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.

  • Letter
  • Published:

Characterization of two classes of small molecule inhibitors of Arp2/3 complex

Abstract

Polymerization of actin filaments directed by the actin-related protein (Arp)2/3 complex supports many types of cellular movements1. However, questions remain regarding the relative contributions of Arp2/3 complex versus other mechanisms of actin filament nucleation to processes such as path finding by neuronal growth cones; this is because of the lack of simple methods to inhibit Arp2/3 complex reversibly in living cells. Here we describe two classes of small molecules that bind to different sites on the Arp2/3 complex and inhibit its ability to nucleate actin filaments. CK-0944636 binds between Arp2 and Arp3, where it appears to block movement of Arp2 and Arp3 into their active conformation. CK-0993548 inserts into the hydrophobic core of Arp3 and alters its conformation. Both classes of compounds inhibit formation of actin filament comet tails by Listeria and podosomes by monocytes. Two inhibitors with different mechanisms of action provide a powerful approach for studying the Arp2/3 complex in living cells.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Figure 1: Two classes of small molecules inhibit nucleation of actin filaments by Arp2/3 complex.
Figure 2: Inhibition of actin assembly in live cells by CK-548, CK-636 and CK-666.
Figure 3: Crystal structure of BtArp2/3 complex with bound CK-636.
Figure 4: Crystal structure of BtArp2/3 complex with bound CK-548.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Structural data have been deposited in the Protein Data Bank under accession codes 3DXK (CK-0944636) and 3DXM (CK-0993548).

References

  1. Goley, E. D. & Welch, M. D. The Arp2/3 complex: an actin nucleator comes of age. Nature Rev. Mol. Cell Biol. 7, 713–726 (2006)

    Article  CAS  Google Scholar 

  2. Welch, M. D., Rosenblatt, J., Skoble, J., Portnoy, D. A. & Mitchison, T. J. Interaction of human Arp2/3 complex and the Listeria monocytogenes ActA protein in actin filament nucleation. Science 281, 105–108 (1998)

    Article  ADS  CAS  Google Scholar 

  3. Linder, S. et al. The polarization defect of Wiskott–Aldrich syndrome macrophages is linked to dislocation of the Arp2/3 complex. J. Immunol. 165, 221–225 (2000)

    Article  CAS  Google Scholar 

  4. Gimona, M., Buccione, R., Courtneidge, S. A. & Linder, S. Assembly and biological role of podosomes and invadopodia. Curr. Opin. Cell Biol. 20, 235–241 (2008)

    Article  CAS  Google Scholar 

  5. Robinson, R. C. et al. Crystal structure of Arp2/3 complex. Science 294, 1679–1684 (2001)

    Article  ADS  CAS  Google Scholar 

  6. Nolen, B. J., Littlefield, R. S. & Pollard, T. D. Crystal structures of actin-related protein 2/3 complex with bound ATP or ADP. Proc. Natl Acad. Sci. USA 101, 15627–15632 (2004)

    Article  ADS  CAS  Google Scholar 

  7. Nolen, B. J. & Pollard, T. D. Insights into the influence of nucleotides on actin family proteins from seven structures of Arp2/3 complex. Mol. Cell 26, 449–457 (2007)

    Article  CAS  Google Scholar 

  8. Jabs, A., Weiss, M. S. & Hilgenfeld, R. Non-proline cis peptide bonds in proteins. J. Mol. Biol. 286, 291–305 (1999)

    Article  CAS  Google Scholar 

  9. Brunger, A. T. et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998)

    Article  CAS  Google Scholar 

  10. Rouiller, I. et al. The structural basis of actin filament branching by the Arp2/3 complex. J. Cell Biol. 180, 887–895 (2008)

    Article  CAS  Google Scholar 

  11. Holmes, K. C., Popp, D., Gebhard, W. & Kabsch, W. Atomic model of the actin filament. Nature 347, 44–49 (1990)

    Article  ADS  CAS  Google Scholar 

  12. Tomasevic, N. et al. Differential regulation of WASP and N-WASP by Cdc42, Rac1, Nck, and PI(4,5)P2 . Biochemistry 46, 3494–3502 (2007)

    Article  CAS  Google Scholar 

  13. Nolen, B. J. & Pollard, T. D. Structure and biochemical properties of fission yeast Arp2/3 complex lacking the Arp2 subunit. J. Biol. Chem. 283, 26490–26498 (2008)

    Article  CAS  Google Scholar 

  14. MacLean-Fletcher, S. & Pollard, T. D. Identification of a factor in conventional muscle actin preparations which inhibits actin filament self-association. Biochem. Biophys. Res. Commun. 96, 18–27 (1980)

    Article  CAS  Google Scholar 

  15. Kovar, D. R. & Pollard, T. D. Insertional assembly of actin filament barbed ends in association with formins produces piconewton forces. Proc. Natl Acad. Sci. USA 101, 14725–14730 (2004)

    Article  ADS  CAS  Google Scholar 

  16. Cooper, J. A., Walker, S. B. & Pollard, T. D. Pyrene actin: documentation of the validity of a sensitive assay for actin polymerization. J. Muscle Res. Cell Motil. 4, 253–262 (1983)

    Article  CAS  Google Scholar 

  17. Blanchoin, L. et al. Direct observation of dendritic actin filament networks nucleated by Arp2/3 complex and WASP/Scar proteins. Nature 404, 1007–1011 (2000)

    Article  ADS  CAS  Google Scholar 

  18. Dayel, M. J., Holleran, E. A. & Mullins, R. D. Arp2/3 complex requires hydrolyzable ATP for nucleation of new actin filaments. Proc. Natl Acad. Sci. USA 98, 14871–14876 (2001)

    Article  ADS  CAS  Google Scholar 

  19. Lee, J., Ishihara, A., Theriot, J. A. & Jacobson, K. Principles of locomotion for simple-shaped cells. Nature 362, 167–171 (1993)

    Article  ADS  CAS  Google Scholar 

  20. Higgs, H. N., Blanchoin, L. & Pollard, T. D. Influence of the C terminus of Wiskott-Aldrich syndrome protein (WASp) and the Arp2/3 complex on actin polymerization. Biochemistry 38, 15212–15222 (1999)

    Article  CAS  Google Scholar 

  21. Pollard, T. D. Polymerization of ADP-actin. J. Cell Biol. 99, 769–777 (1984)

    Article  CAS  Google Scholar 

  22. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

    Article  CAS  Google Scholar 

  23. Dodson, E. J., Winn, M. & Ralph, A. Collaborative Computational Project, number 4: providing programs for protein crystallography. Methods Enzymol. 277, 620–633 (1997)

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

Download references

Acknowledgements

This work was supported by Cytokinetics, Inc., NIH research grant GM-066311 (to T.D.P.), an NSF graduate research fellowship (C.D.M.) and a Ruth Kirschstein postdoctoral fellowship (GM074374-02 to B.J.N.). We thank L. Belmont, Z. Khurshid, O. Ezizika, J. Lee, S. Leuenroth, Z. Cournia and H. Chen for help with the project.

Author Contributions B.J.N., N.T., A.R., D.W.P., Z.J. and J.H. designed and carried out experiments; C.D.M. analysed data; R.S. and T.D.P. supervised research; and B.J.N., N.T., A.R., J.H. and T.D.P. wrote the paper.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to T. D. Pollard.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-7 with Legends and Supplementary Tables S1-S5. (PDF 439 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Nolen, B., Tomasevic, N., Russell, A. et al. Characterization of two classes of small molecule inhibitors of Arp2/3 complex. Nature 460, 1031–1034 (2009). https://doi.org/10.1038/nature08231

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature08231

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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