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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.

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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.

Accession codes

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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).

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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.

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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). https://doi.org/10.1038/nature08231

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