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Small molecules discovered in a pathway screen target the Rho pathway in cytokinesis

Abstract

We report the discovery of small molecules that target the Rho pathway, which is a central regulator of cytokinesis—the final step in cell division. We have developed a way of targeting a small molecule screen toward a specific pathway, which should be widely applicable to the investigation of any signaling pathway. In a chemical genetic variant of a classical modifier screen, we used RNA interference (RNAi) to sensitize cells and identified small molecules that suppressed or enhanced the RNAi phenotype. We discovered promising candidate molecules, which we named Rhodblock 18, and we identified the target of Rhodblock 6 as Rho kinase. Several Rhodblocks inhibited one function of the Rho pathway in cells: the correct localization of phosphorylated myosin light chain during cytokinesis. Rhodblocks differentially perturb Rho pathway proteins in cells and can be used to dissect the mechanism of the Rho pathway during cytokinesis.

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Figure 1: Screening strategy to identify small molecules that target the Rho pathway.
Figure 2: Rhodblock 6 inhibits Rho kinase.
Figure 3: Several Rhodblocks prevent the accumulation of phospho-myosin regulatory light chain and/or Anillin at the cleavage furrow.
Figure 4: Effect of Rhodblocks 1a, 3 and 6 on cytokinesis protein localization.
Figure 5: Movie stills of GFP-MRLC S2 cells treated with 100 μM Rhodblock 1a after overnight Rho RNAi sensitization.

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Acknowledgements

We thank the staff at ICCB-Longwood, the Nikon Imaging Center at Harvard Medical School and the Broad Institute's Imaging Platform for their assistance and F. Roth for helpful discussions. We thank C. Field (Harvard Medical School), R. Saint (Australian National University) and E. Griffis (University of California, San Francisco) for reagents. Funding for M.S.V., T.R.J. and A.E.C. was from the Life Sciences Research Foundation (Novartis), L'Oreal for Women in Science, the Society for Biomolecular Screening and the US National Institutes of Health (NIH) 5 RL1 CA133834-03. A.B.C., Y.S., A.D.T. and U.S.E. were supported by NIH grant R01 GM082834, the Claudia Adams Barr Program and the Dana-Farber Cancer Institute.

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A.B.C. and U.S.E. designed the study. A.B.C., Y.S., A.D.T. and U.S.E. designed and conducted experiments and analyzed data. A.B.C., Y.S., M.S.V., T.R.J. and A.E.C. designed and performed automated image analysis. U.S.E. wrote the manuscript, with input from the other authors.

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Correspondence to Ulrike S Eggert.

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Castoreno, A., Smurnyy, Y., Torres, A. et al. Small molecules discovered in a pathway screen target the Rho pathway in cytokinesis. Nat Chem Biol 6, 457–463 (2010). https://doi.org/10.1038/nchembio.363

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