Modulation of small GTPases-mediated signaling processes by conformation-selective protein binders

Abstract

Small GTPases are important signaling node related in cytoskeleton and have critical role in oncogenesis and metastasis. However, specifically direct targeting of small GTPases have been challenging, such as KRas. Many researchers have been tried to develop an inhibitor against KRas but there are no FDA approved drug direct targeting KRas. Also, there have been developed various types of visualizing experimental tools about Rho GTPases (Rho, Rac, Cdc42) However, existing tools can visualize the artificially expressed target through new construct. Therefore, direct targeting agents are required for small GTPase research. Conformation-selective binding also has advantages at therapeutic strategies or imaging. It can reduce side effects or background signals, while all type of cells has small GTPases and basal level of their activities. In chapter 1, we developed active-KRas specific repebodies. The selected protein binder, repebody can distinguish active KRas from inactive KRas and it can inhibit the protein-protein interaction between KRas and BRaf-RBD. In chapter 2, we developed active-Cdc42 specific repebodies to discriminate Cdc42 activity and their signaling network. Conformation selective repebodies were selected through modified phage display screening, called soluble biopanning. Soluble biopanning using biotinylated target protein and streptavidin coated magnetic beads, was effective method to expose the loop conformation change of small GTPases. The selected repebodies were evaluated by immunoassay, pull-down assay, and co-immunoprecipitation. Additionally, the computational method, EpiScope, was applied to predict their epitope. The predicted epitope can explain the biochemical results. The selected conformation-selective repebodies have been evaluated through biochemical methods, but for future application, further experiments are required to discriminate their therapeutic ability in cancer cells and tumor xenografted model animals. Additionally, dye optimization should be prior to apply live cell imaging.