Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.786001
Title: Analysis and inhibition of oncogenic RET-dependent signalling
Author: Sala, Elisa
Awarding Body: Open University
Current Institution: Open University
Date of Award: 2007
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Abstract:
The aim of this PhD project is to analyse the RET-dependent signalling pathway and to block it with small molecule inhibitors, thus promoting innovative approaches to cure papillary thyroid carcinoma (PTC). The first goal was to determine the three-dimensional structure of RET catalytic domain to obtain detailed knowledge of the target molecule. The Baculovirus expression system and purification procedure was optimised to produce high amount of recombinant protein (rRET) to perform biochemical characterization and crystallization attempts. rRET was active and properly folded, thus resulting a suitable reagent for inhibitors screening and crystallization experiments. The crystallization screening was performed using rRET (complete kinase domain) and three other constructs, in which different deletions were introduced. No crystals were obtained after many attempts in two independent laboratories. Concurrently, the members of 2-indolinone family, a promising class of compounds studied with molecular modelling, were screened in an ELISA-based assay to identify the best inhibitor of RET. SU5416 demonstrated an efficient inhibition of RET and was further analysed both in vitro and cellular systems, showing good specificity and selectivity. In animal model SU5416 blocked RET phosphorylation, indicating feasible pharmacological properties also in in vivo system. Since B-RAF hyperactivation leads to transformation of thyroid cells, we also used siRNA technology to investigate whether B-RAF represented a target for selective treatment and tested a new inhibitor that showed activity in B-RAF positive melanoma cell lines, in B-RAF and RET/PTC thyroid models. The current study demonstrated that B-RAF inhibition by siRNA and small molecule inhibitor determined re-expression of thyroid specific proteins, thus indicating a restoring of the differentiation process, and block of the proliferation. This work has proposed the rational identification of RET and B-RAF inhibitors by analysing their signalling pathway. These results raise the possibility that PTC could be treated effectively with small molecule inhibitors.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.786001  DOI:
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