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


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.

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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.

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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).


  1. 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)

    CAS  Article  Google Scholar 

  2. 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)

    ADS  CAS  Article  Google Scholar 

  3. 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)

    CAS  Article  Google Scholar 

  4. 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)

    CAS  Article  Google Scholar 

  5. 5

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

    ADS  CAS  Article  Google Scholar 

  6. 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)

    ADS  CAS  Article  Google Scholar 

  7. 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)

    CAS  Article  Google Scholar 

  8. 8

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

    CAS  Article  Google Scholar 

  9. 9

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

    CAS  Article  Google Scholar 

  10. 10

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

    CAS  Article  Google Scholar 

  11. 11

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

    ADS  CAS  Article  Google Scholar 

  12. 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)

    CAS  Article  Google Scholar 

  13. 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)

    CAS  Article  Google Scholar 

  14. 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)

    CAS  Article  Google Scholar 

  15. 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)

    ADS  CAS  Article  Google Scholar 

  16. 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)

    CAS  Article  Google Scholar 

  17. 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)

    ADS  CAS  Article  Google Scholar 

  18. 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)

    ADS  CAS  Article  Google Scholar 

  19. 19

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

    ADS  CAS  Article  Google Scholar 

  20. 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)

    CAS  Article  Google Scholar 

  21. 21

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

    CAS  Article  Google Scholar 

  22. 22

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

    CAS  Article  Google Scholar 

  23. 23

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

    CAS  Article  Google Scholar 

  24. 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 

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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.

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Correspondence to T. D. Pollard.

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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).

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