In vitro activity: Rac inhibition by EHop-016 increases the activity of the closely related Rho GTPase RhoA by a compensatory mechanism. In MDA-MB-435 cells, EHop-016 (2-5 μM) inhibits association of active Vav2 with Rac1(G15A) mutant fusion protein, and reduces Rac-regulated cell functions including lamellipodia formation and cell migration. In addition, EHop-016 inhibits cell viability of MDA-MB-435 cells with IC50 of 10 μM. EHop-016 also inhibits KITD814V-induced growth in SM and AML patients-derived cells.

Kinase Assay: Rac activity is determined from lysates of the MDA-MB-435 and MDA-MB-231 human metastatic cancer cell lines (from ATCC). Cancer cells in culture medium (DMEM, 10% FBS, pH 7.5) are treated with vehicle (0.1% DMSO) or varying concentrations of EHop-016 (0–10 μM) for 24 h. Rac1 activity is determined using the G-LISA Rac1 activation assay kit.

Cell Assay: MDA-MB-231, MDA-MB-435, or MCF-10A mammary epithelial cells (from ATCC) are incubated in vehicle (0.1% DMSO) or varying concentrations of EHop-016 (0–10 μM) for 24 h. Cell viability is measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell survival and proliferation kit according to the manufacturer's instructions.

J Biol Chem. 2012 Apr 13;287(16):13228-38; J Clin Invest. 2013 Oct;123(10):4449-63.