Letter | Published:

Synergistic blockade of mitotic exit by two chemical inhibitors of the APC/C

Nature volume 514, pages 646649 (30 October 2014) | Download Citation

Abstract

Protein machines are multi-subunit protein complexes that orchestrate highly regulated biochemical tasks. An example is the anaphase-promoting complex/cyclosome (APC/C), a 13-subunit ubiquitin ligase that initiates the metaphase–anaphase transition and mitotic exit by targeting proteins such as securin and cyclin B1 for ubiquitin-dependent destruction by the proteasome1,2. Because blocking mitotic exit is an effective approach for inducing tumour cell death3,4, the APC/C represents a potential novel target for cancer therapy. APC/C activation in mitosis requires binding of Cdc20 (ref. 5), which forms a co-receptor with the APC/C to recognize substrates containing a destruction box (D-box)6,7,8,9,10,11,12,13,14. Here we demonstrate that we can synergistically inhibit APC/C-dependent proteolysis and mitotic exit by simultaneously disrupting two protein–protein interactions within the APC/C–Cdc20–substrate ternary complex. We identify a small molecule, called apcin (APC inhibitor), which binds to Cdc20 and competitively inhibits the ubiquitylation of D-box-containing substrates. Analysis of the crystal structure of the apcin–Cdc20 complex suggests that apcin occupies the D-box-binding pocket on the side face of the WD40-domain. The ability of apcin to block mitotic exit is synergistically amplified by co-addition of tosyl-l-arginine methyl ester, a small molecule that blocks the APC/C–Cdc20 interaction15,16. This work suggests that simultaneous disruption of multiple, weak protein–protein interactions is an effective approach for inactivating a protein machine.

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Accessions

Primary accessions

Protein Data Bank

Data deposits

Structure coordinates have been deposited in Protein Data Bank under accession number 4N14.

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Acknowledgements

We thank W. Harper for providing constructs for WD40-containing proteins, T. Gahman for assistance with apcin synthesis and D. Tomchick for assistance with structure refinement. Results shown in this report are derived from work performed at Argonne National Laboratory, Structural Biology Center at the Advanced Photon Source. Argonne is operated by UChicago Argonne, LLC, for the US Department of Energy, Office of Biological and Environmental Research under contract DE-AC02-06CH11357. This work was supported by grants from the National Institutes of Health (GM085004 to X.L. and GM066492 to R.W.K.) and by a grant from the Lynch Foundation to R.W.K.

Author information

Author notes

    • Katharine L. Sackton
    • , Nevena Dimova
    • , Xing Zeng
    •  & Wei Tian

    These authors contributed equally to this work.

    • Wei Tian
    • , Kathleen L. Pfaff
    •  & Frederic Sigoillot

    Present addresses: Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China (W.T.); Department of Stem Cell and Regenerative Biology, Harvard University, 7 Divinity Avenue, Cambridge, Massachusetts 02138, USA (K.L.P.); Novartis Institutes for Biomedical Research, 250 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA (F.S.).

Affiliations

  1. Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA

    • Katharine L. Sackton
    • , Nevena Dimova
    • , Xing Zeng
    • , Mengmeng Zhang
    • , Johnathan Meaders
    • , Kathleen L. Pfaff
    • , Frederic Sigoillot
    •  & Randall W. King
  2. Department of Pharmacology, University of Texas Southwestern Medical Center, 6001 Forest Park Road, Dallas, Texas 75390, USA

    • Wei Tian
    • , Hongtao Yu
    •  & Xuelian Luo
  3. Department of Organismic and Evolutionary Biology, Harvard University, 26 Oxford Street, Cambridge, Massachusetts 02138, USA

    • Timothy B. Sackton
  4. Howard Hughes Medical Institute, 4000 Jones Bridge Road, Chevy Chase, Maryland 20815, USA

    • Hongtao Yu

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Contributions

K.L.S. and X.Z. performed experiments to identify Cdc20 as the target of apcin. K.L.S. characterized binding of apcin to Cdc20, and evaluated effects of Cdc20 mutations on apcin binding and cyclin proteolysis. W.T. purified Cdc20 and performed crystallization and structure determination of the Cdc20–apcin complex. H.Y. and X.L. contributed to structure determination and data analysis. N.D. and X.Z. characterized effects of apcin and TAME on substrate degradation, ubiquitylation and Cdc20 binding to APC/C in Xenopus extract. N.D. evaluated binding of apcin to Cdc20 using the thermal shift assay. M.Z. characterized effects of apcin and proTAME on mitotic index in fixed cell assays. K.L.S. and X.Z. characterized effects of apcin and proTAME by live cell imaging. F.S. developed the high-throughput mitotic index assay. K.L.S., X.Z., J.M., K.L.P. and F.S. analysed time-lapse videos. T.B.S. developed statistical models and performed statistical analysis. R.W.K. conceived the project, assisted with experimental design and data analysis, and wrote the manuscript, with assistance from all authors.

Competing interests

There is a patent application on this work, filed by Harvard University on behalf of the authors.

Corresponding author

Correspondence to Randall W. King.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains Supplementary Methods (synthesis of apcin and apcin-A, statistical methods for analysis of fixed cell and time lapase data), a Supplementary Discussion, and Supplementary References.

Text files

  1. 1.

    Supplementary Data

    R code to generate statistical analysis of combined action of proTAME and apcin in fixed cell imaging assay.

  2. 2.

    Supplementary Data

    R code to generate statistical analysis of live cell imaging data.

Videos

  1. 1.

    Effects of apcin and proTAME in control-siRNA treated cells

    Apcin + proTAME prolongs mitotic duration more than either drug alone in cells treated with control siRNA. RPE1-H2B-GFP cells were treated with 25 μM apcin and/or 6 μM proTAME. The apcin-treated cell divides. The proTAME-treated cell demonstrates abnormal mitotic exit, forming a single mononucleated daughter cell after an attempt at anaphase. The apcin + proTAME –treated cell dies in mitosis. Top row shows Histone H2B-GFP signal, bottom row shows differential interference contrast (DIC). Scale bar indicates 20 μm.

  2. 2.

    Effects of apcin and proTAME in Mad2-siRNA treated cells

    Apcin + proTAME prolongs mitotic duration more than either drug alone in cells treated with Mad2 siRNA. RPE1-H2B-GFP cells were treated with 25 μM apcin and/or 6 μM proTAME. The apcin-treated cell and the proTAME-treated cell divide. The apcin + proTAME –treated cell demonstrates abnormal mitotic exit: the condensed chromatin decondenses, forming a single mononucleated daughter. Another cell in the top left of the same frame dies in mitosis. Top row shows Histone H2B-GFP signal, bottom row shows differential interference contrast (DIC). Scale bar indicates 20 μm.

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DOI

https://doi.org/10.1038/nature13660

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