Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.771743
Title: Understanding molecular mechanisms of protein kinases regulation and inhibition
Author: Pucheta-Martinez, Encarna
ISNI:       0000 0004 7659 6621
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2018
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Abstract:
Protein kinases (PKs) play a key role in regulating cellular processes. Kinase dysfunction can lead to disease, thus kinases are important targets for drug design and a fundamental class of pharmacological targets for anti-cancer therapy. Among protein kinases, B-Raf and c-Src are remarkably interesting as anticancer drug targets because of their important role in cancer onset (B-Raf) and progression (c-Src). This thesis is mainly focused on the characterization of the molecular mechanism at the basis of the regulation and inhibition of these remarkable PKs. By using nuclear magnetic resonance (NMR) and molecular dynamics simulations (MD) we have studied in great details their activation dynamics, their inhibition and the effect of clinically-relevant oncogenic mutations on their structure and dynamics. C-Scr was the first viral oncogenic protein discovered, is involved in metastasis and is mutated in 50% of colon, liver, lung, breast and pancreas tumours. Upon phosphorylation, various conserved structural elements, including the activation loop, switch from an inactive to an active form able to bind ATP and phosphorylate a substrate in a cellular signalling process leading to cell replication. In this thesis, we will discuss how phosphorylation drastically changes the dynamics of the C-lobe in c-Src by NMR analysis, a phenomenon not easily accessible by static crystallographic studies. The second part of the thesis will be focused on B-Raf, a protein serine/threonine kinase. B-Raf kinase is a key target for the treatment of melanoma, since a single mutation (V600E) is found in more than 50% of all malignant melanomas. Despite their importance, the molecular mechanisms explaining the increased kinase activity in this mutant remains elusive. As kinase activity is often tightly regulated by one or more conformational transitions between an active and an inactive state, which are difficult to be observed experimentally, molecular dynamics simulations are often useful to interpret the experimental results. In this project, we will examine the mechanism by which the V600E mutation enhances the activity of the B-Raf monomer. We will also employ a combination of MD techniques with NMR experiments to fully map the effects of the mutation on the conformational landscape of B-Raf. An understanding at the atomic level of the mechanisms leading to their activation and inhibition is an extremely important goal in anti-cancer drug discovery. A better understanding of these proteins' mechanisms might lead to more potent and less toxic drugs. Finally, I report on the studies of a much small domain often associated with PKs in regulatory pathways: the WW domain. By using a combination of MD simulations and NMR, we have characterized the effect of a pathogenic mutation on its folding landscape.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.771743  DOI: Not available
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