Covalent inhibition of NSD1 histone methyltransferase


The nuclear receptor-binding SET domain (NSD) family of histone methyltransferases is associated with various malignancies, including aggressive acute leukemia with NUP98-NSD1 translocation. While NSD proteins represent attractive drug targets, their catalytic SET domains exist in autoinhibited conformation, presenting notable challenges for inhibitor development. Here, we employed a fragment-based screening strategy followed by chemical optimization, which resulted in the development of the first-in-class irreversible small-molecule inhibitors of the nuclear receptor-binding SET domain protein 1 (NSD1) SET domain. The crystal structure of NSD1 in complex with covalently bound ligand reveals a conformational change in the autoinhibitory loop of the SET domain and formation of a channel-like pocket suitable for targeting with small molecules. Our covalent lead—compound BT5—demonstrates on-target activity in NUP98-NSD1 leukemia cells, including inhibition of histone H3 lysine 36 dimethylation and downregulation of target genes, and impaired colony formation in an NUP98-NSD1 patient sample. This study will facilitate the development of the next generation of potent and selective inhibitors of the NSD histone methyltransferases.

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Fig. 1: Development of NSD1 ligands using a fragment-based approach.
Fig. 2: Crystal structure of NSD1 with covalently bound BT3.
Fig. 3: Characterization of the covalent engagement of irreversible NSD1 inhibitors.
Fig. 4: Characterization of the activity of NSD1 inhibitors in HMT assays.
Fig. 5: Treatment with BT5 demonstrates on-target activity in cells.
Fig. 6: Activity of BT5 in primary samples.

Data availability

The crystal structure of NSD1 (PDB code no. 3OOI) was used as the search model to determine the structures reported in this study. The crystal structures of NSD1 and NSD1 in complex with BT3 have been deposited in the PDB under the accession codes 6KQP and 6KQQ, respectively. Source data are provided with this paper.


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This work was funded by National Institute of Health (NIH) grant nos. R01 CA226759 and CA207272 to T.C., and grant nos. 1R01 CA160467 and R01 CA244254 to J.G.; Leukemia & Lymphoma Society (LLS) Translational Research Program grant nos. 6111-14 and 6564-19 to T.C., 6485-16 and 6579-20 to J.G. and LLS Scholar grant nos. 1340-17 to T.C. and 1215-14 to J.G. C.A.H. is supported by Chemistry Biology Interface Training Program (no. T32GM008597). J.G. is Rogel Scholar at the University of Michigan. This work was also supported by the Chemotherapy Foundation (A.F.) and NIH grant nos. R35 CA210065 (to A.F.) and P30 CA013696 (Flow Cytometry Shared Resource). This research used resources of the Advanced Photon Source, a U.S. 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. Use of the LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (grant no. 085P1000817). The NUP98-NSD1 construct was a kind gift from A. J. Deshpande. The MOZ-TIF2 construct was provided by I. Kitabayashi and the NUP98-HOXA9 cells were provided by H. Xu.

Author information




T.C. and J.G. are responsible for initiating and supervising the entire project. H.H., S.Z. and M.A.P. synthesized the compounds. H.J.C. carried out the structural biology studies. C.A.H., H.L., J.N., D.S.R., C.N., J.A. and A.H. performed the biochemical studies. S.S., K.K., P.G.A., H.M. and T.P. performed the cell biology studies. A.M., M.L.S. and A.F. provided the reagents and advised the study. All authors contributed to data analysis and the writing of the manuscript.

Corresponding authors

Correspondence to Jolanta Grembecka or Tomasz Cierpicki.

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Competing interests

H.H., C.A.H., S.Z., H.J.C., M.A.P., J.G. and T.C. are coinventors on a patent application for NSD1 inhibitors. T.C. and J.G. received prior support from Kura Oncology for an unrelated project and received licensing royalties from Kura Oncology. A.F. is consulting for Ayala Pharmaceuticals and SpringWorks Therapeutics; he received previous research support from Pfizer, Bristol Myers Squibb, Merck and Eli Lilly, as well as patent and reagent licensing royalties from Novartis, EMD Millipore and Applied Biological Materials.

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Extended data

Extended Data Fig. 1 Crystal structure of NSD1 SET domain and mapping of the binding of BT2 using NMR.

a, Superposition of the crystal structure of NSD1 SET domain (residues 1863–2085) determined in this work (magenta) onto the previously described crystal structure of NSD1 SET (PDB code 3OOI, green). Positions of the N- and C- termini are labeled and SAM is in blue sticks. b, Ribbon (left) and surface (right) representations of NSD1 SET domain (residues 1863–2085) with mapped residues undergoing strong chemical shift perturbations (ΔσHN > 0.05ppm or ΔσN > 0.5ppm) upon binding of BT2 (shown in red).

Extended Data Fig. 2 Crystal structure of NSD1 SET domain in complex with covalently bound compound BT3.

a, The crystal structure of NSD1 SET domain (residues 1863–2085) with bound BT3 (blue), SAM (pale blue) and mapped residues undergoing strong chemical shift perturbations (ΔσHN > 0.05ppm or ΔσN > 0.5ppm) upon binding of BT3 (shown in red). Disulfide-bond dimer of BT3 found in the structure (pale green). The dimer binds in a site that is distant from inhibitor binding and lack of chemical shift perturbations in this area indicates that this represents a crystallization artifact. b, Same as in panel a rotated by 90 deg.

Extended Data Fig. 3 BT3 and BT5 bind to the same site on NSD1 SET domain.

a, b, Comparison of the two different fragments of 1H-15N HSQC spectra of 150 μM NSD1 SET domain (black), with 500 μM compound BT3 (blue) or 300 μM compound BT5 (red). Selected residues perturbed by both compounds BT3 and BT5 are boxed. c, The crystal structure of NSD1-BT3 complex showing chemical shift perturbations (ΔσHN > 0.05ppm or ΔσN > 0.5ppm) upon binding of BT3 (shown in red). Position of two cysteine residues (C2070 and C2072) involved in Zn coordination and strongly perturbed upon binding of BT3 is shown.

Extended Data Fig. 4 Compound BT5 does not bind covalently to NSD1 C2062A SET domain.

a, MS spectra of 1 μM wild-type NSD1 SET domain incubated with DMSO (left) or 16 μM BT5 (right) for 2 h showing 51% covalent engagement with BT5. b, MS spectra of 1 μM NSD1 C2062A SET domain incubated with DMSO (left) or 16 μM BT5 (right) for 2 h. No covalent engagement of BT5 is observed (expected mass 30,066 Da). Representative spectra shown are from two independent experiments (n = 2).

Extended Data Fig. 5 Treatment with BT5 lacks engagement with NSD2 and NSD3 in cells.

CETSA assay in HEK293T cells transfected with FLAG-NSD2 SET (top) or FLAG-NSD3 SET (bottom) constructs treated with DMSO or 5 μM BT5. Cell lysates were incubated for 3 min at indicated temperatures. Experiments were performed two times. Source data

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Supplementary Information

Supplementary Figs. 1–11, Table 1 and Notes

Reporting Summary

Source data

Source Data Fig. 5

Uncropped gels for panels a, d, e, f

Source Data Extended Data Fig. 5

Uncropped gels

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Huang, H., Howard, C.A., Zari, S. et al. Covalent inhibition of NSD1 histone methyltransferase. Nat Chem Biol (2020).

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