Abstract

N-terminal acetylation is an abundant modification influencing protein functions. Because 80% of mammalian cytosolic proteins are N-terminally acetylated, this modification is potentially an untapped target for chemical control of their functions. Structural studies have revealed that, like lysine acetylation, N-terminal acetylation converts a positively charged amine into a hydrophobic handle that mediates protein interactions; hence, this modification may be a druggable target. We report the development of chemical probes targeting the N-terminal acetylation–dependent interaction between an E2 conjugating enzyme (UBE2M or UBC12) and DCN1 (DCUN1D1), a subunit of a multiprotein E3 ligase for the ubiquitin-like protein NEDD8. The inhibitors are highly selective with respect to other protein acetyl-amide–binding sites, inhibit NEDD8 ligation in vitro and in cells, and suppress anchorage-independent growth of a cell line with DCN1 amplification. Overall, our data demonstrate that N-terminal acetyl-dependent protein interactions are druggable targets and provide insights into targeting multiprotein E2–E3 ligases.

  • Compound

    1-(1-benzylpiperidin-4-yl)-3-(3-(trifluoromethyl)phenyl)urea

  • Compound

    1-benzyl-1-(1-butylpiperidin-4-yl)-3-(3,4-dichlorophenyl)urea

  • Compound

    1-benzyl-1-(1-butylpiperidin-4-yl)-3-(pyridin-3-yl)urea

  • Compound

    N-(2-((1-(1-(pentan-2-yl)piperidin-4-yl)-3-(3-(trifluoromethyl)phenyl)ureido)methyl)phenyl)acrylamide

  • Compound

    2-chloro-N-(2-((1-(1-(pentan-2-yl)piperidin-4-yl)-3-(3-(trifluoromethyl)phenyl)ureido)methyl)phenyl)acetamide

  • Compound

    N-(3-((1-(1-(pentan-2-yl)piperidin-4-yl)-3-(3-(trifluoromethyl)phenyl)ureido)methyl)phenyl)acrylamide

  • Compound

    2-chloro-N-(3-((1-(1-(pentan-2-yl)piperidin-4-yl)-3-(3-(trifluoromethyl)phenyl)ureido)methyl)phenyl)acetamide

  • Compound

    N-(2-((1-(1-(pentan-2-yl)piperidin-4-yl)-3-(3-(trifluoromethyl)phenyl)ureido)methyl)benzyl)acrylamide

  • Compound

    2-chloro-N-(2-((1-(1-(pentan-2-yl)piperidin-4-yl)-3-(3-(trifluoromethyl)phenyl)ureido)methyl)benzyl)acetamide

  • Compound

    N-(3-((1-(1-(pentan-2-yl)piperidin-4-yl)-3-(3-(trifluoromethyl)phenyl)ureido)methyl)benzyl)acrylamide

  • Compound

    2-chloro-N-(3-((1-(1-(pentan-2-yl)piperidin-4-yl)-3-(3-(trifluoromethyl)phenyl)ureido)methyl)benzyl)acetamide

  • Compound

    N-(2-(1-(1-(pentan-2-yl)piperidin-4-yl)-3-(3-(trifluoromethyl)phenyl)ureido)ethyl)acrylamide

  • Compound

    2-chloro-N-(2-(1-(1-(pentan-2-yl)piperidin-4-yl)-3-(3-(trifluoromethyl)phenyl)ureido)ethyl)acetamide

  • Compound

    N-(3-(1-(1-(pentan-2-yl)piperidin-4-yl)-3-(3-(trifluoromethyl)phenyl)ureido)propyl)acrylamide

  • Compound

    2-chloro-N-(3-(1-(1-(pentan-2-yl)piperidin-4-yl)-3-(3-(trifluoromethyl)phenyl)ureido)propyl)acetamide

  • Compound

    N-(2-((1-(1-(pentan-2-yl)piperidin-4-yl)-3-(3-(trifluoromethyl)phenyl)ureido)methyl)phenyl)propionamide

  • Compound

    1-butylpiperidin-4-one

  • Compound

    N-benzyl-1-butylpiperidin-4-amine

  • Compound

    piperidin-4-one

  • Compound

    1-(pentan-2-yl)piperidin-4-one

  • Compound

    tert-butyl (2-(((1-(pentan-2-yl)piperidin-4-yl)amino)methyl)phenyl)carbamate

  • Compound

    tert-butyl (2-((1-(1-(pentan-2-yl)piperidin-4-yl)-3-(3-(trifluoromethyl)phenyl)ureido)methyl)phenyl)carbamate

  • Compound

    1-(2-aminobenzyl)-1-(1-(pentan-2-yl)piperidin-4-yl)-3-(3-(trifluoromethyl)phenyl)urea

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Acknowledgements

B.A.S., ALSAC, HHMI, and NIH R37GM069530, P30CA021765; J.T.H., NIH F32GM113310; J.W.H., NIH AG011085; J.A.P., NIH DK098285; J.P., NIH GM114260; SJCRH Proteomics Facility, NIH P30CA021765; American Lebanese Syrian Associated Charities. We acknowledge the High Throughput Biosciences Center, Medicinal Chemistry Center, Compound Management, and High Throughput Analytical Chemistry Centers in Chemical Biology and Therapeutics; Hartwell Center for use of their personnel and facilities. We thank J. Lee (Harvard Medical School) for assistance with TMT statistics, E.R. Watson (St. Jude Children's Research Hospital) for purified hNatC enzyme, J. Earl (St. Jude Children's Research Hospital) for photography, R. Marmorstein (Perelman School of Medicine, University of Pennsylvania) and T. Arnesen (University of Bergen) for NAT constructs, and staff at the BL8.2.1 and 22-ID beamlines at the Advanced Light Source and Advanced Photon Source.

Author information

Author notes

    • Jared T Hammill
    • , Yizhe Chen
    • , Ho Shin Kim
    •  & R Kip Guy

    Present address: Department of Pharmaceutical Sciences, University of Kentucky, Lexington, Kentucky, USA.

    • Daniel C Scott
    •  & Jared T Hammill

    These authors contributed equally to this work.

Affiliations

  1. Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.

    • Daniel C Scott
    • , Vladislav O Sviderskiy
    • , Julie K Monda
    • , Junmin Peng
    •  & Brenda A Schulman
  2. Howard Hughes Medical Institute, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.

    • Daniel C Scott
    •  & Brenda A Schulman
  3. Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.

    • Jared T Hammill
    • , Jaeki Min
    • , Michele Connelly
    • , Deepak Bhasin
    • , Yizhe Chen
    • , Su-Sien Ong
    • , Sergio C Chai
    • , Asli N Goktug
    • , Jonathan Low
    • , Ho Shin Kim
    • , Anang A Shelat
    • , Taosheng Chen
    •  & R Kip Guy
  4. Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA.

    • David Y Rhee
    • , Joao A Paulo
    • , Joe R Cannon
    •  & J Wade Harper
  5. Laboratory of Epithelial Cancer Biology, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.

    • Guochang Huang
    •  & Bhuvanesh Singh
  6. MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, UK.

    • Ian R Kelsall
  7. Department of Molecular Machines and Signaling, Max Planck Institute of Biochemistry, Martinsried, Germany.

    • Arno F Alpi
  8. St. Jude Proteomics Facility, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.

    • Vishwajeeth Pagala
    • , Xusheng Wang
    •  & Junmin Peng

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Contributions

D.C.S., J.T.H., B.S., J.W.H., B.A.S., and R.K.G. designed the research project and analyzed data; D.C.S., J.K.M., S.C.C., A.N.G., and J.M. contributed to assay development; D.C.S., V.O.S., and J.K.M. performed X-ray crystallography; J.T.H., D.B., and H.S.K. designed and performed compound optimization and syntheses; A.A.S. and T.C. contributed to the HTS campaign; D.C.S. performed in vitro neddylation experiments and western blotting; M.C. and D.Y.R. generated cell lines; S.-S.O. and M.C. generated the DCN1 shRNA line; I.R.K. and A.F.A. generated DCN1 Flp-In cell lines; Y.C. performed in vitro ADME-tox studies to aid in compound optimization; M.C., G.H., and J.L. performed cell-based experiments; D.Y.R., J.A.P., and J.R.C. performed cell-based AP-MS experiments; V.P., X.W., and J.P. performed TMT analysis on proteome samples; all authors contributed to specific parts of the manuscript, and D.C.S., J.T.H., B.A.S. and R.K.G. assume responsibility for the manuscript in its entirety.

Competing interests

The authors have filed a provisional application with the US Patent and Trademark Office in relation to this work.

Corresponding authors

Correspondence to Brenda A Schulman or R Kip Guy.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Results, Supplementary Tables 1–3 and Supplementary Figures 1–16

  2. 2.

    Supplementary Note

    Synthetic procedures

Excel files

  1. 1.

    Supplementary Data Set 1

    Hit validation set

  2. 2.

    Supplementary Data Set 2

    Related analogs

  3. 3.

    Supplementary Data Set 3

    Histone acetyltransferase off target profiling

  4. 4.

    Supplementary Data Set 4

    Bromodomain off target profiling

  5. 5.

    Supplementary Data Set 5

    Histone deacetylase and sirtuin off target profiling

  6. 6.

    Supplementary Data Set 6

    UBE2M TMT interaction proteomics

  7. 7.

    Supplementary Data Set 7

    TMT total proteome data. 8-plex TMT-LC/LC-MS/MS analysis of N-terminal acetylated peptides

  8. 8.

    Supplementary Data Set 8

    Raw data for TMT total proteomics in the absence or presence of NAcM inhibitors

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DOI

https://doi.org/10.1038/nchembio.2386

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