RBC8 is a specific GTPases RalA/RalB inhibitor by inhibiting the binding of Ral to its effector RALBP1, no inhibition on the GTPases RhoA and Ras.

GTPase (H2122 cells):3.5 μM, Ral GTPase (H358 cells):3.4 μM

In mice bearing H358 xenografts, RBC8 administered intraperitoneally (i.p.) at a dosage of 50 mg/kg specifically inhibits RalA and RalB, thereby suppressing tumor growth.

Within living cells, RBC8k can induce a chemical shift change in RalB-GDP, thereby diminishing the activation of RalA. In Ral-dependent H2122 (IC50: 3.5 μM) and H358 (IC50: 3.4 μM) models, RBC8 leads to the inhibition of anchorage-independent growth.

Growth inhibition of human lung cancer cells by the compounds is measured under anchorage-independent conditions in soft agar. Cells are seeded into 6-well plates (coated with a base layer made of 2.0 ml of 1% low-melting-point agarose) at 15,000 cells per well in 3.0 mL of 0.4% low-melting-point agarose containing various concentration of drug. Two to four weeks (depending on the cell line) after incubation, the cells are stained with 1.0 mg ml?1 nitroblue tetrazolium, and colonies are counted under a microscope. The IC50 values are defined as the concentration of drug that resulted in a 50% reduction in colony number compared with the DMSO-treated control. (Only for Reference)