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The SUV4-20 inhibitor A-196 verifies a role for epigenetics in genomic integrity

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Abstract

Protein lysine methyltransferases (PKMTs) regulate diverse physiological processes including transcription and the maintenance of genomic integrity. Genetic studies suggest that the PKMTs SUV420H1 and SUV420H2 facilitate proficient nonhomologous end-joining (NHEJ)-directed DNA repair by catalyzing the di- and trimethylation (me2 and me3, respectively) of lysine 20 on histone 4 (H4K20). Here we report the identification of A-196, a potent and selective inhibitor of SUV420H1 and SUV420H2. Biochemical and co-crystallization analyses demonstrate that A-196 is a substrate-competitive inhibitor of both SUV4-20 enzymes. In cells, A-196 induced a global decrease in H4K20me2 and H4K20me3 and a concomitant increase in H4K20me1. A-196 inhibited 53BP1 foci formation upon ionizing radiation and reduced NHEJ-mediated DNA-break repair but did not affect homology-directed repair. These results demonstrate the role of SUV4-20 enzymatic activity in H4K20 methylation and DNA repair. A-196 represents a first-in-class chemical probe of SUV4-20 to investigate the role of histone methyltransferases in genomic integrity.

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Figure 1: A-196 is potent peptide-site competitive inhibitor of SUV420H1 and SUV420H2.
Figure 2: A-196 directly binds to SUV420H1.
Figure 3: Crystal structure of A-196 bound to SUV420H1.
Figure 4: A-196 inhibits H4K20 di- and trimethylation in cells.
Figure 5: A-196 inhibits 53BP1 foci formation and NHEJ proficiency.

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  • 31 January 2017

    In the version of this article initially published online, the second paragraph of the introduction contained two unnecessary commas, and "Suv4-20h1" was incorrectly listed in place of "Suv4-20h2" in one instance. The first two sentences of the description of mouse Suv4-20h expression should read: "Mouse Suv4-20h1 is ubiquitously expressed throughout embryogenesis and adult homeostasis, and mice that are homozygous null for this gene are perinatal lethal and have incomplete penetrance10. Suv4-20h2 expression, by comparison, is much less abundant during murine development. It is highly restricted in the adult, and Suv4-20h2 homozygous null mice display no apparent defects." These errors have been corrected in the print, PDF and HTML versions of this article.

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Acknowledgements

AbbVie acknowledgments: We thank V. Abraham and M. Smith of AbbVie for high-content microscopy expertise and S. Kennedy of the Structural Genomics Consortium (SGC) for technical support. The SGC is a registered charity (number 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), Janssen, Merck & Co., Novartis Pharma AG, Pfizer, São Paulo Research Foundation-FAPESP, Takeda, and Wellcome Trust. 7TM, kinase, and ion channel off-target selectivity screening was kindly supplied by Eurofins-Cerep. Further Ki determinations and receptor binding profiles were generously provided by the National Institute of Mental Health's Psychoactive Drug Screening Program, Contract # HHSN-271-2013-00017-C (NIMH PDSP). The NIMH PDSP is directed by B.L. Roth MD, PhD at the University of North Carolina at Chapel Hill and Project Officer J. Driscoll at NIMH, Bethesda, Maryland, USA. Use of the IMCA-CAT beamline 17-ID at the Advanced Photon Source was supported by the companies of the Industrial Macromolecular Crystallography Association through a contract with Hauptman-Woodward Medical Research Institute. Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357. J.C.R. is supported by the American Cancer Society (RSG117619) and an NCI Cancer Center Support Grant (P30CA014089). G.S. is supported by the Deutsche Forschungsgemeinschaft (SFB1064/A11).

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Authors

Contributions

M.A.J. and K.M.C. developed and conducted the high-throughput screen. Y.D., R.F.S., and M.R.M. designed compounds. M.T. generated 3D protein homology models and small-molecule docking/computational models. A.K.U. produced protein and protein crystals and C.G.J. performed X-ray structure determination and analysis. A.K.U. performed ITC analysis and Thermal Shift analyses. N.B.S., V.M, and M.A.A. performed in vitro biochemical studies. K.D.B. and L.M.L. performed high-content microscopy cellular methyl mark and proliferation assays. G.S. and A.N. performed immunofluorescence analyses in MEFs and ES cells. C.T.T. and J.C.R. performed DNA-damage response, 53BP1, NHEJ and HDR reporter assays. C.L. and A.M. performed the class switch recombination assays. T.R.H.M. and D.B.-L. performed histone marks analysis, toxicity and sensitization experiments. F.L. performed all lead optimization screening and IC50 determinations and mechanism-of-action studies. F.L. and M.S.E. performed selectivity assays. M.V. designed experiments, reviewed data and led in vitro assays. P.J.B., V.S., C.H.A., K.D.B., M.A., C.S., A.R.S., G.G.C., J.C.R., and W.N.P. designed studies and interpreted results. T.M., D.B.-L., K.D.B., and W.N.P. wrote the paper.

Corresponding authors

Correspondence to Dalia Barsyte-Lovejoy or William N Pappano.

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Competing interests

This study was sponsored by AbbVie. AbbVie contributed to the study design, research, and interpretation of data, writing, reviewing, and approving the publication. K.D.B., A.K.U., C.G.K., M.A.J., K.M.C., L.M.L., Y.D., N.B.S., V.M., M.A., R.F.S., M.T., C.S., M.R.M., A.R.S., G.G.C. and W.N.P. were employees of AbbVie at the time of the study.

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Bromberg, K., Mitchell, T., Upadhyay, A. et al. The SUV4-20 inhibitor A-196 verifies a role for epigenetics in genomic integrity. Nat Chem Biol 13, 317–324 (2017). https://doi.org/10.1038/nchembio.2282

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