The biological and pharmacological allosteric modulation of inositol monophosphatase

Noble, James William (2020) The biological and pharmacological allosteric modulation of inositol monophosphatase. Doctoral thesis (PhD), University of Sussex.

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Abstract

The purpose of this project was to develop novel pharmacological tools against Inositol Monophosphatase (IMPase) and gain new insights into the Calbindin-IMPase interaction. IMPase is an enzyme that has been of significant therapeutic interest since the development of the inositol depletion hypothesis of lithium’s efficacy in the treatment of bipolar disorder. More recent studies have implicated IMPase inhibition in the induction of autophagy and the clearance of peptides involved in the pathogenesis of neurodegenerative diseases. Despite this there is a lack of specific inhibitors and those that have been developed lack favorable physiochemical properties to explore specific IMPase inhibition in vivo. My aim was to find novel inhibiters and binding chemical fragments that could later be developed into potent specific inhibitors of IMPase that would allow for the specific evaluation of IMPase inhibition in vivo. To achieve this, two approaches were tried: first a crystallographic fragment soaking screen was used to identify novel binding sites and chemical matter. This resulted in the discovery of a novel fragment binding site at the homodimer interface and mutagenesis at this site inhibited IMPase activity, indicating that the site could be therapeutically relevant. The second approach was to screen a specially curated isothiazolone library based on the previously identified covalent inhibitor Ebselen. Here I identified a new class of isothiazolone inhibitors of IMPase.

The calcium binding protein Calbindin-D28k can allosterically modulate IMPase, this results in the increase in the catalytic activity of IMPase. I aimed to explore this interaction by fusing the proteins using a flexible amino acid linker. The resulting complexes had activity in excess of that of the wild type IMPase, confirming that Calbindin-D28k activated IMPase. Low resolution structural analysis using small angle X-ray scattering (SAXS) revealed a V-shaped IMPase-calbindin complex which was in contrast to the more linear shape predicted by computational docking models. Here we also obtained the first crystal structure of Calbindin-D28k. This crystal structure gave the first direct visualization of the calcium binding sites of the protein and is a better model of Calbindin-D28k in solution than the previous NMR structure.

Item Type: Thesis (Doctoral)
Schools and Departments: School of Life Sciences > Biochemistry
Subjects: Q Science > QP Physiology > QP0501 Animal biochemistry > QP0550 Organic substances > QP771 Vitamins > QP0772.A-Z Special, A-Z > QP0772.I5 Inositol
Depositing User: Library Cataloguing
Date Deposited: 23 Mar 2020 14:47
Last Modified: 23 Mar 2020 14:47
URI: http://sro.sussex.ac.uk/id/eprint/90252

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