Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Disruption of endocytosis through chemical inhibition of clathrin heavy chain function

Nature Chemical Biology volume 15, pages 641–649 (2019)Cite this article

Subjects

Abstract

Clathrin-mediated endocytosis (CME) is a highly conserved and essential cellular process in eukaryotic cells, but its dynamic and vital nature makes it challenging to study using classical genetics tools. In contrast, although small molecules can acutely and reversibly perturb CME, the few chemical CME inhibitors that have been applied to plants are either ineffective or show undesirable side effects. Here, we identify the previously described endosidin9 (ES9) as an inhibitor of clathrin heavy chain (CHC) function in both Arabidopsis and human cells through affinity-based target isolation, in vitro binding studies and X-ray crystallography. Moreover, we present a chemically improved ES9 analog, ES9-17, which lacks the undesirable side effects of ES9 while retaining the ability to target CHC. ES9 and ES9-17 have expanded the chemical toolbox used to probe CHC function, and present chemical scaffolds for further design of more specific and potent CHC inhibitors across different systems.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: ES9 binds CHC.
Fig. 2: ES9 binds the terminal domain of CHC.
Fig. 3: ES9-17 is not a protonophore.
Fig. 4: ES9-17 is a CME inhibitor.
Fig. 5: ES9-17 targets CHC.

Similar content being viewed by others

Data availability

All accession codes supporting the findings of this study are available within the paper and its Supplementary Information. Structures are accessible under the PDB code 6E4L. There is no restriction on data availability.

References

  1. Reynolds, G. D., Wang, C., Pan, J. & Bednarek, S. Y. Inroads into internalization: five years of endocytic exploration. Plant Physiol. 176, 208–218 (2018).

    Article  CAS  Google Scholar 

  2. Kaksonen, M. & Roux, A. Mechanisms of clathrin-mediated endocytosis. Nat. Rev. Mol. Cell Biol. 19, 313–326 (2018).

    Article  CAS  Google Scholar 

  3. Mettlen, M., Chen, P.-H., Srinivasan, S., Danuser, G. & Schmid, S. L. Regulation of clathrin-mediated endocytosis. Annu. Rev. Biochem. 87, 871–896 (2018).

    Article  CAS  Google Scholar 

  4. Mishev, K., Dejonghe, W. & Russinova, E. Small molecules for dissecting endomembrane trafficking: a cross-systems view. Chem. Biol. 20, 475–486 (2013).

    Article  CAS  Google Scholar 

  5. von Kleist, L. et al. Role of the clathrin terminal domain in regulating coated pit dynamics revealed by small molecule inhibition. Cell 146, 471–484 (2011).

    Article  Google Scholar 

  6. Macia, E. et al. Dynasore, a cell-permeable inhibitor of dynamin. Dev. Cell 10, 839–850 (2006).

    Article  CAS  Google Scholar 

  7. McCluskey, A. et al. Building a better dynasore: the Dyngo compounds potently inhibit dynamin and endocytosis. Traffic 14, 1272–1289 (2013).

    Article  CAS  Google Scholar 

  8. Elkin, S. R. et al. Ikarugamycin: a natural product inhibitor of clathrin-mediated endocytosis. Traffic 17, 1139–1149 (2016).

    Article  CAS  Google Scholar 

  9. Dejonghe, W. et al. Mitochondrial uncouplers inhibit clathrin-mediated endocytosis largely through cytoplasmic acidification. Nat. Commun. 7, 11710 (2016).

    Article  CAS  Google Scholar 

  10. Jafari, R. et al. The cellular thermal shift assay for evaluating drug target interactions in cells. Nat. Protoc. 9, 2100 (2014).

    Article  CAS  Google Scholar 

  11. Lomenick, B. et al. Target identification using drug affinity responsive target stability (DARTS). Proc. Natl Acad. Sci. USA 106, 21984–21989 (2009).

  12. Jelínková, A. et al. Probing plant membranes with FM dyes: tracking, dragging or blocking? Plant J. 61, 883–892 (2010).

    Article  Google Scholar 

  13. Ludwig, A., Stolz, J. & Sauer, N. Plant sucrose‐H+ symporters mediate the transport of vitamin H. Plant J. 24, 503–509 (2000).

  14. Van Leene, J. et al. An improved toolbox to unravel the plant cellular machinery by tandem affinity purification of Arabidopsis protein complexes. Nat. Protoc. 10, 169–187 (2015).

    Article  Google Scholar 

  15. Mishev, K. et al. Nonselective chemical inhibition of Sec7 domain-containing ARF GTPase exchange factors. Plant Cell 30, 2573–2593 (2018).

    Article  Google Scholar 

  16. Kitakura, S. et al. Clathrin mediates endocytosis and polar distribution of PIN auxin transporters in Arabidopsis. Plant Cell 23, 1920–1931 (2011).

    Article  CAS  Google Scholar 

  17. Popova, N. V., Deyev, I. E. & Petrenko, A. G. Clathrin-mediated endocytosis and adaptor proteins. Acta Naturae 5, 62–73 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Onizuka, S., Ikewaki, N. & Shiraishi, S. A mechanism by which propofol induces cytotoxicity. J. Drug Metab. Toxicol. 8, 230 (2017).

    Article  Google Scholar 

  19. Poot, M. et al. Analysis of mitochondrial morphology and function with novel fixable fluorescent stains. J. Histochem. Cytochem. 44, 1363–1372 (1996).

    Article  CAS  Google Scholar 

  20. Dolman, N. J., Kilgore, J. A. & Davidson, M. W. A review of reagents for fluorescence microscopy of cellular compartments and structures, part I: BacMam labeling and reagents for vesicular structures. Curr. Protoc. Cytom. 65, 12.30.1–12.30.27 (2013).

  21. Wang, Y.-S., Yoo, C.-M. & Blancaflor, E. B. Improved imaging of actin filaments in transgenic Arabidopsis plants expressing a green fluorescent protein fusion to the C‐ and N‐termini of the fimbrin actin‐binding domain 2. New Phytol. 177, 525–536 (2008).

    CAS  PubMed  Google Scholar 

  22. Marc, J. et al. GFP-MAP4 reporter gene for visualizing cortical microtubule rearrangements in living epidermal cells. Plant Cell 10, 1927–1940 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Teh, O.-k & Moore, I. An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells. Nature 448, 493–496 (2007).

    Article  CAS  Google Scholar 

  24. Dettmer, J., Hong-Hermesdorf, A., Stierhof, Y.-D. & Schumacher, K. Vacuolar H+-ATPase activity is required for endocytic and secretory trafficking in Arabidopsis. Plant Cell 18, 715–730 (2006).

    Article  CAS  Google Scholar 

  25. Irani, N. G. et al. Fluorescent castasterone reveals BRI1 signaling from the plasma membrane. Nat. Chem. Biol. 8, 583–589 (2012).

    Article  CAS  Google Scholar 

  26. Friedrichsen, D. M., Joazeiro, C. A. P., Li, J., Hunter, T. & Chory, J. Brassinosteroid-insensitive-1 is a ubiquitously expressed leucine-rich repeat receptor serine/threonine kinase. Plant Physiol. 123, 1247–1255 (2000).

    Article  CAS  Google Scholar 

  27. Ortiz-Morea, F. A. et al. Danger-associated peptide signaling in Arabidopsis requires clathrin. Proc. Natl Acad. Sci. USA 113, 11028–11033 (2016).

    Article  CAS  Google Scholar 

  28. Gadeyne, A. et al. The TPLATE adaptor complex drives clathrin-mediated endocytosis in plants. Cell 156, 691–704 (2014).

    Article  CAS  Google Scholar 

  29. Di Rubbo, S. et al. The clathrin adaptor complex AP-2 mediates endocytosis of brassinosteroid insensitive1 in Arabidopsis. Plant Cell 25, 2986–2997 (2013).

    Article  Google Scholar 

  30. Dhonukshe, P. et al. Clathrin-mediated constitutive endocytosis of PIN auxin efflux carriers in Arabidopsis. Curr. Biol. 17, 520–527 (2007).

    Article  CAS  Google Scholar 

  31. Adamowski, M. et al. A functional study of AUXILIN-LIKE1 and 2, two putative clathrin uncoating factors in Arabidopsis. Plant Cell 30, 700–716 (2018).

    Article  CAS  Google Scholar 

  32. Robinson, D. G. & Pimpl, P. Clathrin and post-Golgi trafficking: a very complicated issue. Trends Plant Sci. 19, 134–139 (2014).

    Article  CAS  Google Scholar 

  33. Hicks, G. R. & Raikhel, N. V. Small molecules present large opportunities in plant biology. Annu. Rev. Plant Biol. 63, 261–282 (2012).

    Article  CAS  Google Scholar 

  34. Drakakaki, G. et al. Clusters of bioactive compounds target dynamic endomembrane networks in vivo. Proc. Natl Acad. Sci. USA 108, 17850–17855 (2011).

    Article  CAS  Google Scholar 

  35. Zhang, C. et al. Endosidin2 targets conserved exocyst complex subunit EXO70 to inhibit exocytosis. Proc. Natl Acad. Sci. USA 113, E41–E50 (2016).

    Article  CAS  Google Scholar 

  36. Kania, U. et al. The inhibitor Endosidin 4 targets SEC7 domain-type ARF GTPase exchange factors and interferes with subcellular trafficking in eukaryotes. Plant Cell 30, 2553–2572 (2018).

    Article  CAS  Google Scholar 

  37. Li, R. et al. Different endomembrane trafficking pathways establish apical and basal polarities. Plant Cell 29, 90–108 (2017).

    Article  CAS  Google Scholar 

  38. Sharfman, M. et al. Endosomal signaling of the tomato leucine-rich repeat receptor-like protein LeEix2. Plant J. 68, 413–423 (2011).

    Article  CAS  Google Scholar 

  39. Helsens, K. et al. ms_lims, a simple yet powerful open source laboratory information management system for MS-driven proteomics. Proteomics 10, 1261–1264 (2010).

    Article  CAS  Google Scholar 

  40. Scott, W. R. P. et al. The GROMOS biomolecular simulation program package. J. Phys. Chem. 103, 3596–3607 (1999).

    Article  CAS  Google Scholar 

  41. Guex, N. & Peitsch, M. C. SWISS-MODEL and the Swiss-Pdb Viewer: an environment for comparative protein modeling. Electrophoresis 18, 2714–2723 (1997).

    Article  CAS  Google Scholar 

  42. Hanwell, M. D. et al. Avogadro: an advanced semantic chemical editor, visualization, and analysis platform. J. Cheminform. 4, 17 (2012).

    Article  CAS  Google Scholar 

  43. Halgren, T. A. & MMFF, V. I. MMFF94s option for energy minimization studies. J. Comput. Chem. 20, 720–729 (1999).

    Article  CAS  Google Scholar 

  44. Sanner, M. F. Python: a programming language for software integration and development. J. Mol. Graph. Model. 17, 57–61 (1999).

    CAS  PubMed  Google Scholar 

  45. Trott, O. & Olson, A. J. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading. J. Comput. Chem. 31, 455–461 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Kabsch, W. XDS. Acta Crystallogr. D 66, 125–132 (2010).

    Article  CAS  Google Scholar 

  47. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  CAS  Google Scholar 

  48. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010).

  49. Schüttelkopf, A. W. & van Aalten, D. M. PRODRG: a tool for high-throughput crystallography of protein-ligand complexes. Acta Crystallogr. D 60, 1355–1363 (2004).

    Article  Google Scholar 

  50. Huynh, K. & Partch, C. L. Analysis of protein stability and ligand interactions by thermal shift assay. Curr. Protoc. Protein Sci. 79, 28.9.1-28.9.14 (2015).

    Google Scholar 

Download references

Acknowledgements

We thank S. Vanneste for fruitful discussions, R. Kumar for providing the pDONR211-AP2S plasmid, D. Martinez Molina for help with the CETSA protocol and M. De Cock for help in preparing the manuscript. This work was supported by the Research Foundation-Flanders (project No. G022516N to E.R., project No. G009415N to D.V.D and project No. G0E5718N to E.R. and J.F.); the European Research Council (ERC Co T-Rex, grant No. 682436 to D.V.D); the Deutsche Forschungsgemeinschaft (No. TRR186/A08 to V.H.); the Agency for Innovation by Science and Technology for postdoctoral (K.M.) and predoctoral (W.D. and S.D.M) fellowships; the China Science Council for a predoctoral fellowship (Q.L.); the joint research projects (Nos. VS.025.13N and VS.095.16N) within the framework of cooperation between the Research Foundation-Flanders and the Bulgarian Academy of Sciences (K.M.); and the Belgian Science Policy Office for a postdoctoral fellowship to non-EU researchers (I.S.).

Author information

Author notes

  1. Wim Dejonghe

    Present address: Department of Botany and Plant Sciences, Institute of Integrative Genome Biology, University of California, Riverside, CA, USA

  2. These authors contributed equally: Wim Dejonghe, Isha Sharma.

Authors and Affiliations

  1. Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium

    Wim Dejonghe, Isha Sharma, Qing Lu, Kiril Mishev, Evelien Mylle, Riet De Rycke, Daniel V. Savatin, Klaas Yperman, Daniël Van Damme & Eugenia Russinova

  2. Center for Plant Systems Biology, VIB, Ghent, Belgium

    Wim Dejonghe, Isha Sharma, Qing Lu, Kiril Mishev, Evelien Mylle, Riet De Rycke, Daniel V. Savatin, Klaas Yperman, Daniël Van Damme & Eugenia Russinova

  3. Department of Organic and Macromolecular Chemistry, Ghent University, Ghent, Belgium

    Bram Denoo, Annemieke Madder & Johan Winne

  4. Unit for Structural Biology, Department of Biochemistry and Microbiology, Ghent University, Ghent, Belgium

    Steven De Munck & Savvas N. Savvides

  5. Unit for Structural Biology, Center for Inflammation Research, VIB, Ghent, Belgium

    Steven De Munck & Savvas N. Savvides

  6. Institute of Plant Physiology and Genetics, Bulgarian Academy of Sciences, Sofia, Bulgaria

    Kiril Mishev

  7. Department of Biology, Chemistry and Pharmacy, Freie Universität Berlin, Berlin, Germany

    Haydar Bulut

  8. Ghent University Expertise Centre for Transmission Electron Microscopy and VIB BioImaging Core, Ghent, Belgium

    Riet De Rycke

  9. Institute of Science and Technology, Klosterneuburg, Austria

    Mina Vasileva & Jiří Friml

  10. Center for Medical Biotechnology, VIB, Ghent, Belgium

    Wim Nerinckx, An Staes & Kris Gevaert

  11. Department of Biochemistry and Microbiology, Ghent University, Ghent, Belgium

    Wim Nerinckx

  12. Department of Biomolecular Medicine, Ghent University, Ghent, Belgium

    An Staes & Kris Gevaert

  13. VIB Screening Core, Ghent, Belgium

    Andrzej Drozdzecki & Dominique Audenaert

  14. Expertise Centre for Bioassay Development and Screening, Ghent University, Ghent, Belgium

    Andrzej Drozdzecki & Dominique Audenaert

  15. Department of Molecular Pharmacology and Cell Biology, Leibniz Research Institute for Molecular Pharmacology, Berlin, Germany

    Volker Haucke

Authors
  1. Wim Dejonghe
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Isha Sharma
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Bram Denoo
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Steven De Munck
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Qing Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Kiril Mishev
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Haydar Bulut
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Evelien Mylle
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Riet De Rycke
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Mina Vasileva
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Daniel V. Savatin
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Wim Nerinckx
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. An Staes
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Andrzej Drozdzecki
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Dominique Audenaert
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Klaas Yperman
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Annemieke Madder
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Jiří Friml
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. Daniël Van Damme
    View author publications

    You can also search for this author in PubMed Google Scholar

  20. Kris Gevaert
    View author publications

    You can also search for this author in PubMed Google Scholar

  21. Volker Haucke
    View author publications

    You can also search for this author in PubMed Google Scholar

  22. Savvas N. Savvides
    View author publications

    You can also search for this author in PubMed Google Scholar

  23. Johan Winne
    View author publications

    You can also search for this author in PubMed Google Scholar

  24. Eugenia Russinova
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

W.D. and E.R. initiated the work. W.D., I.S. and E.R. designed the experiments. W.D., I.S., B.D., A.M. and J.W. performed SAR. W.D., K.M., A.S. and K.G. performed affinity purification and MS analysis. H.B. and V.H. performed the X-ray crystallography. I.S., S.D.M. and S.N.S. performed the in vitro binding assay. W.N. did the molecular docking. W.D., I.S. and Q.L. performed CETSA. W.D., I.S. and Q.L. performed DARTS. W.D., I.S., E.M., D.V.S., and D.V.D. carried out the imaging and data analysis. I.S. performed the cloning and generated transgenic Arabidopsis cell cultures. W.D., I.S., A.D. and D.A. performed ATP measurements. M.V. and J.F. contributed to the HeLa cell assays. K.Y. generated the TPLATE antibody. Q.L. and R.D.R performed TEM. W.D., I.S. and E.R. wrote the manuscript. All authors commented on the results and the manuscript.

Corresponding author

Correspondence to Eugenia Russinova.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

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

Supplementary information

Supplementary Information

Supplementary Figures 1–12, Supplementary Tables 1–3

Reporting Summary

Supplementary Note 1

Synthetic Procedures

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Dejonghe, W., Sharma, I., Denoo, B. et al. Disruption of endocytosis through chemical inhibition of clathrin heavy chain function. Nat Chem Biol 15, 641–649 (2019). https://doi.org/10.1038/s41589-019-0262-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41589-019-0262-1

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing