Discovery of a chemical probe for PRDM9

Allali-Hassani, Abdellah; Szewczyk, Magdalena M.; Ivanochko, Danton; Organ, Shawna; Bok, Jabez; Ho, Jessica; Li, Fengling; Blazer, Levi L.; Eram, Mohammad S.; Halabelian, L.; Dilworth, David; Luciani, Genna M.; Lima‐Fernandes, Evelyne; Wu, Qin; Loppnau, P.; Palmer, Nathan; Talib, S. Zakiah A.; Brown, Peter J.; Schapira, Matthieu; Kaldis, Philipp; O’Hagan, Ronan; Guccione, Ernesto; Baršytė-Lovejoy, Dalia; Arrowsmith, C.H.; Sanders, John M.; Kattar, Solomon D.; Bennett, David Jonathan; Nicholson, Benjamin; Vedadi, Masoud

doi:10.1038/s41467-019-13652-x

Cited by 34 publications

(33 citation statements)

References 37 publications

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“…A wide variety of methyltransferase assays have been described in the literature (38,44,(61)(62)(63)(64)(65)(66); however, few optimized assays have been reported for NSD2 in plate formats to enable HTS. Recently a radiolabeled [ 3 H]SAM assay was optimized for the NSD2 SET domain with both histone octamer (Z' = 0.69) and nucleosome (Z' = 0.75) substrates in a 384-well format (39).…”

Section: Discussionmentioning

confidence: 99%

Small Molecule Inhibitors of the Human Histone Lysine Methyltransferase NSD2 / WHSC1 / MMSET Identified from a Quantitative High-Throughput Screen with Nucleosome Substrate

Henderson

et al. 2017

Preprint

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The activity of the histone lysine methyltransferase NSD2 is thought to play a driving role in oncogenesis. Both overexpression of NSD2 and point mutations that increase its catalytic activity are associated with a variety of human cancers. While NSD2 is an attractive therapeutic target, no potent, selective and cell-active inhibitors have been reported to date, possibly due to the challenging nature of developing high-throughput assays for NSD2. To establish a platform for the discovery and development of selective NSD2 inhibitors, multiple assays were optimized and implemented. Quantitative high-throughput screening was performed with full-length wildtype NSD2 and a nucleosome substrate against a diverse collection of known bioactives comprising 16,251 compounds. Actives from the primary screen were further interrogated with orthogonal and counter assays, as well as activity assays with the clinically relevant NSD2 mutants E1099K and T1150A. Five confirmed inhibitors were selected for follow-up, which included a radiolabeled validation assay, surface plasmon resonance studies, methyltransferase profiling, and histone methylation in cells. The identification of NSD2 inhibitors that bind the catalytic SET domain and demonstrate activity in cells validates the workflow, providing a template for identifying selective NSD2 inhibitors.

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Section: Discussionmentioning

confidence: 99%

Small Molecule Inhibitors of the Human Histone Lysine Methyltransferase NSD2 / WHSC1 / MMSET Identified from a Quantitative High-Throughput Screen with Nucleosome Substrate

Henderson

et al. 2017

Preprint

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“…Selectivity of UNC6934 for NSD2-PWWP1 over 14 other PWWP domains was tested using differential scanning fluorimetry (DSF) as previously described 35 The diffraction data for NSD2-PWWP1+UNC6934 was collected at 100K on the home source Rigaku FR-E superbright and data set was processed using the HKL-3000 suite 38 . The structure was solved by molecular replacement methods with PHASER using PDB entry 5VC8 as search template.…”

Section: Selectivity Assaysmentioning

confidence: 99%

Pharmacological targeting of a PWWP domain demonstrates cooperative control of NSD2 localization

Dilworth

et al. 2021

Preprint

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NSD2 is the primary enzyme responsible for the dimethylation of lysine 36 of histone 3 (H3K36me2), a mark associated with active gene transcription and intergenic DNA methylation. In addition to a methyltransferase domain, NSD2 harbors two PWWP and five PHD domains believed to serve as chromatin reading modules, but their exact function in the regulation of NSD2 activity remains underexplored. Here we report a first-in-class chemical probe targeting the N-terminal PWWP (PWWP1) domain of NSD2. UNC6934 binds potently (Kd of 91 +/- 8 nM) to PWWP1, antagonizes its interaction with nucleosomal H3K36me2, and selectively engages endogenous NSD2 in cells. Crystal structures show that UNC6934 occupies the canonical H3K36me2-binding pocket of PWWP1 which is juxtaposed to the DNA-binding surface. In cells, UNC6934 induces accumulation of endogenous NSD2 in the nucleolus, phenocopying the localization defects of NSD2 protein isoforms lacking PWWP1 as a result of translocations prevalent in multiple myeloma. Mutation of other NSD2 chromatin reader domains also increases NSD2 nucleolar localization, and enhances the effect of UNC6934. Finally we identified two C-terminal nucleolar localization sequences in NSD2 that appear to drive nucleolar accumulation when one or more chromatin reader domains are disabled. These data support a model in which NSD2 chromatin engagement is achieved in a cooperative manner and subcellular localization is controlled by multiple competitive structural determinants. This chemical probe and the accompanying negative control, UNC7145, will be useful tools in defining NSD2 biology.

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“…Finally, the recent discovery of MRK-740 as a potent, selective and cell-active substrate-competitive PRDM9 inhibitor, opens the way for targeting the PRDM subfamily of SAM-dependent methyltransferases [298].…”

Section: Clinical Value Of Prdms In Cancer and Concluding Remarksmentioning

confidence: 99%

Multifaceted Role of PRDM Proteins in Human Cancer

Casamassimi

Rienzo

Zazzo

et al. 2020

IJMS

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The PR/SET domain family (PRDM) comprise a family of genes whose protein products share a conserved N-terminal PR [PRDI-BF1 (positive regulatory domain I-binding factor 1) and RIZ1 (retinoblastoma protein-interacting zinc finger gene 1)] homologous domain structurally and functionally similar to the catalytic SET [Su(var)3-9, enhancer-of-zeste and trithorax] domain of histone methyltransferases (HMTs). These genes are involved in epigenetic regulation of gene expression through their intrinsic HMTase activity or via interactions with other chromatin modifying enzymes. In this way they control a broad spectrum of biological processes, including proliferation and differentiation control, cell cycle progression, and maintenance of immune cell homeostasis. In cancer, tumor-specific dysfunctions of PRDM genes alter their expression by genetic and/or epigenetic modifications. A common characteristic of most PRDM genes is to encode for two main molecular variants with or without the PR domain. They are generated by either alternative splicing or alternative use of different promoters and play opposite roles, particularly in cancer where their imbalance can be often observed. In this scenario, PRDM proteins are involved in cancer onset, invasion, and metastasis and their altered expression is related to poor prognosis and clinical outcome. These functions strongly suggest their potential use in cancer management as diagnostic or prognostic tools and as new targets of therapeutic intervention.

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Discovery of a chemical probe for PRDM9

Cited by 34 publications

References 37 publications

Small Molecule Inhibitors of the Human Histone Lysine Methyltransferase NSD2 / WHSC1 / MMSET Identified from a Quantitative High-Throughput Screen with Nucleosome Substrate

Small Molecule Inhibitors of the Human Histone Lysine Methyltransferase NSD2 / WHSC1 / MMSET Identified from a Quantitative High-Throughput Screen with Nucleosome Substrate

Pharmacological targeting of a PWWP domain demonstrates cooperative control of NSD2 localization

Multifaceted Role of PRDM Proteins in Human Cancer

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