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Potent and selective bivalent inhibitors of BET bromodomains

Nature Chemical Biology volume 12, pages 1097–1104 (2016)Cite this article

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Abstract

Proteins of the bromodomain and extraterminal (BET) family, in particular bromodomain-containing protein 4 (BRD4), are of great interest as biological targets. BET proteins contain two separate bromodomains, and existing inhibitors bind to them monovalently. Here we describe the discovery and characterization of probe compound biBET, capable of engaging both bromodomains simultaneously in a bivalent, in cis binding mode. The evidence provided here was obtained in a variety of biophysical and cellular experiments. The bivalent binding results in very high cellular potency for BRD4 binding and pharmacological responses such as disruption of BRD4–mediator complex subunit 1 foci with an EC50 of 100 pM. These compounds will be of considerable utility as BET/BRD4 chemical probes. This work illustrates a novel concept in ligand design—simultaneous targeting of two separate domains with a drug-like small molecule—providing precedent for a potentially more effective paradigm for developing ligands for other multi-domain proteins.

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Figure 1: Discovery of a novel class of BRD inhibitor.
Figure 2: X-ray crystal structure of 2 bound to BRD4(1) (PDB ID 5AD2).
Figure 3: Cellular SARs and optimization of bivalent binding leading to 6.
Figure 4: Biophysical characterization of bivalent binding.
Figure 5: Characterization of binding mode in cells using NanoBRET systems.
Figure 6: Potency in cellular assays of BRD4 pharmacology.

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Acknowledgements

The authors thank G. Carr, S. Lamont, T. Moss, A. Patel and G. Wrigley for the synthesis of test compounds; H. Ashton for coordinating biological testing at DiscoverX; K. Eckersley and P. McArdle for supporting cellular imaging studies; and P. Filippakopoulos for BRD4 mutant vector constructs. We thank K. Embrey for helpful comments on the compilation of the manuscript.

Author information

Author notes

  1. Michael J Waring, Huawei Chen, Alfred A Rabow and Graeme Walker: These authors contributed equally to this work.

Authors and Affiliations

  1. AstraZeneca, Alderley Park, UK

    Michael J Waring, Alfred A Rabow, Graeme Walker, Romel Bobby, Rob H Bradbury, Rowena Callis, Ian Dale, Liz Flavell, Geoff Holdgate, Mark McAlister, Derek Ogg, Graeme R Robb, Natalie Stratton, David Whittaker & David M Wilson

  2. Northern Institute for Cancer Research, School of Chemistry, Newcastle University, Newcastle upon Tyne, UK

    Michael J Waring

  3. AstraZeneca, Waltham, Massachusetts, USA

    Huawei Chen, Scott Boiko, Edwin Clark, Austin Dulak, Joe Patel, Philip Petteruti, Sakina Saif, Wenxian Wang & Yi Yao

  4. Promega Corporation, Madison, Wisconsin, USA

    Danette L Daniels, Jacqui Méndez & Matthew B Robers

  5. Wellcome Trust Centre for Cell-Matrix Research, University of Manchester, Manchester, UK

    Thomas A Jowitt

  6. European Molecular Biology Laboratory, Hamburg, Germany

    Alexey Kikhney & Dmitri I Svergun

Authors
  1. Michael J Waring
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Contributions

M.J.W. designed compounds, coordinated chemistry, analyzed and interpreted data and coordinated manuscript preparation. H.C. coordinated cell biology work and analyzed and interpreted biological and biophysical data. A.A.R. designed compounds, carried out computational modeling and structural analysis and interpreted data. G.W. coordinated biophysical testing and AR/ER-based cell experiments and conceived of and coordinated NanoBRET experiments. R.B. designed and carried out NMR studies. S.B. carried out cell biology experiments. R.H.B. designed compounds, coordinated chemistry and analyzed and interpreted data. R.C. carried out ER cell biology experiments. E.C. was the project leader. I.D. carried out NanoBRET experiments. D.L.D. coordinated NanoBRET target engagement experiments and construct production. A.D. coordinated transciptomic profiling and interpreted data. L.F. designed, expressed and purified protein constructs. G.H. analyzed and interpreted biophysical data. T.A.J. carried out AUC experiments and ab initio modeling. A.K. carried out SAXS experiments. M.M. coordinated AUC experiments and analyzed and interpreted biophysical data. J.M. conducted NanoBRET target engagement experiments. D.O. coordinated SAXS experiments and analyzed and interpreted biophysical data. J.P. carried out protein X-ray structure determination. P.P. carried out the c-Myc flow cell assay. G.R.R. carried out computational modeling of tandem domain proteins. M.B.R. carried out NanoBRET target engagement experiments. S.S. carried out transcriptomics experments. N.S. carried out AR cell biology experiments. D.I.S. coordinated and carried out SAXS experiments. W.W. carried out cell biology experiments. D.W. synthesized compounds. D.M.W. analyzed and interpreted the data. Y.Y. carried out transcriptomics experiments. All authors discussed results and commented on the manuscript.

Corresponding authors

Correspondence to Michael J Waring or Edwin Clark.

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Competing interests

The work described here was initiated as a project within AstraZeneca. M.J.W., A.A.R., G.W., R.H.B., D.O. and J.P. are former employees of AstraZeneca and may hold stock in the company. H.C., R.B., S.B., R.C., E.C., I.D., A.D., L.F., G.H., M.M., P.P., G.R.R., S.S., N.S., W.W., D.W., D.M.W. and Y.Y. are current employees of AstraZeneca and may hold stock in the company. Promega Corp. carried out the NanoBRET experiments as part of a collaboration with AstraZeneca. D.L.D., J.M. and M.B.R. are employees of Promega Corp. and may hold stock in the company. The University of Manchester carried out the AUC studies at the request of AstraZeneca. T.A.J. is an employee of the University of Manchester. The European Molecular Biology Laboratory carried out the SAXS studies at the request of AstraZeneca. A.K. and D.I.S. are employees of the European Molecular Biology Laboratory.

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Supplementary Results, Supplementary Tables 1–10, Supplementary Figures 1–19 and Supplementary Note 2. (PDF 3096 kb)

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Waring, M., Chen, H., Rabow, A. et al. Potent and selective bivalent inhibitors of BET bromodomains. Nat Chem Biol 12, 1097–1104 (2016). https://doi.org/10.1038/nchembio.2210

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