In vitro activity: In living cells, RBC8 also reduces the activation of RalA by inducing chemical shift changes in RalB–GDP. In Ral-dependent lines H2122 and H358, RBC8 causes anchorage-independent growth inhibition with IC50 of 3.5 μM and 3.4 μM, respectively

Kinase Assay:His-RalA (100 ng) was incubated with gamma-labeled 32P-GTP (8 nM assay concentration) and either DMSO or individual compounds (50 μM assay concentration) dissolved in DMSO in the presence of EDTA (20 mM) for 15 min at 30°C. The reaction was stopped by dilution into excess MgCl2, and the incorporation of radiolabeled nucleotide was measured by filter binding. 32P-GTP (alpha-labeled) was converted to 32P-GDP by nucleotide diphosphokinase, and used for the binding assay with GDP.

Cell Assay: 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.