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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Gustafson, J. L. et al. Small-molecule-mediated degradation of the androgen receptor through hydrophobic tagging. Angew. Chem. Int. Edn. Engl. 54, 9659–9662 (2015).
Bondeson, D. P. et al. Catalytic in vivo protein knockdown by small-molecule PROTACs. Nat. Chem. Biol. 11, 611–617 (2015).
Buckley, D. L. et al. HaloPROTACS: use of small molecule PROTACs to induce degradation of HaloTag fusion proteins. ACS Chem. Biol. 10, 1831–1837 (2015).
Lu, J. et al. Hijacking the E3 ubiquitin ligase cereblon to efficiently target BRD4. Chem. Biol. 22, 755–763 (2015).
Gadd, M. S. et al. Structural basis of PROTAC cooperative recognition for selective protein degradation. Nat. Chem. Biol. 13, 514–521 (2017).
Winter, G. E. et al. DRUG DEVELOPMENT. Phthalimide conjugation as a strategy for in vivo target protein degradation. Science 348, 1376–1381 (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. USA 98, 8554–8559 (2001).
Kenten, J. H. & Roberts, S. F. Controlling protein levels in eucaryotic organisms. US patent 6306663, B1 (2001).
Huang, H. T. et al. A chemoproteomic approach to query the degradable kinome using a multi-kinase degrader. Cell Chem. Biol. 25, 88–99.e6 (2017).
Winter, G. E. et al. BET bromodomain proteins function as master transcription elongation factors independent of CDK9 recruitment. Mol. Cell 67, 5–18.e19 (2017).
Fischer, E. S., Park, E., Eck, M. J. & Thomä, N. H. SPLINTS: small-molecule protein ligand interface stabilizers. Curr. Opin. Struct. Biol. 37, 115–122 (2016).
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 causes selective degradation of IKZF1 and IKZF3 in multiple myeloma cells. Science 343, 301–305 (2014).
Gandhi, A. K. et al. Immunomodulatory agents lenalidomide and pomalidomide co-stimulate T cells by inducing degradation of T cell repressors Ikaros and Aiolos via modulation of the E3 ubiquitin ligase complex CRL4(CRBN.). Br. J. Haematol. 164, 811–821 (2014).
An, J. et al. pSILAC mass spectrometry reveals ZFP91 as IMiD-dependent substrate of the CRL4CRBNubiquitin ligase. Nat. Commun. 8, 15398 (2017).
Krönke, J. et al. Lenalidomide induces ubiquitination and degradation of CK1α in del(5q) MDS. Nature 523, 183–188 (2015).
Petzold, G., Fischer, E. S. & Thomä, N. H. Structural basis of lenalidomide-induced CK1α degradation by the CRL4(CRBN) ubiquitin ligase. Nature 532, 127–130 (2016).
Chamberlain, P. P. et al. Structure of the human Cereblon-DDB1-lenalidomide complex reveals basis for responsiveness to thalidomide analogs. Nat. Struct. Mol. Biol. 21, 803–809 (2014).
Ito, T. et al. Identification of a primary target of thalidomide teratogenicity. Science 327, 1345–1350 (2010).
Fischer, E. S. et al. Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide. Nature 512, 49–53 (2014).
Buckley, D. L. et al. Small-molecule inhibitors of the interaction between the E3 ligase VHL and HIF1α. Angew. Chem. Int. Edn. Engl. 51, 11463–11467 (2012).
Raina, K. & Crews, C. M. Targeted protein knockdown using small molecule degraders. Curr. Opin. Chem. Biol. 39, 46–53 (2017).
Toure, M. & Crews, C. M. Small-Molecule PROTACS: New Approaches to Protein Degradation. Angew. Chem. Int. Edn. Engl. 55, 1966–1973 (2016).
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).
Lai, A. C. et al. Modular PROTAC design for the degradation of oncogenic BCR-ABL. Angew. Chem. Int. Edn. Engl. 55, 807–810 (2016).
Remillard, D. et al. Degradation of the BAF complex factor BRD9 by heterobifunctional ligands. Angew. Chem. Int. Edn. Engl. 56, 5738–5743 (2017).
Bondeson, D. P. et al. Lessons in PROTAC design from selective degradation with a promiscuous warhead. Cell Chem. Biol. 25, 78–87.e5 (2017).
Filippakopoulos, P. et al. Selective inhibition of BET bromodomains. Nature 468, 1067–1073 (2010).
Zengerle, M., Chan, K. H. & Ciulli, A. Selective small molecule induced degradation of the BET bromodomain protein BRD4. ACS Chem. Biol. 10, 1770–1777 (2015).
Filippakopoulos, P. et al. Histone recognition and large-scale structural analysis of the human bromodomain family. Cell 149, 214–231 (2012).
Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (2007).
Matyskiela, M. E. et al. A novel cereblon modulator recruits GSPT1 to the CRL4(CRBN) ubiquitin ligase. Nature 535, 252–257 (2016).
Douglass, E. F. Jr., Miller, C. J., Sparer, G., Shapiro, H. & Spiegel, D. A. A comprehensive mathematical model for three-body binding equilibria. J. Am. Chem. Soc. 135, 6092–6099 (2013).
Sircar, A., Chaudhury, S., Kilambi, K. P., Berrondo, M. & Gray, J. J. A generalized approach to sampling backbone conformations with RosettaDock for CAPRI rounds 13-19. Proteins 78, 3115–3123 (2010).
Kuriyan, J. & Eisenberg, D. The origin of protein interactions and allostery in colocalization. Nature 450, 983–990 (2007).
Chau, N. G. et al. Intensive treatment and survival outcomes in NUT midline carcinoma of the head and neck. Cancer 122, 3632–3640 (2016).
Zuber, J. et al. RNAi screen identifies Brd4 as a therapeutic target in acute myeloid leukaemia. Nature 478, 524–528 (2011).
Stathis, A. et al. Clinical response of carcinomas harboring the BRD4-NUT oncoprotein to the targeted bromodomain inhibitor OTX015/MK-8628. Cancer Discov. 6, 492–500 (2016).
Buchdunger, E. et al. Abl protein-tyrosine kinase inhibitor STI571 inhibits in vitro signal transduction mediated by c-kit and platelet-derived growth factor receptors. J. Pharmacol. Exp. Ther. 295, 139–145 (2000).
Ritchie, M. E. et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 43, e47 (2015).
Abdulrahman, W. et al. A set of baculovirus transfer vectors for screening of affinity tags and parallel expression strategies. Anal. Biochem. 385, 383–385 (2009).
Zakeri, B. et al. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proc. Natl Acad. Sci. USA 109, E690–E697 (2012).
Cavadini, S. et al. Cullin-RING ubiquitin E3 ligase regulation by the COP9 signalosome. Nature 531, 598–603 (2016).
Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).
Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 (2011).
McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).
Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D Biol. Crystallogr. 68, 352–367 (2012).
BUSTER v. 2.10.2 (Global Phasing Ltd., Cambridge, United Kingdom, 2011).
Moriarty, N. W., Grosse-Kunstleve, R. W. & Adams, P. D. electronic Ligand Builder and Optimization Workbench (eLBOW): a tool for ligand coordinate and restraint generation. Acta Crystallogr. D Biol. Crystallogr. 65, 1074–1080 (2009).
Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).
Marks, B. D. et al. Multiparameter analysis of a screen for progesterone receptor ligands: comparing fluorescence lifetime and fluorescence polarization measurements. Assay Drug Dev. Technol. 3, 613–622 (2005).
McAlister, G. C. et al. MultiNotch MS3 enables accurate, sensitive, and multiplexed detection of differential expression across cancer cell line proteomes. Anal. Chem. 86, 7150–7158 (2014).
R Development Core Team. A language and environment for statistical computing. (R Foundation for Statistical Computing, Vienna, Austria, 2013).
Morin, A. et al. Collaboration gets the most out of software. eLife 2, e01456 (2013).
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.
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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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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