Inhibition of Ras using Affimer reagents.

Ras proteins are small GTPases that are mutationally activated in around 30% of all human cancers. Oncogenic mutations in Ras trigger uncontrolled cellular differentiation and division through uninhibited Ras-GTP signalling. Despite major efforts in developing inhibitors, lack of treatments directly targeting Ras in cancer led to the current assumption that Ras is undruggable. A new approach, which involves use of biologics, have shown great potential for development of Ras inhibitors, as demonstrated by recent increase in the number of antibody mimetic reagents targeting Ras, including single domain antibodies, monobodies and DARPins. Research presented in this thesis has developed modulators of Ras activity using novel artificial binding proteins, termed Affimers. Affimer reagents are small 91 amino acid scaffold proteins that constrain one or two randomised nine amino acid loop regions for molecular recognition. Affimers isolated against KRas, the most commonly mutated Ras family member, were shown to be effective at inhibiting nucleotide exchange and blocked interaction between Ras and its effector Raf. When expressed in mammalian cells, Affimers bound with endogenous Ras and inhibited Ras-mediated signalling. Site-directed mutagenesis of the Affimer variable regions revealed three residues critical for binding and inhibition. X-ray crystal structure of Affimer in complex with KRas demonstrated that these residues bind into a hydrophobic pocket on Ras, previously described with small molecules. Molecular docking was used to generate mimics of the Affimer residues, therefore providing insights into new mode of therapeutic development. These Affimer-derived compounds were shown to be effective in biochemical and cell-based assays, with comparable properties to previously published small molecule inhibitors of Ras, but without any optimisation of the compounds. Therefore, these molecules hold great potential for development of even more potent inhibitors through employment of medicinal chemistry. The work presented in this thesis established a novel use of Affimer reagents as valuable tools for the development of small molecule inhibitors.