A chemical probe selectively inhibits G9a and GLP methyltransferase activity in cells

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  • An Erratum to this article was published on 17 August 2011


Protein lysine methyltransferases G9a and GLP modulate the transcriptional repression of a variety of genes via dimethylation of Lys9 on histone H3 (H3K9me2) as well as dimethylation of non-histone targets. Here we report the discovery of UNC0638, an inhibitor of G9a and GLP with excellent potency and selectivity over a wide range of epigenetic and non-epigenetic targets. UNC0638 treatment of a variety of cell lines resulted in lower global H3K9me2 levels, equivalent to levels observed for small hairpin RNA knockdown of G9a and GLP with the functional potency of UNC0638 being well separated from its toxicity. UNC0638 markedly reduced the clonogenicity of MCF7 cells, reduced the abundance of H3K9me2 marks at promoters of known G9a-regulated endogenous genes and disproportionately affected several genomic loci encoding microRNAs. In mouse embryonic stem cells, UNC0638 reactivated G9a-silenced genes and a retroviral reporter gene in a concentration-dependent manner without promoting differentiation.

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Figure 1: UNC0638 competes with the peptide substrate but not with the cofactor SAM.
Figure 2: UNC0638 inhibits cellular H3K9 dimethylation and shows good separation of functional potency and toxicity in MDA-MB-231 cells.
Figure 3: Quantitative MS analysis of histone post-translational modifications in MDA-MB-231 cells.
Figure 4: Effects of UNC0638 on H3K9me2 and DNA methylation.
Figure 5: UNC0638 reactivates a silent EGFP retrovirus vector and G9a-regulated endogenous genes in mES cells.

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We thank A. Tumber for JMJD2E assay support; J. Moffat (University of Toronto) for the gift of shRNAs; R. Bristow (University Health Network) for RV221 and PC3 cells; T. Hajian and F. Syeda for protein purification; G. Senisterra for contributing to DSF and DSLS data analysis; M. Herold for graphical design and illustration; I. Korboukh, M. Herold and J. Yost for critical reading of the manuscript; and R. Trump and C. Yates for helpful discussion. The research described here was supported by the National Institute of General Medical Sciences, US National Institutes of Health (NIH; grant RC1GM090732), the Carolina Partnership and University Cancer Research Fund from the University of North Carolina at Chapel Hill, the US National Science Foundation (NSF), the Ontario Research Fund, the Ontario Ministry of Health and Long-term Care and the Structural Genomics Consortium. The Structural Genomics Consortium is a registered charity (number 1097737) that receives funds from the Canadian Institutes for Health Research (CIHR), the Canada Foundation for Innovation, Genome Canada through the Ontario Genomics Institute, GlaxoSmithKline, Karolinska Institutet, the Knut and Alice Wallenberg Foundation, the Ontario Innovation Trust, the Ontario Ministry for Research and Innovation, Merck & Co. Inc., the Novartis Research Foundation, the Swedish Agency for Innovation Systems, the Swedish Foundation for Strategic Research and the Wellcome Trust. C.H.A. holds a Canada Research Chair in Structural Genomics. V.L. is supported by a CIHR fellowship. A.P. is supported by grants from the CIHR (199170 and 186007) and from the NIH (MH074127, MH088413, DP3DK085698 and HG004535). A.P. is Tapscott Chair in Schizophrenia Studies and a Senior Fellow of the Ontario Mental Health Foundation. J.E. is supported by CIHR grant IG1-102956. B.A.G. is supported by grants from the NSF (Early Faculty CAREER award and CBET-0941143), the American Society for Mass Spectrometry and the NIH Office of the Director (DP2OD007447). P.A.D. is supported by NIH postdoctoral fellowship F32 NRSA.

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M.V., A.A.-H., A.S. and I.C. performed SAHH-coupled, fluorescence-polarization, DSF, DSLS and DNMT1 assays; D.B.-L. developed and performed in-cell western, MTT, ChIP, gene expression, clonogenicity, western blotting and immunofluorescence studies; F.L. developed the synthetic route to UNC0638 and UNC0737 and synthesized the compounds; S.R.-G. and J.E. developed and performed mES cell studies; V.L., S.-C.W. and A.P. performed H3K9me2 genomic localization and DNA methylation analysis; T.J.W. and W.P.J. performed mechanism-of-action studies; P.A.D. and B.A.G. performed MS-based proteomics studies; M.V., G.A.W., A.D., W.T., D.B.K. and C.H.A. solved and analyzed the X-ray crystal structure of the G9a-UNC0638-SAH complex; T.J.W. and A.T. performed SPR studies; X.C. and S.G.P. performed UNC0638 stability studies; S.G.P. performed RT-qPCR studies; T.J.M., X.-p.H. and B.L.R. performed GPCR selectivity studies; C.D.S. and W.P.J. performed kinase selectivity studies; J.L.N. purified proteins; J.J. designed UNC0638 and UNC0737; C.H.A., J.J., S.V.F., M.V., D.B.-L., P.J.B., J.E., S.R.-G., A.P., V.L., B.A.G., T.J.W. and A.E. designed studies and discussed results; J.J., C.H.A., D.B.-L., S.R.-G., M.V., V.L., S.V.F., J.E., A.P., B.A.G., P.J.B. and T.J.W. wrote the paper.

Correspondence to Cheryl H Arrowsmith or Jian Jin.

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