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Discovery of a chemical probe for the L3MBTL3 methyllysine reader domain

Abstract

We describe the discovery of UNC1215, a potent and selective chemical probe for the methyllysine (Kme) reading function of L3MBTL3, a member of the malignant brain tumor (MBT) family of chromatin-interacting transcriptional repressors. UNC1215 binds L3MBTL3 with a Kd of 120 nM, competitively displacing mono- or dimethyllysine-containing peptides, and is greater than 50-fold more potent toward L3MBTL3 than other members of the MBT family while also demonstrating selectivity against more than 200 other reader domains examined. X-ray crystallography identified a unique 2:2 polyvalent mode of interaction between UNC1215 and L3MBTL3. In cells, UNC1215 is nontoxic and directly binds L3MBTL3 via the Kme-binding pocket of the MBT domains. UNC1215 increases the cellular mobility of GFP-L3MBTL3 fusion proteins, and point mutants that disrupt the Kme-binding function of GFP-L3MBTL3 phenocopy the effects of UNC1215 on localization. Finally, UNC1215 was used to reveal a new Kme-dependent interaction of L3MBTL3 with BCLAF1, a protein implicated in DNA damage repair and apoptosis.

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Figure 1: UNC1215 is a potent antagonist of L3MBTL3.
Figure 2: X-ray crystal structure of the UNC1215–3MBT complex.
Figure 3: UNC1215 binds a small set of Kme reader proteins with lower affinity than L3MBTL3.
Figure 4: UNC1215 potently antagonizes 3MBT localization in cells.
Figure 5: UNC1215 binds and colocalizes with full-length L3MBTL3.
Figure 6: Identification of BCLAF1 as a new L3MBTL3 protein interactor.

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Acknowledgements

We thank M. Vedadi, G. Wasney and F. Li for support with the protein lysine methyltransferase selectivity screening; O. Fedorov for support with the bromodomain selectivity screening; A. Tumber for support with the lysine demethylase selectivity screening; E. Hull-Ryde for support with the CellTiter-Glo cell viability assay; K. Hahn (University of North Carolina (UNC)) for providing mero76; B. Roth for helpful discussion regarding the GPCR selectivity studies; and G. Wang (UNC) for providing PHF23 and JARID1 proteins. Results shown in this report are derived from work performed at the Structural Biology Center at the Advanced Photon Source at Argonne National Laboratory. Argonne is operated by UChicago Argonne, LLC, for the US Department of Energy, Office of Biological and Environmental Research, under contract DE-AC02-06CH11357. The research described here was supported by the US National Institute of General Medical Sciences; US National Institutes of Health (grant RC1GM090732 and R01GM100919); the Carolina Partnership and the University Cancer Research Fund; University of North Carolina at Chapel Hill; the Center for Environmental and Molecular Carcinogenesis at the M.D. Anderson Cancer Center; the National Institute of Mental Health Psychoactive Drug Screening Program; the Ontario Research Fund (grant ORF-GL2); the Natural Sciences and Engineering Research Council of Canada; the Ontario Ministry of Health and Long-Term Care; the American Cancer Society (C.J.M.; 119169-PF-10-183-01-TBE); and the Structural Genomics Consortium, which is a registered charity (number 1097737) that receives funds from Canadian Institutes of Health Research; Eli Lilly Canada; Genome Canada; GlaxoSmithKline; the Ontario Ministry of Economic Development and Innovation; the Novartis Research Foundation; Pfizer; Abbott; Takeda; and the Wellcome Trust. M.T.B. is supported by an institutional grant from the US National Institute of Environmental Health Sciences (ES007784) and Cancer Prevention Research Institute of Texas funding (RP110471). C.H.A. holds a Canada Research Chair in Structural Genomics.

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L.I.J. synthesized all compounds and related analogs and performed ITC studies; D.B.-L. and L.K. performed immunofluorescence FRAP, affinity purification and coimmunoprecipitation studies; N.Z. and W.T. solved and analyzed the X-ray crystal structure of the UNC1215–L3MBTL3 complex; V.K.K. and W.P.J. performed and analyzed AlphaScreen studies; C.J.M. synthesized the mero76-UNC1215 conjugate; J.L.N. purified proteins and performed mutagenesis; C.A.S. and M.T.B. performed protein array and protein pull-down experiments; E.M., H.G., A.E. and J.F.G. performed MS-based studies; S.D. cloned mammalian expression vectors for all cellular studies; X.-P.H. performed and analyzed GPCR selectivity studies; L.I.J., D.B.-L., N.Z., L.K., J.M.H., V.K.K., C.G., D.B.K., J.J., W.P.J., P.J.B., M.T.B, C.H.A. and S.V.F. designed studies and discussed results; L.I.J., C.H.A. and S.V.F. wrote the paper.

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Correspondence to Cheryl H Arrowsmith or Stephen V Frye.

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James, L., Barsyte-Lovejoy, D., Zhong, N. et al. Discovery of a chemical probe for the L3MBTL3 methyllysine reader domain. Nat Chem Biol 9, 184–191 (2013). https://doi.org/10.1038/nchembio.1157

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