Article

Inhibition of Delta-induced Notch signaling using fucose analogs

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Published online:

Abstract

Notch is a cell-surface receptor that controls cell-fate decisions and is regulated by O-glycans attached to epidermal growth factor-like (EGF) repeats in its extracellular domain. Protein O-fucosyltransferase 1 (Pofut1) modifies EGF repeats with O-fucose and is essential for Notch signaling. Constitutive activation of Notch signaling has been associated with a variety of human malignancies. Therefore, tools that inhibit Notch activity are being developed as cancer therapeutics. To this end, we screened L-fucose analogs for their effects on Notch signaling. Two analogs, 6-alkynyl and 6-alkenyl fucose, were substrates of Pofut1 and were incorporated directly into Notch EGF repeats in cells. Both analogs were potent inhibitors of binding to and activation of Notch1 by Notch ligands Dll1 and Dll4, but not by Jag1. Mutagenesis and modeling studies suggest that incorporation of the analogs into EGF8 of Notch1 markedly reduces the ability of Delta ligands to bind and activate Notch1.

  • Compound

    Guanosine 5'-(diphosphate) P'-(β-L-fucosyl) ester

  • Compound

    Guanosine 5'-(diphosphate) P'-(α-D-arabinosyl) ester

  • Compound

    Guanosine 5'-(diphosphate) P'-(6,7-dideoxy-β-L-galacto-hept-6-ynopyranosyl) ester

  • Compound

    Guanosine 5'-(diphosphate) P'-(6,7-dideoxy-β-L-galacto-hept-6-enopyranosyl) ester

  • Compound

    Guanosine 5'-(diphosphate) P'-(6-deoxy-6-fluoro-β-L-galactosyl) ester

  • Compound

    Guanosine 5'-(diphosphate) P'-(β-L-galactosyl) ester

  • Compound

    Guanosine 5'-(diphosphate) P'-(6-O-methyl-β-L-galactosyl) ester

  • Compound

    Guanosine 5'-(diphosphate) P'-(6-azido-6-deoxy-β-L-galactosyl) ester

  • Compound

    1,2,3,4-Tetraacetate-L-fucopyranose

  • Compound

    6,7-Dideoxy-1,2,3,4-tetraacetate-L-galacto-hept-6-ynopyranose

  • Compound

    6,7-Dideoxy-1,2,3,4-tetraacetate-L-galacto-hept-6-enopyranose

  • Compound

    1,2,3,4-Tetraacetate-D-arabinopyranose

  • Compound

    6-Deoxy-6-fluoro-1,2,3,4-tetraacetate-L-galactosepyranose

  • Compound

    1,2,3,4,6-pentaacetate-L-galactosepyranose

  • Compound

    6-O-Methyl-1,2,3,4-tetraacetate-L-galactosepyranose

  • Compound

    6-Azido-6-deoxy-1,2,3,4-tetraacetate-L-galactosepyranose

  • Compound

    2,6-Dideoxy-2-fluoro-1,3,4-triacetate-L-galactosepyranose

  • Compound

    1,2:3,4-Di-O-isopropylidene-α-L-galactopyranose

  • Compound

    1,2:3,4-Di-O-isopropylidene-α-L-galactohexodialdo-1,5-pyranose

  • Compound

    6,7-Dideoxy-1,2:3,4-di-O-isopropylidene-α-L-galacto-hept-6-ynopyranose

  • Compound

    6,7-Dideoxy-1,2:3,4-di-O-isopropylidene-α-L-galacto-hept-6-enopyranose

  • Compound

    1,2;3,4-Di-O-isopropylidene-6-O-tosyl-α-L-galactopyranose

  • Compound

    6-Azido-6-deoxy-1,2:3,4-di-O-isopropylidene-α-L-galactopyranose

  • Compound

    1,2:3,4-Di-O-isopropylidene-6-O-methyl-α-L-galactopyranose

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Acknowledgements

This work was supported by NIH grants R01GM061126 (R.S.H.), R01GM093282 (P.W.), R01GM106417 (P.S.) and K99CA204738 (V.C.L.). M.S. was partially supported by T32GM00844. We thank the following for providing materials: J. Nye (Northwestern University Medical School), S. Chiba (University of Tokyo), G. Bornkamm (Helmholtz Zentrum Munchen), G. Weinmater (UCLA), S. Blacklow (Harvard), S. Kakuda (Stony Brook University), and C. Guidos (University of Toronto).

Author information

Author notes

    • Vincent C Luca

    Present address: Department of Drug Discovery, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, USA.

    • Vivek Kumar
    •  & Lars Ulrik Nordstrøm

    These authors contributed equally to this work.

Affiliations

  1. Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York, USA.

    • Michael Schneider
    • , Hideyuki Takeuchi
    •  & Robert S Haltiwanger
  2. Department of Cell Biology, Albert Einstein College of Medicine, New York, USA.

    • Vivek Kumar
    •  & Pamela Stanley
  3. Department of Biochemistry, Albert Einstein College of Medicine, New York, USA.

    • Lars Ulrik Nordstrøm
    • , Lei Feng
    •  & Peng Wu
  4. Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia, USA.

    • Hideyuki Takeuchi
    • , Huilin Hao
    •  & Robert S Haltiwanger
  5. Departments of Molecular and Cellular Physiology and Structural Biology, Stanford University School of Medicine, Stanford, California, USA.

    • Vincent C Luca
    •  & K Christopher Garcia
  6. Howard Hughes Medical Institute, Stanford, California, USA.

    • Vincent C Luca
    •  & K Christopher Garcia
  7. Department of Molecular Medicine, The Scripps Research Institute, La Jolla, California, USA.

    • Peng Wu

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Contributions

M.S., P.W. and R.S.H. conceived experiments, analyzed and interpreted data and wrote the paper. L.U.N. synthesized fucose analogs. M.S., V.K., L.F., H.T. and H.H. designed and performed experiments. V.C.L. and K.C.G. constructed structural models. P.S. and P.W. helped design experiments, interpret data and revise the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Peng Wu or Robert S Haltiwanger.

Supplementary information

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    Supplementary Results, Supplementary Figures 1–9

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  3. 3.

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    Supplementary Note 1

  4. 4.

    Supplementary Note 2

    Synthetic protocols