Plasticity in binding confers selectivity in ligand-induced protein degradation

Abstract

Heterobifunctional small-molecule degraders that induce protein degradation through ligase-mediated ubiquitination have shown considerable promise as a new pharmacological modality. However, we currently lack a detailed understanding of the molecular basis for target recruitment and selectivity, which is critically required to enable rational design of degraders. Here we utilize a comprehensive characterization of the ligand-dependent CRBN–BRD4 interaction to demonstrate that binding between proteins that have not evolved to interact is plastic. Multiple X-ray crystal structures show that plasticity results in several distinct low-energy binding conformations that are selectively bound by ligands. We demonstrate that computational protein–protein docking can reveal the underlying interprotein contacts and inform the design of a BRD4 selective degrader that can discriminate between highly homologous BET bromodomains. Our findings that plastic interprotein contacts confer selectivity for ligand-induced protein dimerization provide a conceptual framework for the development of heterobifunctional ligands.

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Fig. 1: Structure of the DDB1∆B-CRBN-dBET23-BRD4BD1 complex.
Fig. 2: Degrader-mediated BRD4 recruitment is governed by negative cooperativity.
Fig. 3: Quantitative assessment of cellular degradation for BRD4BD1 and BRD4BD2.
Fig. 4: Plasticity of CRBN–substrate interactions.
Fig. 5: In silico docking to design degrader molecules.
Fig. 6: Selective degradation of BRD4.

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Acknowledgements

We thank N. Thomä and G. Petzold (Friedrich Miescher Institute for Biomedical Research) for providing constructs and purified protein for some of the CRBN mutants. We are grateful to S. Dhe-Paganon and H.-S. Seo (Dana-Farber Cancer Institute) for providing purified BRD4BD1 and BRD4BD2 protein and the BRD4BD1 construct, and to S. Dastjerdi for help in the synthesis of dBET55. We thank M. Eck for critical feedback on the manuscript. Financial support for this work was provided by NIH grant NCI R01CA214608 (grant to E.S.F.), The Harvard University William F. Milton Fund (grant to E.S.F.), the Friends of Dana Farber (grant to E.S.F.), the Claudia Adams Barr Program for Innovative Cancer Research and the Linde Family Foundation (both start-up funds to E.S.F.), and the Damon Runyon Cancer Research foundation (DRG-2196-14, fellowship to D.L.B). This work is based upon research conducted at the Northeastern Collaborative Access Team beamlines, which are funded by the National Institute of General Medical Sciences from the National Institutes of Health (P41 GM103403). The Pilatus 6 M detector on 24-ID-C beamline is funded by a NIH-ORIP HEI grant (S10 RR029205). This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. The authors would like to thank Diamond Light Source for beamtime, and the staff of beamlines I04-1 for assistance with crystal testing and data collection.

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Contributions

R.P.N, S.L.D. and E.S.F. initiated the project. R.P.N. and S.L.D. with help from C.M.P. conducted the protein purification and crystallization. R.P.N. collected, processed and refined X-ray data. R.P.N. conceived and performed biochemical assays. D.B. and M.I. synthesized the dBET series of compounds. Z.H. and T.Z. synthesized other small molecules used in this study. K.A.D. and M.P.J. conducted the mass spectrometry experiments. J.A., N.S., C.M.P. and R.P.N. designed, constructed and performed the cellular reporter assays. R.P.N. and E.S.F. conceived and performed protein-docking experiments. J.D.M., N.S.G., J.E.B., and E.S.F. supervised all aspects of the project. R.P.N. and E.S.F. wrote the manuscript with input from all authors. All authors read, revised, and approved the manuscript.

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Correspondence to Eric S. Fischer.

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Competing interests

E.S.F. is a member of the scientific advisory board of C4 Therapeutics and is a consultant to Novartis Pharmaceuticals. N.S.G. is a founder and scientific advisory board member of C4 Therapeutics. J.E.B. is an executive and shareholder of Novartis Pharmaceuticals.

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Supplementary Figures 1–10, Supplementary Table 1

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Supplementary Note 1

Synthetic Procedures

Supplementary Dataset 1

Proteomics data of dBET23, dBET70 and ZXH-3-26 cellular effects

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Nowak, R.P., DeAngelo, S.L., Buckley, D. et al. Plasticity in binding confers selectivity in ligand-induced protein degradation. Nat Chem Biol 14, 706–714 (2018). https://doi.org/10.1038/s41589-018-0055-y

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