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Fragment-based discovery of a chemical probe for the PWWP1 domain of NSD3

Nature Chemical Biology volume 15, pages 822–829 (2019)Cite this article

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Abstract

Here, we report the fragment-based discovery of BI-9321, a potent, selective and cellular active antagonist of the NSD3-PWWP1 domain. The human NSD3 protein is encoded by the WHSC1L1 gene located in the 8p11-p12 amplicon, frequently amplified in breast and squamous lung cancer. Recently, it was demonstrated that the PWWP1 domain of NSD3 is required for the viability of acute myeloid leukemia cells. To further elucidate the relevance of NSD3 in cancer biology, we developed a chemical probe, BI-9321, targeting the methyl-lysine binding site of the PWWP1 domain with sub-micromolar in vitro activity and cellular target engagement at 1 µM. As a single agent, BI-9321 downregulates Myc messenger RNA expression and reduces proliferation in MOLM-13 cells. This first-in-class chemical probe BI-9321, together with the negative control BI-9466, will greatly facilitate the elucidation of the underexplored biological function of PWWP domains.

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Fig. 1: 15N TROSY NMR confirmation and structural analysis of fragment hits of the NSD3-PWWP1 domain.
Fig. 2: Structure-based optimization of methylimidazoles as NSD3-PWWP1 antagonists:
Fig. 3: Biophysical validation and selectivity profiling of the chemical probe BI-9321.
Fig. 4: Cellular target engagement of the NSD3-PWWP1 Probe BI-9321.
Fig. 5: BI-9321 downregulates MYC mRNA and reduces proliferation in MOLM-13 and RN2 cells.

Data availability

The authors declare that the data supporting the findings of this study are available within the publication and its Supplementary Information files or have been deposited in the RCSB Protein Data Bank (PDB, http://www.rcsb.org) with the following accession numbers: selenomethionine labeled (6G3P); unlabeled (6G3T); X-ray structure of NSD3-PWWP1 in complex with compound 3 (6G24); X-ray structure of NSD3-PWWP1 in complex with compound 4 (6G25); X-ray structure of NSD3-PWWP1 in complex with compound 5 (6G27); X-ray structure of NSD3-PWWP1 in complex with compound 6 (6G29); X-ray structure of NSD3-PWWP1 in complex with compound 8 (6G2B); X-ray structure of NSD3-PWWP1 in complex with compound 9 (6G2C); X-ray structure of NSD3-PWWP1 in complex with compound 13 (6G2E); X-ray structure of NSD3-PWWP1 in complex with compound 16 (6G2F); X-ray structure of NSD3-PWWP1 in complex with compound BI-9321 (6G2O).

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Acknowledgements

The Structural Genomics Consortium is a registered charity (no. 1097737) that receives funds from AbbVie; Bayer Pharma AG; Boehringer Ingelheim; Canada Foundation for Innovation; Eshelman Institute for Innovation; Genome Canada; Innovative Medicines Initiative (EU/EFPIA) (ULTRA-DD grant no. 115766); Janssen; Merck & Co.; Novartis Pharma AG; Ontario Ministry of Economic Development and Innovation; Pfizer; São Paulo Research Foundation-FAPESP; Takeda and the Wellcome Trust. We thank the Expose team for data collection at the Swiss Light Source beamlines X06SA and X06DA. We thank D. Daniels, M. Robers and C. Corona from Promega for advising on the NanoBRET and target engagement assays, and acknowledge the Natural Sciences and Engineering Research Council of Canada (NSERC) for a postdoctoral fellowship awarded to D.D.

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Author notes

  1. These authors contributed equally: David Dilworth, Ulrich Reiser, Ralph A. Neumüller

Authors and Affiliations

  1. Boehringer Ingelheim RCV GmbH & Co KG, Vienna, Austria

    Jark Böttcher, Ulrich Reiser, Ralph A. Neumüller, Michael Schleicher, Mark Petronczki, Nikolai Mischerikow, Klaus Rumpel, Andreas Zoephel, Moriz Mayer, Tobias Wunberg, Dietrich Böse, Stephan Zahn, Heribert Arnhof, Helmut Berger, Christoph Reiser, Alexandra Hörmann, Teresa Krammer, Maja Corcokovic, Bernadette Sharps, Sandra Winkler, Daniela Häring, Xiao-Ling Cockcroft, Julian E. Fuchs, Barbara Müllauer, Alexander Weiss-Puxbaum, Thomas Gerstberger, Guido Boehmelt, Mark Pearson & Darryl B. McConnell

  2. Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada

    David Dilworth, Abdellah Allali-Hassani, Magdalena M. Szewczyk, Fengling Li, Steven Kennedy, Masoud Vedadi, Dalia Barsyte-Lovejoy, Peter J. Brown & Cheryl H. Arrowsmith

  3. Boehringer Ingelheim Pharma GmbH & Co KG, Biberach, Germany

    Markus Zeeb

  4. Department of Pharmacology and Toxicology, University of Toronto, Toronto, Ontario, Canada

    Masoud Vedadi

  5. Structural Genomics Consortium, Nuffield Department of Medicine, University of Oxford, Oxford, UK

    Kilian V. M. Huber, Catherine M. Rogers & Oleg Fedorov

  6. Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Oxford, UK

    Kilian V. M. Huber, Catherine M. Rogers & Oleg Fedorov

  7. Structural Genomics Consortium, Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Carrow I. Wells

  8. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA

    Christopher R. Vakoc

  9. Princess Margaret Cancer Centre and Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada

    Cheryl H. Arrowsmith

Authors
  1. Jark Böttcher
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  2. David Dilworth
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Contributions

J.B. and U.R. supervised the chemistry team. U.R., T.W., S.Z. and D.B. designed synthetic strategies. D.D., R.A.N., M.S., M. Petronczki, D.B.-L., M.V., K.V.M.H., C.R.V. and M. Pearson designed biological experiments and supervised the biology teams. M.Z. supervised the DSF, NMR measurements of the FBS screening. A.Z. supervised protein production and ITC experiments. J.B., B.M. and A.W.-P. performed structural analysis. K.R., S.K. and S.W. performed SPR assays. O.F., F.L. and A.A.-H. performed selectivity assays. A.A.-H. contributed to performing SPR and ITC experiments. C.M.R. performed FRAP assays. M.M.S. performed NanoBRET assays. D.D. and M.C. performed cellular experiments. C.W. synthesized tracer ligands. M.M. and H.B. performed analytics and wrote the Supplementary Note. N.M. performed quantitative proteomics experiments. H.A. performed quantitative tandem mass spectroscopy. C.R., A.H. and T.K. performed biological experiments. B.S., D.H. and T.G. performed biochemical assays. X.-L.C. and J.E.F. provided Compchem support. G.B. supervised the collaboration with SGC. P.J.B. managed the project for SGC. C.H.A. provided supervision and funding. D.B.M. was responsible for the medicinal chemistry strategy. J.B., D.D., R.A.N. and U.R. prepared the manuscript with input from all authors.

Corresponding author

Correspondence to Jark Böttcher.

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Competing interests

J.B., U.R., R.A.N., M. Petronczki, M.Z., N.M., K.R., A.Z., M.M., T.W., S.Z., H.A., H.B., C.M.R., A.H., T.K., M.C., B.S., S.W., D.H., X.-L.C., J.E.F., B.M., A.W.-P., T.G., G.B., M. Pearson and D.B.M. are employees of Boehringer Ingelheim.

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Supplementary information

Supplementary Information

Supplementary Tables 1–13, Supplementary Figs. 1–13

Reporting Summary

Supplementary Note

Synthetic procedures

Data Set 1

Data Set 2

Data Set 3

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Böttcher, J., Dilworth, D., Reiser, U. et al. Fragment-based discovery of a chemical probe for the PWWP1 domain of NSD3. Nat Chem Biol 15, 822–829 (2019). https://doi.org/10.1038/s41589-019-0310-x

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