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Small-molecule inhibitors of the AAA+ ATPase motor cytoplasmic dynein

Abstract

The conversion of chemical energy into mechanical force by AAA+ (ATPases associated with diverse cellular activities) ATPases is integral to cellular processes, including DNA replication, protein unfolding, cargo transport and membrane fusion1. The AAA+ ATPase motor cytoplasmic dynein regulates ciliary trafficking2, mitotic spindle formation3 and organelle transport4, and dissecting its precise functions has been challenging because of its rapid timescale of action and the lack of cell-permeable, chemical modulators. Here we describe the discovery of ciliobrevins, the first specific small-molecule antagonists of cytoplasmic dynein. Ciliobrevins perturb protein trafficking within the primary cilium, leading to their malformation and Hedgehog signalling blockade. Ciliobrevins also prevent spindle pole focusing, kinetochore–microtubule attachment, melanosome aggregation and peroxisome motility in cultured cells. We further demonstrate the ability of ciliobrevins to block dynein-dependent microtubule gliding and ATPase activity in vitro. Ciliobrevins therefore will be useful reagents for studying cellular processes that require this microtubule motor and may guide the development of additional AAA+ ATPase superfamily inhibitors.

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Figure 1: Ciliobrevins disrupt primary cilium-dependent Gli regulation.
Figure 2: Ciliobrevins disrupt spindle pole assembly and kinetochore–microtubule attachment.
Figure 3: Ciliobrevins inhibit melanosome aggregation and peroxisome motility.
Figure 4: Ciliobrevins inhibit cytoplasmic dynein-dependent microtubule gliding and ATPase activity.

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Acknowledgements

We thank T. Caspary for anti-Arl13b antibodies, W. Brinkley for human CREST anti-serum, T. Yen for anti-CENP-E antibodies, U. Peters for purified bovine dynein, S. Wacker for human kinesin-5 motor domain, R. Vallee for a pVL1393 baculovirus expression vector containing the rat dynein motor domain, and K. Bersuker and R. Kopito for TCR-α–GFP-expressing cells. This work was supported by funding from the National Institutes of Health (R01 CA136574 to J.K.C.; R01 GM65933 to T.M.K.; R01 GM71772 to T.M.K. and V.I.G.; R01 GM52111 to V.I.G.)

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Contributions

J.K.C. and T.M.K. conceived and directed the study. A.J.F. performed chemical syntheses and assays of Hedgehog signaling, primary cilia formation and function, ATPase activity, vanadate-dependent dynein photocleavage and p97-dependent protein degradation. A.J.F. and M.M. performed mitotic spindle analyses. K.B. and V.I.G. designed and interpreted the melanophore and peroxisome trafficking assays. J.S.W. performed microtubule gliding and dynein/microtubule binding assays. L.D.L. and M.O. designed and interpreted the Mcm2–7 helicase assays. A.J.F. and J.K.C. wrote the manuscript with contributions from all other authors.

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Correspondence to Tarun M. Kapoor or James K. Chen.

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Firestone, A., Weinger, J., Maldonado, M. et al. Small-molecule inhibitors of the AAA+ ATPase motor cytoplasmic dynein. Nature 484, 125–129 (2012). https://doi.org/10.1038/nature10936

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