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Investigating K-Ras inhibition by α-helical peptides https://doi.org/10.17863/CAM.116542 Comfort, Hannah Megan Freeman Mott, Helen Mutations in the KRAS gene are common drivers of human cancer and inhibition of KRas is a key goal in drug discovery. Recently, the development of covalent K-Ras(G12C) inhibitors has presented a breakthrough in the field and challenged the belief that K-Ras was "undruggable". Despite this, there are currently no approved inhibitors directly targeting other K-Ras variants and long-term efficacy of K-Ras(G12C) inhibitors is compromised by resistance. This necessitates investigation into alternative methods to target K-Ras. α-helical peptides present an effective method to inhibit protein-protein interactions such as those between K-Ras and its effectors. So far, K-Ras-targeting α-helical peptides have been resticted to SOS1 mimetics. This work aimed to investigate inhibition of K-Ras by α-helical peptides discovered by de novo screening or from a Ral GTPase effector protein domain (RLIP76 RBD) template. A single hit from a selection of an α-helical peptide library against K-Ras(G12D) was found to bind K-Ras(G12D) with low micromolar affinity. Further characterisation revealed that this peptide (P39) displayed selectivity for the GTP-bound state of K-Ras(G12D) and that binding was competitive with the K-Ras effector Raf. The P39 binding site on K-Ras(G12D) was mapped using a combination of NMR titrations and molecular dynamics simulations, revealing a novel binding pose between switch I and II of K-Ras(G12D). In addition to de novo screening, design of a K-Ras-binding α-helical peptide from RLIP76 RBD was attempted. RLIP76 RBD variants previously selected to bind K-Ras were validated biochemically, however their dimerisation prevented K-Ras binding. A point mutation successfully reversed dimerisation, although K-Ras binding of the resulting RBD could not be verified. Rational design of RLIP76 RBD point mutations enabled K-Ras binding in a single case, suggesting K-Ras affinity can be achieved by design. Finally, cellular studies revealed that when co-expressed in HEK-293T cells, the P39- K-Ras interaction could be seen. Surprisingly, treatment of a KRAS(G12D)-mutant colon cancer cell line with octa-Arg-appended P39 resulted in an increase in K-Ras activity and downstream MAPK and PI3K signalling. P39-induced hyperactivation of Ras signalling led to apoptosis in these cells. P39 therefore represents a useful starting point for further development. University of Cambridge 2024-09-27 Thesis Doctoral Doctor of Philosophy (PhD) eng https://www.repository.cam.ac.uk/handle/1810/381309 embargo 2026-03-11 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/f63c473b-8fb2-4a5a-b190-5a9220231de7/download 87eda9de84448d1f82354d60eee3eb5f https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/e033ea7a-f29b-4486-8fdf-cf3d87caf6b7/download b2e5980cd19e6d8c2080b2dfc0829816 http://purl.org/NET/rdflicense/allrightsreserved K-Ras peptide therapeutic