The use of Affimers to explore RAS biology and inform small molecule design

Mutations in RAS are the most abundant oncogenic drivers, contributing to ~25% of all human cancers. However, therapeutically targeting this small GTPase has remained an elusive challenge due to its high nucleotide affinity and paucity of deep, druggable pockets. Here, a panel of RAS binding Affimers are shown to differentially inhibit RAS activation through nucleotide exchange and nanoclustering. Co-crystallisation of the most potent nucleotide exchange inhibitors showed that Affimers K3 and K6 use their small binding interface to exquisitely select druggable hotspots on this hard-to-drug protein. Affimer K3 presents the first non-covalent binder of the SII-α3 region of RAS, one of the most dynamic regions of the protein. This pocket is the binding site of the only FDA approved covalent KRASG12C inhibitors, thus informing its therapeutic potential. Binding here allosterically inhibits effector interactions and ablates protein dynamics necessary for effective nucleotide exchange. This project aimed to translate the inhibitory activity of Affimer K3 into therapeutically suitable small molecules through Affimer-induced structure-guided, and ligand-based drug discovery approaches. The development of competitive NanoBRET and competitive surface plasmon resonance assays for future high throughput drug-screening approaches are also described, alongside the optimisation of KRAS crystallisation conditions to validate novel small molecule binding.