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


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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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, 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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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.

Author information

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.

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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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.