood 137, 661–677 (2021).

Hagner, P. R. et al. CC-122, a pleiotropic pathway modifier, mimics

an interferon response and has antitumor activity in DLBCL. Blood

126, 779–789 (2015).

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. U.S.A. 17, 8554–8559 (2001).

Winter, G. E. et al. Phthalimide conjugation as a strategy for in vivo

target protein degradation. Science 348, 1376–1381 (2015).

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

Silva, M. C. et al. Targeted degradation of aberrant tau in frontotemporal dementia patient-derived neuronal cell models. eLife 8,

e45457 (2019).

Sun, X. et al. PROTACs: great opportunities for academia and

industry. Signal Transduct. Target Ther. 4, 64–33 (2019).

Donovan, K. A. et al. Mapping the degradable kinome provides a

resource for expedited degrader development. Cell 183,

1714–1731 (2020).

Dale, B. et al. Advancing targeted protein degradation for cancer

therapy. Nat. Rev. Cancer 21, 638–654 (2021).

16

Article

22. Békés, M., Langley, D. R. & Crews, C. M. PROTAC targeted protein

degraders: the past is prologue. Nat. Rev. Drug Discov. 21,

181–200 (2022).

23. Lee, J. et al. Discovery of E3 ligase ligands for target protein

degradation. Molecules 27, 6515 (2021).

24. 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).

25. Itoh, Y., Ishikawa, M., Naito, M. & Hashimoto, Y. Protein knockdown

using methyl bestatin−ligand hybrid molecules: design and synthesis of inducers of ubiquitination-mediated degradation of cellular

retinoic acid-binding proteins. J. Am. Chem. Soc. 132,

5820–5826 (2010).

26. 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).

27. 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).

28. Zhang, X. et al. DCAF11 supports targeted protein degradation by

electrophilic proteolysis-targeting chimeras. J. Am. Chem. Soc. 143,

5141–5149 (2021).

29. Dobrovolsky, D. et al. Bruton tyrosine kinase degradation as a

therapeutic strategy for cancer. Blood 133, 952–961 (2019).

30. Yamanaka, S. et al. A proximity biotinylation-based approach to

identify protein-E3 ligase interactions induced by PROTACs and

molecular glues. Nat. Commun. 13, 183 (2022).

31. Fischer, E. S. et al. Structure of the DDB1-CRBN E3 ubiquitin ligase in

complex with thalidomide. Nature 512, 49–53 (2014).

32. Chamberlain, P. P. et al. Structure of the human

cereblon–DDB1–lenalidomide complex reveals basis for responsiveness to thalidomide analogs. Nat. Struc. Mol. Biol. 21,

803–809 (2014).

33. 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).

34. Matyskiela, M. E. et al. A novel cereblon modulator recruits GSPT1 to

the CRL4(CRBN) ubiquitin ligase. Nature 535, 252–257 (2016).

35. Furihata, H. et al. Structural bases of IMiD selectivity that emerges

by 5-hydroxythalidomide. Nat. Commun. 11, 4578 (2020).

36. Yamamoto, J. et al. ARID2 is a pomalidomide-dependent

CRL4(CRBN) substrate in multiple myeloma cells. Nat. Chem. Biol.

16, 1208–1217 (2020).

37. Martinez-Høyer, S. et al. Loss of lenalidomide-induced megakaryocytic differentiation leads to therapy resistance in del(5q)

myelodysplastic syndrome. Nat. Cell Biol. 22, 526–533 (2020).

38. Kido, K. et al. AirID, a novel proximity biotinylation enzyme, for

analysis of protein-protein interactions. eLife 9, e54983 (2020).

39. Sievers, Q. L. et al. Defining the human C2H2 zinc finger degrome

targeted by thalidomide analogs through CRBN. Science 362,

eaat0572 (2018).

40. Trott, O. & Olson, A. J. AutoDock Vina: improving the seed and

accuracy of docking with a new scoring function, efficient optimization, and multithreading. J. Comput. Chem. 31, 455–461 (2010).

41. An, J. et al. pSILAC mass spectrometry reveals ZFP91 as IMiDdependent substrate of the CRL4CRBN ubiquitin ligase. Nat. Commun. 8, 15398 (2017).

42. Renneville, A. et al. Avadomide induces degradation of ZMYM2

fusion oncoproteins in hematologic malignancies. Blood Cancer

Discov 2, 250–265 (2021).

43. Wang, E. S. et al. Acute pharmacological degradation of Helios

destabilizes regulatory T cells. Nat. Chem. Biol. 17, 711–717 (2021).

Nature Communications | (2023)14:4683

https://doi.org/10.1038/s41467-023-40385-9

44. Bonazzi, S. et al. Discovery and characterization of a selective IKZF2

glue degrader for cancer immunotherapy. Cell Chem. Biol. 30,

235–247 (2023).

45. Neubert, D., Heger, W., Merker, H. J., Sames, K. & Meister, R.

Embryotoxic effects of thalidomide derivatives in the non-human

primate Callithrix jacchus. II. Elucidation of the susceptible period

and of the variability of embryonic stages. Arch. Toxicol 61,

180–191 (1988).

46. Vickers, T. H. The thalidomide embryopathy in hybrid rabbits. Br. J.

Exp. Pathol. 61, 180–191 (1988).

47. Fratta, I. D., Sigg, E. B. & Maiorana, K. Teratogenic effects of thalidomide in rabbits, rats, hamsters, and mice. Toxicol. Appl. Pharmacol. 7, 268–286 (1965).

48. Okumura-Asatsuma, T. et al. p63 is a cereblon substrate involved in

thalidomide teratogenicity. Nat. Chem. Biol. 15, 1077–1084 (2019).

49. Mahony, C. et al. Pomalidomide is nonteratogenic in chicken and

zebrafish embryos and nonneurotoxic in vitro. Proc. Natl. Acad. Sci.

U.S.A. 110, 12703–12708 (2013).

50. Puissant, A. et al. Targeting MYCN in neuroblastoma by BET bromodomain inhibition. Cancer Discov. 3, 309–323 (2013).

51. Prinjha, R. K., Witherington, J. & Lee, K. Place your BETs: The therapeutic potential of bromodomains. Trends Pharmacol. Sci. 33,

146–153 (2012).

52. Matyskiela, M. E. et al. A cereblon modulator (CC-220) with

improved degradation of ikaros and aiolos. J. Med. Chem. 61,

535–542 (2018).

53. Hansen, J. D. et al. A discovery of CRBN E3 ligase modulator CC92480 for the treatment of relapsed and refractory multiple myeloma. J. Med. Chem. 63, 6648–6676 (2020).

54. Reichermeier, K. M. et al. PIKES analysis reveals response to

degraders and key regulatory mechanisms of the CRL4 network.

Moll. Cell 77, 1092–1106 (2020).

55. Goracci, L. et al. Understanding the metabolism of proteolysis targeting chimeras (PROTACs): the next step toward pharmaceutical

applications. J. Med. Chem. 63, 11615–11638 (2020).

56. Nguyen, T. T. L. et al. Development of an LC-MS/MS method for

ARV-110, a PROTAC molecule, and applications to pharmacokinetic

studies. Molecules 27, 1977 (2022).

57. Troup, R. I., Fallan, C. & Baud, M. G. J. Current strategies for the

design of PROTAC linkers: a critical review. Explor. Target. Antitumor Ther. 1, 273–312 (2020).

58. Sawasaki, T., Ogasawara, T., Morishita, R. & Endo, Y. A cell-free

protein synthesis system for high-throughput proteomics. Proc.

Natl. Acad. Sci. U.S.A. 99, 14652–14657 (2002).

59. Nagase, T. et al. Exploration of human ORFeome- high-throughput

preparation of ORF clones and efficient characterization of their

protein products. DNA Res. 15, 137–149 (2008).

60. Nakamura, S. et al. Retrovirus-mediated gene transfer of granulocyte colony-stimulating factor receptor (G-CSFR) cDNA into MDS

cells and induction of their differentiation by G-CSF. Cytokines Cell

Mol. Ther. 6, 61–70 (2000).

61. Yano, T. et al. AGIA tag system based on a high affinity rabbit

monoclonal antibody against human dopamine receptor D1 for

protein analysis. PLoS ONE 11, e0156716 (2016).

62. Sawasaki, T. et al. Arabidopsis HY5 protein functions as a DNAbinding tag for purification and functional immobilization of proteins on agarose/DNA microplate. FEBS Lett 582, 221–228 (2008).

63. Schüttelkopf, A. W. & Aalten, Van D.M.F. PRODRG: a tool for highthroughput crystallography of protein-ligand complexes. Acta

Crystallogr. D: Biol. Crystallogr. 60, 1355–1363 (2004).

64. Pettersen, E. F. et al. UCSF Chimera–a visualization system for

exploratory research and analysis. J. Comput. Chem. 25,

1605–1625 (2004).

17

Article

65. Brenes, A. J. et al. Erosion of human X chromosome inactivation

causes major remodeling of the iPSC proteome. Cell Rep. 35,

109032 (2021).

https://doi.org/10.1038/s41467-023-40385-9

Competing interests

The authors declare no competing interests.

Additional information

Acknowledgements

The authors thank C. Takahashi, Y. Horiuchi and T. Nakagawa for technical assistance and the Applied Protein Research Laboratory of Ehime

University. We also thank Prof. K. Tohyama (Kawasaki Medical School) for

providing the MDS-L cell line. We also thank Prof. F. Tokunaga and Dr D.

Oikawa (Osaka Metropolitan University) for providing the B-cell lymphoma and DLBCL cell lines. This work was mainly supported by the

Project for Cancer Research and Therapeutic Evolution (P-CREATE) from

the Japan Agency for Medical Research and Development (AMED) under

grant number JP21cm0106181h0006 (S.Yamanaka), Project for Promotion of Cancer Research and Therapeutic Evolution (P-PROMOTE) from

AMED under grant number JP22cm0106181h0002 (S.Yamanaka), the

Platform Project for Supporting Drug Discovery and Life Science

Research (Basis for Supporting Innovative Drug Discovery and Life Science Research [BINDS]) from AMED under Grant Number

22ama121010j0001 (T.S.), a Grant-in-Aid for Scientific Research on

Innovative Areas (21H00285 for S.Yamanaka, JP16H06579 for T.S. and

JP19H04966 for H.K.) from the Japan Society for the Promotion of Science (JSPS). This work was also partially supported by JSPS KAKENHI

(21K15076 for S.Y., JP19H03218 for T.S., and JP17H06112 for N.S.), Takeda

Science Foundation, and Joint Usage and Joint Research Programs of the

Institute of Advanced Medical Sciences, Tokushima University (T.S).

Author contributions

S.Yamanaka, K.N. and Y.S. performed the biochemical, molecular, and

cellular biology experiments. H.F., M.T. and T.M. performed ITC experiments and docking simulation. A.T., T.N., M.U. and N.S. synthesised and

analysed the thalidomide derivatives and PROTACs. S.Yamanaka, Y.Y.,

S.Yoshida, and Y.I. analysed anti-proliferative effect of PROTACs. K.N.

and H.K. performed TMT-based proteomics analyses. S.Yamanaka and

T.S. analysed the data, designed the study, and wrote the paper, and all

authors contributed to the manuscript.

Nature Communications | (2023)14:4683

Supplementary information The online version contains

supplementary material available at

https://doi.org/10.1038/s41467-023-40385-9.

Correspondence and requests for materials should be addressed to

Tatsuya Sawasaki.

Peer review information Nature Communications thanks the anonymous reviewers for their contribution to the peer review of this work. A

peer review file is available.

Reprints and permissions information is available at

http://www.nature.com/reprints

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Open Access This article is licensed under a Creative Commons

Attribution 4.0 International License, which permits use, sharing,

adaptation, distribution and reproduction in any medium or format, as

long as you give appropriate credit to the original author(s) and the

source, provide a link to the Creative Commons licence, and indicate if

changes were made. The images or other third party material in this

article are included in the article’s Creative Commons licence, unless

indicated otherwise in a credit line to the material. If material is not

included in the article’s Creative Commons licence and your intended

use is not permitted by statutory regulation or exceeds the permitted

use, you will need to obtain permission directly from the copyright

holder. To view a copy of this licence, visit http://creativecommons.org/

licenses/by/4.0/.

© The Author(s) 2023

18

...