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Catalytic in vivo protein knockdown by small-molecule PROTACs

Abstract

The current predominant therapeutic paradigm is based on maximizing drug-receptor occupancy to achieve clinical benefit. This strategy, however, generally requires excessive drug concentrations to ensure sufficient occupancy, often leading to adverse side effects. Here, we describe major improvements to the proteolysis targeting chimeras (PROTACs) method, a chemical knockdown strategy in which a heterobifunctional molecule recruits a specific protein target to an E3 ubiquitin ligase, resulting in the target's ubiquitination and degradation. These compounds behave catalytically in their ability to induce the ubiquitination of super-stoichiometric quantities of proteins, providing efficacy that is not limited by equilibrium occupancy. We present two PROTACs that are capable of specifically reducing protein levels by >90% at nanomolar concentrations. In addition, mouse studies indicate that they provide broad tissue distribution and knockdown of the targeted protein in tumor xenografts. Together, these data demonstrate a protein knockdown system combining many of the favorable properties of small-molecule agents with the potent protein knockdown of RNAi and CRISPR.

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Figure 1: Proteolysis targeting chimeras (PROTACs).
Figure 2: PROTACs downregulate the protein levels of their respective targets.
Figure 3: PROTACs induce the catalytic ubiquitination of their target protein in a reconstituted E1-E2-VHL assay.
Figure 4: PROTACs are highly specific for their respective target.
Figure 5: PROTAC_ERRα is efficacious in mice.

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Acknowledgements

We thank K. Mueller and D. Poeckel for cell culture support; S. Melchert, E. Stonehouse, A. Lachert, J. Cox, M. Leveridge, C. Pancevac and M. Jundt for biochemistry and sample preparation support; M. Boesche, T. Rudi, M. Kloes-Hudak and K. Kammerer for mass spectrometry support; and S. Gade for data analysis support. This research was partially supported by US National Institutes of Health grants AI084140, T32GM067543 and T32GM007223.

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Authors and Affiliations

Authors

Contributions

I.E.D.S., E.K., S.C., A.H.M., J.D.H., D.L.B., J.L.G., L.N.C. and B.J.V. contributed to the design and synthesis of compounds. D.P.B., A.M., K.E.M., N.R., C.C., D.A.G., R.R.W., J.J.F. and W.d.B. contributed to running of in vitro, cellular and in vivo experiments. N.Z., P.G., S.S., G.B., M.F.-S. and M.B. designed, performed and interpreted proteomic analyses. D.P.B., A.M., M.B., P.G., G.B., J.J.F., K.F., L.K., P.S.C., J.D.H., I.C. and C.M.C. designed studies and interpreted results. D.P.B., C.M.C. and I.C. wrote the manuscript.

Corresponding authors

Correspondence to Ian Churcher or Craig M Crews.

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Competing interests

A.M., I.E.D.S., S.C., A.M., N.Z., P.G., S.S., G.B., M.F.-S., M.B., L.N.C., B.J.V., K.F., L.K., P.S.C., J.D.H. and I.C. are employees of GlaxoSmithKline. D.A.G., R.R.W. and J.J.F. are employees of Arvinas, LLC. C.M.C. is a shareholder/consultant for Arvinas, LLC and consults for Canaan Partners.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–10 and Supplementary Note. (PDF 11920 kb)

Supplementary Table 1

Relative abundance of proteins immunoprecipitated with the active or inactive VHL ligands (XLSX 46 kb)

Supplementary Table 2

Results summary for expression proteomics (XLSX 568 kb)

Supplementary Table 3

Proteomic analysis of RIPK2 phosphorylation sites (XLSX 12 kb)

Supplementary Table 4

Results summary for ternary complex formation (XLSX 414 kb)

Supplementary Table 5

Results summary for Kinobead competition binding experiments (XLSX 11061 kb)

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Bondeson, D., Mares, A., Smith, I. et al. Catalytic in vivo protein knockdown by small-molecule PROTACs. Nat Chem Biol 11, 611–617 (2015). https://doi.org/10.1038/nchembio.1858

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