Targeting SH3 Domains to Study Protein-Protein Interactions in Cell Signalling Pathways Implicated in Human Cancers

Src homology 3 (SH3) domains play a critical role in mediating protein-protein interactions (PPIs) involved in cell proliferation, migration, and the cytoskeleton. Despite their abundance in the human proteome, the functions and molecular interactions of many SH3 domains remain unknown, and this is in part due to the lack of SH3 domain-specific reagents available for their study. Affimer proteins have been developed as affinity reagents targeting a diverse range of targets, including those involved in PPIs. The work presented in this thesis investigated the potential of using Affimer proteins to specifically bind SH3 domains to inhibit their interactions and functions. The Grb2 SH3 domains were chosen as proof-of-concept targets due to their role in linking activated RTKs to Ras-MAPK signalling via recruitment of SOS. Thus, the ability to impede Grb2 binding of SOS to restrict downstream signalling events was used to evaluate Affimer efficacy in inhibiting Grb2 SH3 interactions.

In this project, Affimer proteins were isolated against the SH3 domains of Grb2 and PLCG1 by phage display. New methods for screening against individual SH3 domains tagged with either biotin acceptor peptide or glutathione S-transferase were developed in this project. The Affimer proteins were capable of binding target within full-length, endogenous protein and demonstrated binding specificity when tested in ELISAs against other SH3 domains. Furthermore, Affimer proteins targeting the N-terminal SH3 domain (SH3N) of Grb2 demonstrated inhibition of the interaction between Grb2 and a SOS-derived peptide. Affimer expression and function was also tested in mammalian cells. Affimer C-C12, a binder of the C-terminal SH3 domain (SH3C) of Grb2, demonstrated inhibition of Ras-MAPK and PI3K/Akt signalling by reducing levels of phosphorylated ERK and phosphorylated Akt in HEK-293 and U-2 OS cells activated with epidermal growth factor. Overall, the work presented in this thesis has provided evidence that Affimer reagents can be used to study the function of SH3 domain interactions in cells.