Figure 3

R2 bound to the FAK-N-terminal domain and disrupted interaction of FAK and p53 proteins. A. Upper panel. R2 compound docked into the FAK-NT protein. Lower panel: Zoomed image. The Blue color shows area of interaction. In the R2 compound, the blue color shows nitrogen and the red-oxygen and grey color shows carbon. The amino-acids of FAK-NT involved in interaction with R2 are shown in blue color. Hydrogen bonds are marked by yellow dashed color are between R2 compound and FAK amino-acids, Asp154 and Arg252. B. The R2 compound directly bound FAK-N-terminal domain by Octet Binding assay. Binding is observed with R2 and FAK-NT, but not with the negative control buffer. C. Immunoprecipitation showed that R2 disrupted binding of FAK and p53 proteins. The immunoprecipitatioon of p53 was performed after treatment of HCT116 cells with R2 at 100 μM for 24 h. Then Western blotting was performed with FAK antibody to detect complex of p53 with FAK. The binding was present in untreated cells, but not in R2-treated cells. Plus (+) marked immunoprecipitation; and minus (−) marked no immunoprecipitation. D. Pull-down assay demonstrated that R2 disrupted FAK and p53 complex. Left panel: Recombinant proteins andbaculoviral FAK (marked by arrows). Right panel: Pull-down assay with recombinant GST-p53 and FAK protein demonstrated binding of FAK and p53 proteins. The R2 compound disrupted the binding of FAK and p53 proteins. Upper panel: Western blotting with FAK antibody. Lower panel: Western blotting with GST antibody. E. R2 disrupted the binding of FAK and p53 proteins in a dose-dependent manner, while a negative control compound (A18), which was not targeting FAK-p53 interaction did not. The pull-down assay was performed as in Figure 3D with 1 and 10 μM of R2 and with 10 μM of A18 (negative control).