Abstract

The ubiquitin system regulates essential cellular processes in eukaryotes. Ubiquitin is ligated to substrate proteins as monomers or chains and the topology of ubiquitin modifications regulates substrate interactions with specific proteins. Thus ubiquitination directs a variety of substrate fates including proteasomal degradation1. Deubiquitinase enzymes cleave ubiquitin from substrates and are implicated in disease2; for example, ubiquitin-specific protease-7 (USP7) regulates stability of the p53 tumour suppressor and other proteins critical for tumour cell survival3. However, developing selective deubiquitinase inhibitors has been challenging4 and no co-crystal structures have been solved with small-molecule inhibitors. Here, using nuclear magnetic resonance-based screening and structure-based design, we describe the development of selective USP7 inhibitors GNE-6640 and GNE-6776. These compounds induce tumour cell death and enhance cytotoxicity with chemotherapeutic agents and targeted compounds, including PIM kinase inhibitors. Structural studies reveal that GNE-6640 and GNE-6776 non-covalently target USP7 12 Å distant from the catalytic cysteine. The compounds attenuate ubiquitin binding and thus inhibit USP7 deubiquitinase activity. GNE-6640 and GNE-6776 interact with acidic residues that mediate hydrogen-bond interactions with the ubiquitin Lys48 side chain5, suggesting that USP7 preferentially interacts with and cleaves ubiquitin moieties that have free Lys48 side chains. We investigated this idea by engineering di-ubiquitin chains containing differential proximal and distal isotopic labels and measuring USP7 binding by nuclear magnetic resonance. This preferential binding protracted the depolymerization kinetics of Lys48-linked ubiquitin chains relative to Lys63-linked chains. In summary, engineering compounds that inhibit USP7 activity by attenuating ubiquitin binding suggests opportunities for developing other deubiquitinase inhibitors and may be a strategy more broadly applicable to inhibiting proteins that require ubiquitin binding for full functional activity.

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Biological Magnetic Resonance Data Bank

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Acknowledgements

We thank W. Fairbrother, L. Frick, S. Fong, E. Helgason, T. Hunsaker, C. Lam, B. Liederer, M. Merchant, J. Nonomiya, J. Peng, T. Pham, L. Rangell, R. Rodriguez, U. Segal, R. Tong, L. Wang, R. Gennis, T. Iwasaki, the Genentech Protein Expression, Cell Central, gCSI, and Sequencing groups, and the Boston Biochem team for reagents and collaborations. Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under contract number DE-AC02-76SF00515. The SSRl Structural Molecular Biology Program was supported by the DOE Office of Biological and Environmental Research, and by the National Institutes of Health, National Institute of General Medical Sciences (including P41GM103393). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of National Institute of General Medical Sciences or the National Institutes of Health.

Author information

Author notes

    • Lorna Kategaya
    • , Paola Di Lello
    •  & Lionel Rougé

    These authors contributed equally to this work.

Affiliations

  1. Department of Discovery Oncology, Genentech, South San Francisco, California 94080, USA

    • Lorna Kategaya
    • , Eva Lin
    • , John-Paul Upton
    • , Sumit Prakash
    • , Johanna Heideker
    • , Mark McCleland
    • , Scott E. Martin
    •  & Ingrid E. Wertz
  2. Department of Early Discovery Biochemistry, Genentech, South San Francisco, California 94080, USA

    • Lorna Kategaya
    • , John-Paul Upton
    • , Sumit Prakash
    • , Johanna Heideker
    • , Mark McCleland
    •  & Ingrid E. Wertz
  3. Department of Structural Biology, Genentech, South San Francisco, California 94080, USA

    • Paola Di Lello
    • , Lionel Rougé
    • , Jeremy Murray
    •  & Till Maurer
  4. Department of Discovery Chemistry, Genentech, South San Francisco, California 94080, USA

    • Richard Pastor
    • , Frederick Cohen
    • , Chudi Ndubaku
    • , Xiaojing Wang
    •  & Vickie Tsui
  5. Department of Biochemical and Cellular Pharmacology, Genentech, South San Francisco, California 94080, USA

    • Kevin R. Clark
    • , Jason Drummond
    • , Tracy Kleinheinz
    • , Maureen H. Beresini
    •  & Robert A. Blake
  6. MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, UK

    • Maria Stella Ritorto
    •  & Dario R. Alessi
  7. Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne NE2 1HH, UK

    • Matthias Trost
  8. Department of Protein Chemistry, Genentech, South San Francisco, California 94080, USA

    • Travis W. Bainbridge
    • , Michael C. M. Kwok
    •  & James A. Ernst
  9. Department of Microchemistry, Proteomics, and Lipidomics, Genentech, South San Francisco, California 94080, USA

    • Taylur P. Ma
    •  & Kebing Yu
  10. Boston Biochem, 840 Memorial Drive, Cambridge, Massachusetts 02139, USA

    • Zachary Stiffler
    • , Bradley Brasher
    •  & Carsten Schwerdtfeger
  11. Department of Pharmaceutical Chemistry and Small Molecule Discovery Center, University of California, San Francisco, San Francisco, California 94143, USA

    • Yinyan Tang
    • , Priyadarshini Jaishankar
    • , Brian R. Hearn
    • , Adam R. Renslo
    •  & Michelle R. Arkin
  12. Department of Research Pathology, Genentech, South San Francisco, California 94080, USA

    • Frank Peale
  13. Department of Bioinformatics and Computational Biology, Genentech, South San Francisco, California 94080, USA

    • Florian Gnad
    • , Matthew T. Chang
    • , Christiaan Klijn
    •  & William F. Forrest
  14. Department of Translational Oncology, Genentech, South San Francisco, California 94080, USA

    • Elizabeth Blackwood

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Contributions

L.K., E.L., J.-P.U., S.P., F.P., S.E.M., and I.E.W. performed cell viability and signalling studies, protein turnover and ubiquitination experiments, and independently replicated cellular data. P.D.L. and T.M. performed NMR studies; L.R. and J.M. performed X-ray crystallography and cross-validated data. J.D., T.K., T.W.B., M.C.M.K., Z.S., B.B., Y.T., M.R.A., J.A.E., C.S., and R.A.B. performed in vitro and cellular biochemical studies and independently replicated data. J.H., M.S.R., T.P.M., D.R.A., M.T., and K.Y. performed deubiquitinase selectivity profiling studies, and M.M. and E.B. performed in vivo studies and cross-validated data. K.R.C. and M.H.B. developed assays, and performed and analysed high-throughput screening studies. R.P., P.J., B.R.H., A.R.R., F.C., C.N., X.W., and V.T. performed compound design/synthesis. F.G., M.T.C., C.K., and W.F.F. performed bioinformatics/statistical analysis and replicated data. T.M. and I.E.W. coordinated studies. J.M., T.M., and I.E.W. wrote the manuscript. D.R.A. is supported by the UK Medical Research Council (grant MC_UU_12016/2).

Competing interests

L.K., P.D.L., L.R., R.P., K.R.C., J.D., T.K., E.L., J.-P.U., S.P., J.H., M.M., T.W.B., M.C.M.K., T.P.M., F.C., K.Y., F.P., F.G., M.T.C., C.K., E.B., S.E.M., W.F.F., J.A.E., C.N., X.W., M.H.B., V.T., R.A.B., J.M., T.M., and I.E.W. are or were Genentech employees. M.S.R. is a Pfizer employee.

Corresponding authors

Correspondence to Till Maurer or Ingrid E. Wertz.

Reviewer Information Nature thanks M. Rolfe, S. Scherer and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Supplementary information

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  1. 1.

    Reporting Summary

  2. 2.

    Supplementary Information

    This file contains Supplementary Figures 1-2, Supplementary Tables 1-2 and Supplementary Methods.

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    Supplementary Figures

    This file contains Supplementary Figures containing gel source data and graphs, and Supplementary Tables 1 and 2.

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