Journal of Biological Chemistry
Volume 287, Issue 16, 13 April 2012, Pages 13228-13238
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Molecular Bases of Disease
Characterization of EHop-016, Novel Small Molecule Inhibitor of Rac GTPase*

https://doi.org/10.1074/jbc.M111.334524Get rights and content
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The Rho GTPase Rac regulates actin cytoskeleton reorganization to form cell surface extensions (lamellipodia) required for cell migration/invasion during cancer metastasis. Rac hyperactivation and overexpression are associated with aggressive cancers; thus, interference of the interaction of Rac with its direct upstream activators, guanine nucleotide exchange factors (GEFs), is a viable strategy for inhibiting Rac activity. We synthesized EHop-016, a novel inhibitor of Rac activity, based on the structure of the established Rac/Rac GEF inhibitor NSC23766. Herein, we demonstrate that EHop-016 inhibits Rac activity in the MDA-MB-435 metastatic cancer cells that overexpress Rac and exhibits high endogenous Rac activity. The IC50 of 1.1 μm for Rac inhibition by EHop-016 is ∼100-fold lower than for NSC23766. EHop-016 is specific for Rac1 and Rac3 at concentrations of ≤5 μm. At higher concentrations, EHop-016 inhibits the close homolog Cdc42. In MDA-MB-435 cells that demonstrate high active levels of the Rac GEF Vav2, EHop-016 inhibits the association of Vav2 with a nucleotide-free Rac1(G15A), which has a high affinity for activated GEFs. EHop-016 also inhibits the Rac activity of MDA-MB-231 metastatic breast cancer cells and reduces Rac-directed lamellipodia formation in both cell lines. EHop-016 decreases Rac downstream effects of PAK1 (p21-activated kinase 1) activity and directed migration of metastatic cancer cells. Moreover, at effective concentrations (<5 μm), EHop-016 does not affect the viability of transformed mammary epithelial cells (MCF-10A) and reduces viability of MDA-MB-435 cells by only 20%. Therefore, EHop-016 holds promise as a targeted therapeutic agent for the treatment of metastatic cancers with high Rac activity.

Actin
Cdc42
Enzyme Inhibitors
GTPase
Guanine Nucleotide Exchange Factor (GEF)
Rac
Rho
Rho GTPases
Rac Inhibitor, Ehop-016, Rac GEFs

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*

This work was supported, in whole or in part, by National Institutes of Health, NCRR, Grants G12RR035051 (to the University of Puerto Rico Medical Sciences Campus), 5U54CA096297 (to the University of Puerto Rico Medical Sciences Campus), and G12RR03035 (to Universidad Central del Caribe). This work was also supported by Department of Defense/United States Army Breast Cancer Research Program Grants W81XWH-07-1-0330 and 3SC3GM084824-02S1 (to S. D.). A provisional patent application, number 61/536,069, “Novel Small-Molecule Inhibitors of Rac 1 in Metastatic Breast Cancer,” is pending.

This article contains supplemental Figs. S1–S3.

1

Both authors contributed equally to this work.