Inspired by the usefulness of small molecules to study membrane traffic, we used high-throughput synthesis and phenotypic screening to discover secramine, a molecule that inhibits membrane traffic out of the Golgi apparatus by an unknown mechanism. We report here that secramine inhibits activation of the Rho GTPase Cdc42, a protein involved in membrane traffic, by a mechanism dependent upon the guanine dissociation inhibitor RhoGDI. RhoGDI binds Cdc42 and antagonizes its membrane association, nucleotide exchange and effector binding. In vitro, secramine inhibits Cdc42 binding to membranes, GTP and effectors in a RhoGDI-dependent manner. In cells, secramine mimics the effects of dominant-negative Cdc42 expression on protein export from the Golgi and on Golgi polarization in migrating cells. RhoGDI-dependent Cdc42 inhibition by secramine illustrates a new way to inhibit Rho GTPases with small molecules and provides a new means to study Cdc42, RhoGDI and the cellular processes they mediate.
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note made in HTML and Fig 3a; PDF appended; corrected online date added to PDF
*Note: In the version of this article initially published online, two labels in Figure 3a are incorrect. The label in the square below the arrow for step 2 should read 'Cdc42 GTP' instead of 'Cdc42 GDP', and ‘PI’ in step 4 should be ‘Pi’. These errors have been corrected in the PDF version of the article.
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We thank S.C. Harrison for comments on the manuscript. We also thank D.A. Annis, N. Nazef, G. Hoffman, R. Massol, A. Mata-Fink, and A. Griffin for technical assistance and discussions. This work was supported by US National Institutes of Health (NIH) grant GM62566-05 to T.K. and M.D.S. and NIH New England Regional Center of Excellence (NERCE) grant 5 U54 AI057159-03 to T.K. M.S. and J.C. were supported by NIH grant GM068674.
The authors declare no competing financial interests.
Screening results for galanthamine-like library. (PDF 1035 kb)
Effects of secramine on the secretory pathway. (PDF 3147 kb)
Inhibition of Rac1 activation by secramine. (PDF 153 kb)
BS-C-1 cells expressing VSVGts-EGFP at 40°C were treated with cycloheximide and 1% DMSO (vehicle) for 10 min followed by transfer to 32°C and image acquisition. (MOV 3067 kb)
BS-C-1 cells expressing VSVGts-EGFP at 40°C were treated with cycloheximide and 10 μM secramine A for 10 min followed by transfer to 32°C and image acquisition. (MOV 3071 kb)
This movie is a continuation of Supplementary Video 2; it was recorded at 32°C and at the conclusion of movie 2. (MOV 3074 kb)
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Pelish, H., Peterson, J., Salvarezza, S. et al. Secramine inhibits Cdc42-dependent functions in cells and Cdc42 activation in vitro. Nat Chem Biol 2, 39–46 (2006). https://doi.org/10.1038/nchembio751
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