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Title: Biochemical analysis of Tribbles 2 pseudokinase using repurposed kinase inhibitors
Author: Foulkes, Daniel M.
ISNI:       0000 0004 7656 8137
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2018
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Pseudokinases make up ~10% of the human kinome and lack one or more of the evolutionary conserved amino acids that are typically required to hydrolyse ATP and phosphorylate protein substrates. Overexpression of the Tribbles 2 pseudokinase (TRIB2) has been identified in subsets of acute myeloid and acute lymphoblastic leukaemia as well as drug-resistant solid tumours, marking it out as an important new target for drug discovery. TRIB2 is the most ancestral of the three human TRIB pseudokinase homologues, which have evolved features distinguishing them from all other protein kinases. They are uniquely defined by an unusual aC-helix, which forms a binding site for an E3 ubiquitin ligase binding motif in the Tribbles C-terminal tail. The pseudokinase domain also retains a binding platform for substrates, such as the myeloid transcription factor C/EBPa, which are ubiquitinated by context-specific E3 ligases. TRIB2 has also been reported to induce AKT phosphorylation at Ser473, promoting cell survival. A lack of target-validated small molecule ligands with which to probe signalling functions has hindered the analysis of Tribbles pseudokinases. This thesis describes a kinase inhibitor-repurposing approach to discover novel TRIB2 chemical ligands, in order to probe TRIB2 biochemical mechanisms in vitro and analyse TRIB2 signalling mechanisms in human cells. By employing a simple thermal shift assay, recombinant TRIB2 protein was screened using the Published Kinase Inhibitor Set (PKIS), and distinct families of stabilising and destabilising chemical ligands were discovered and analysed. The clinically-approved covalent EGFR/HER2 inhibitors afatinib and neratinib were found to bind covalently to two unique cysteine residues in the aC-helix of the TRIB2 pseudokinase domain, causing it to become uncoupled from its own C-terminal tail region. Cellular afatinib exposure led to rapid proteasome-mediated degradation of both exogenous and endogenously expressed TRIB2 in human cell lines. Mutation of the two Cys residues responsible for covalent compound-binding in vitro also resulted in TRIB2 stabilisation in cells in the presence of afatinib, alongside the maintenance of high levels of pSer473 AKT. Afatinib and neratinib were also highly cytotoxic in the TRIB2 overexpressing leukaemia U937 cell model, downstream of TRIB2 degradation and the induction of apoptosis. The work reported in this thesis suggests new potential therapeutic approaches for the treatment of TRIB2-associated diseases.
Supervisor: Eyers, Patrick Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral