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Title: Novel ligands for the study of inositol polyphosphate-converting enzymes
Author: Baker, Kendall
ISNI:       0000 0004 6351 0825
Awarding Body: University of East Anglia
Current Institution: University of East Anglia
Date of Award: 2016
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The human genome encodes a number of inositol phosphate phosphatases that act upon inositide or phosphoinositide substrates. Among these, the 5-phosphatases have become targets for therapeutic intervention in human pathologies including cancers and diabetes. Of these, SHIP1 and SHIP2 (SH2-domain-containing inositol 5-phosphatase 1 & 2), which dephosphorylate phosphatidylinositol 3,4,5-trisphophate have received particular attention. In this study, attempts have been made to solve X-ray crystallographic structures of these proteins in complex with physiologically relevant inositide and phosphoinositide ligands or substrate analogs. The substrate analogs that have proved most useful include a family of benzene polyphosphates (BzPs) and biphenyl polyphosphates (BiPhs). This thesis describes the cloning and expression of the catalytic domains of SHIP1 and SHIP2 in E. coli, purification of these proteins and structural studies thereon. A structure was solved to 2.75 Å for apo SHIP2cd and from this, and existing literature, a homology model was made for SHIP1cd. While BiPhs have been reported as ligands of 5-phosphatases, this thesis extends their use to the study of a inositol phosphate kinase; Arabidopsis thaliana inositol 1,3,4,5,6- pentakisphosphate (AtIPK1). An enzyme catalysing reversible phosphotransfer between ATP and IP5. Structures were solved to 2.1 Å of ternary complexes between AtIPK1, BiPh and ADP, the structures reveal the adoption by the protein of a ‘half-closed’ conformation, reported previously only in the presence of nucleotide alone. Close scrutiny of the structures of BiPh/protein complexes prompted their examination as potential inhibitors of SHIP1, SHIP2 and AtIPK1. A fluorescence polarisation ligand displacement assay was constructed with a fluorescein-tagged inositol phosphate and comparisons were made of the efficacy of different biphenyl phosphates in displacement of this ligand. The same polarisation assay was used to screen the NCI Diversity Set II for inhibitors of SHIP2 and AtIPK1. A number of compounds were identified, and characterised for dose response with these proteins. Similar screens, which included SHIP1, were performed with the NCI Approved Oncology Drug Set V. Potential lead compounds were taken forward for characterisation of their ability to inhibit AtIPK1.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available