Galdadas, Ioannis; (2021) Understanding the effect of oncogenic mutations on kinases. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Kinases are a large family of enzymes that catalyse the phosphorylation of diverse substrates. This post-translational modification is crucial for the regulation of different cellular functions, including cell growth and proliferation, among others. Deregulation of kinase activity by the acquisition of mutations is known to be central for the survival and spread of cancer cells. Despite the remarkable progress of experimental techniques in probing the structural implications of oncogenic mutations in kinases, our understanding of the behaviour of many clinically relevant mutations at an atomic level is still limited and underlines the need of using computational methods to study the dynamics of such variants at atomic resolution, complementing experimental findings. In this thesis, we report the structural and dynamical implications of a number of activating, oncogenic mutations within the kinase domain of the epidermal growth factor receptor (EGFR) and the phosphoinositide 3-kinase alpha (PI3Ka). For this purpose, we have applied molecular dynamics simulations complemented by metadynamics simulations to sample rare events of biological interest and calculate the associated free energy landscapes of both the wild type and selected mutants. In particular, we have explored the effects of the exon 19 DELREA deletion and the exon 20 L858R point mutation, which comprise the vast majority of EGFR mutations, as well as the lower frequency D770-N771insNPG, and A763-Y764insFQEA exon 20 insertions, which are associated with diverse activation levels and responses to inhibitors. The differences in the conformational energy landscapes of these mutants unravel the molecular effects of these mutations, which lead to aberrant signalling, and suggest that these mutations do not share any common mechanism of action as different mutations favour different conformations. Given the importance of the dimerisation in the activation of EGFR, we discuss also the differences in the dynamics of these four mutations in the context of symmetric and asymmetric homodimers. Furthermore, we expand the list of the studied mutations to include more mutations that have been reported to affect the dimerisation ability of EGFR. Lastly, out of the known mutations of PI3Ka that are implicated in cancer, we discuss the effect of the hotspot mutation E545K on the dynamics of PI3Ka and elucidate its mechanism of action, which is more complicated than previously thought.

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