Understanding the role of KRAS nanoclustering in signalling and disease

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The mitogen activated protein kinase (MAPK) pathway is involved in a variety of fundamental cellular processes activated by a diverse set of intracellular and extracellular stimuli. Dysregulation of the MAPK pathway has been implicated in the development of many human diseases, including cancer. RAS is an important GDP/GTP regulated molecular switch within the MAPK pathway and gain-of-function missense mutations within RAS are found in approx 25% of all human cancers. Therefore, the inhibition of RAS signalling, either directly or indirectly are major focuses in drug discovery campaigns. Although there had been some recent advances in covalently targeting the G12C mutation there remains the issue of targeting other mutations and drug resistance. As a result, new ways of inhibiting RAS function and identifying mediators of RAS requires further research and novel approaches. Affimer reagents are tools that can be isolated that bind to target proteins to regulate function. Affimers (a biologic with a small probe surface comprised of two variable regions) have been isolated that bind to KRAS, some of which inhibit important biological processes like nanoclustering. This project will utilise Affimer reagents to fully understand the importance of nanoclustering during signalling and cancer progression, as well as, identifying novel druggable mediators of RAS signalling. Using a variety of processes including X-ray crystallography (to solve the structure of Affimer-RAS proteins), EM studies (to image RAS dimerization in cellular membranes), cell biology (to understand how inhibiting RAS nanoclustering regulates cellular signalling and cancer cell survival) and cellular phenotypic screening assays using Affimer-mediate protein interference (to identify effectors of KRAS mutations).