Novel ligands for the study of inositol polyphosphate-converting enzymes

Baker, Kendall (2016) Novel ligands for the study of inositol polyphosphate-converting enzymes. Doctoral thesis, University of East Anglia.

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Abstract

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.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Katie Miller
Date Deposited: 04 Jul 2017 14:24
Last Modified: 30 Sep 2019 00:38
URI: https://ueaeprints.uea.ac.uk/id/eprint/64006
DOI:

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