Inhibition of KRas using Affimers

Ras proteins are small GTP binding and hydrolysing proteins (GTPases) that regulate various signalling pathways responsible for cell proliferation, differentiation, migration and survival. Ras proteins are found to be mutated in 30% of all human cancers. Mutations in Ras or their regulators such as GTPase activating proteins (GAPs) or guanine nucleotide exchange factors (GEFs) render Ras proteins to be in persistently active GTP bound state leading to uncontrolled cell growth and proliferation. Despite three decades of extensive research, no pharmacological inhibitors of Ras have reached the market. Targeting Ras proteins directly has been challenging process because of lack of deep binding pockets for small molecules to bind to with high affinity and specificity. A novel approach of using non-antibody scaffolds for development of Ras inhibitors has shown great promise. This is being illustrated by recent increase in antibody mimetics targeting Ras such as Intrabodies, Monobodies and DARPins. In this thesis the Affimer, a novel binding protein based on a consensus cystatin scaffold has been used to inhibit KRas, which is most mutated Ras isoform. The project aims to identify Affimers that are potent inhibitors of KRas function. Three KRas binding Affimers have been identified via phage display that have shown to inhibit KRas activity by inhibiting nucleotide exchange activity. Out of three Affimers, Affimer K3 was identified to exhibit dual mode of inhibition i.e. inhibit nucleotide exchange as well as Ras-Raf interaction. To further understand the binding and inhibition of KRas by K3 Affimer, pulldown assays and nucleotide exchange assays identified that both variable regions of Affimer K3 are important for binding and inhibition of KRas. Furthermore, molecular details of Affimer K3 in complex with KRas revealed a novel Ras conformation with generation of pocket between Switch II and α3 helix. The pocket created by hydrophobic interactions is stabilised by the W44 indole side chain of K3, orienting itself to form hydrogen bonds with H95 present in α-3 helix. Mutation of KRas specific residue H95 has shown preferential specificity of Affimer K3 towards KRas, as compared to HRas and NRas. The work presented in this thesis shows Affimer K3 can be used as valuable tool to study KRas function via identification of novel Ras conformer with druggable SII/α3 pocket.