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A cellular chemical probe targeting the chromodomains of Polycomb repressive complex 1

Nature Chemical Biology volume 12, pages 180–187 (2016)Cite this article

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An Addendum to this article was published on 06 March 2019

Abstract

We report the design and characterization of UNC3866, a potent antagonist of the methyllysine (Kme) reading function of the Polycomb CBX and CDY families of chromodomains. Polycomb CBX proteins regulate gene expression by targeting Polycomb repressive complex 1 (PRC1) to sites of H3K27me3 via their chromodomains. UNC3866 binds the chromodomains of CBX4 and CBX7 most potently, with a Kd of ∼100 nM for each, and is 6- to 18-fold selective as compared to seven other CBX and CDY chromodomains while being highly selective over >250 other protein targets. X-ray crystallography revealed that UNC3866's interactions with the CBX chromodomains closely mimic those of the methylated H3 tail. UNC4195, a biotinylated derivative of UNC3866, was used to demonstrate that UNC3866 engages intact PRC1 and that EED incorporation into PRC1 is isoform dependent in PC3 prostate cancer cells. Finally, UNC3866 inhibits PC3 cell proliferation, consistent with the known ability of CBX7 overexpression to confer a growth advantage, whereas UNC4219, a methylated negative control compound, has negligible effects.

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Figure 1: Molecular dynamics–guided inhibitor design.
Figure 2: ITC studies of UNC3866 and UNC4219 with CBX7 and selectivity profiling of UNC3866.
Figure 3: Structural studies of UNC3866 with CBX7 and CBX8.
Figure 4: UNC4195 pulldown studies in PC3 cells.
Figure 5: Cellular effects of UNC3866.

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Acknowledgements

We thank K. Pearce (UNC Chapel Hill) for useful discussions and careful reading of the manuscript, O. Fedorov (SGC Oxford) for support with the bromodomain selectivity screening, A. Tumber (SGC Oxford) for support with the lysine demethylase selectivity screening and J.R. Walker for the review of the crystal structures. We thank G. Wang (UNC) for providing PHF1, PHF19, PHF23 and JARID1A protein constructs. We thank the labs of T. Magnuson (UNC) and D. Margolis (UNC) for providing EED and BMI-1 antibodies, respectively. We also thank N. Sciaky (UNC) for help with cell counting using the high content imaging microscope. Results shown in this report are derived from work performed at Argonne National Laboratory, Structural Biology Center at the Advanced Photon Source, a US 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. The research described here was supported by the National Institute of General Medical Sciences, US National Institutes of Health (NIH, grant R01GM100919), the Carolina Partnership and the University Cancer Research Fund, University of North Carolina at Chapel Hill, and the Welch Foundation (G-1847). The SGC is a registered charity (no. 1097737) that receives funds from AbbVie, Boehringer Ingelheim, the Canada Foundation for Innovation (CFI), the Canadian Institutes of Health Research (CIHR), Genome Canada, Ontario Genomics Institute Grant OGI-055, GlaxoSmithKline, Janssen, Lilly Canada, the Novartis Research Foundation, the Ontario Ministry of Economic Development and Innovation, Pfizer, Takeda and Wellcome Trust Grant 092809/Z/10/Z. M.T.B. directs the Protein Array Core, which is supported by the Cancer Prevention Research Institute of Texas (RP130432) and by the Centre for Environmental and Molecular Carcinogenesis at the MD Anderson Cancer Center.

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Authors and Affiliations

  1. Division of Chemical Biology and Medicinal Chemistry, Center for Integrative Chemical Biology and Drug Discovery, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

    Jacob I Stuckey, Bradley M Dickson, Nancy Cheng, Jacqueline L Norris, Stephanie H Cholensky, Katherine G Huber, Brandi M Baughman, Samantha G Pattenden, Lindsey I James & Stephen V Frye

  2. Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada

    Yanli Liu, Wolfram Tempel, Su Qin, Fengling Li, Guillermo Senisterra, Masoud Vedadi, Peter J Brown, Jinrong Min & Cheryl H Arrowsmith

  3. Department of Epigenetics and Molecular Carcinogenesis, University of Texas MD Anderson Cancer Center, Smithville, Texas, USA

    Cari Sagum, Karynne Black & Mark T Bedford

  4. National Institute of Mental Health Psychoactive Drug Screening Program, University of North Carolina at Chapel Hill Medical School, Chapel Hill, North Carolina, USA

    Xi-Ping Huang & Bryan L Roth

  5. Department of Pharmacology, University of North Carolina at Chapel Hill Medical School, Chapel Hill, North Carolina, USA

    Xi-Ping Huang & Bryan L Roth

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  1. Jacob I Stuckey
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Contributions

J.I.S. designed and synthesized all compounds and related analogs, performed ITC studies and pulldown studies, rendered structural images, assisted with AlphaScreen studies and performed all cell culture; B.M.D. performed adaptively biased molecular dynamics studies and assisted in compound design; N.C. performed CellTiter-Glo assays and cellular proliferation assays; Y.L., W.T. and S.Q. solved the X-ray crystal structures of UNC3866 with the various CBX proteins; J.L.N. and S.H.C. expressed purified proteins; K.G.H. performed pulldown studies; C.S. and K.B. performed protein array experiments; F.L. performed methyltransferase assays; X.-P.H. performed GPCR functional assays. B.M.B. performed and analyzed AlphaScreen assays; G.S. performed EED ITC experiments; J.I.S., B.D.M., S.G.P., J.M., B.L.R., M.V., P.J.B., M.T.B., C.H.A, L.I.J. and S.V.F. designed studies and discussed results; J.I.S., L.I.J. and S.V.F. wrote the paper.

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Correspondence to Lindsey I James or Stephen V Frye.

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Supplementary Results, Supplementary Figures 1–20, Supplementary Tables 1–15 and Synthetic Procedures (PDF 3958 kb)

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Stuckey, J., Dickson, B., Cheng, N. et al. A cellular chemical probe targeting the chromodomains of Polycomb repressive complex 1. Nat Chem Biol 12, 180–187 (2016). https://doi.org/10.1038/nchembio.2007

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