Hadfield, Lynsay Claire (2018) Heparin and heparin-like molecules inhibit the Alzheimer’s β-secretase (BACE1): considerations for biological assay and future therapeutic development. Doctoral thesis, Keele University.

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Abstract

The biologically and medically important heparan sulphate and heparin
polysaccharides have previously been shown to modulate the activity of an aspartyl
protease, β-secretase (BACE1), implicated in the aetiology of Alzheimer’s disease. Research
groups investigating the activity of heparin with BACE1 have demonstrated both inhibitory
and stimulatory effects of this glycosaminoglycan, and other analogues. In an attempt to
understand this relationship, a review of the available recombinant BACE1 products was
conducted to determine if protein purification tag had an effect on the heparin/heparan
sulphate interaction with BACE1. FLAG-tagged BACE1 was identified as a suitable proxy for
native, untagged BACE1 for activity studies.
In this study, the common purification tag, IgG Fc region, has been shown to interact
with heparin and other glycosaminoglycans at acid pH (pH 4.0 and pH 5.0) with heparin
affinity of > 800 mM. Further investigation, first of whole immunoglobulin (IgG & IgM) and
then with Fab and F(ab’)2 antibody fragments, identified heparin binding at acid pH in all
fragments tested. Thermal stability studies were conducted to identify heparin/HS
structural requirements for this novel interaction, which suggest sulphate is necessary but
not sufficient for activity.
Finally, a library of chemically sulphated non-heparin polysaccharides were utilised
to identify ‘hit’ BACE1 inhibitors with beneficial BACE1 therapeutic attributes such as low
molecular weight, minimal charge and attenuated off-target effects, such as anti-coagulant
activity. Differential scanning fluorimetry was identified as a potential high-throughput
screening assay to replace fluorescence resonant energy transfer, during the initial nonheparin
polysaccharide library BACE1 inhibitor screening.

Item Type: Thesis (Doctoral)
Subjects: R Medicine > R Medicine (General)
Divisions: Faculty of Natural Sciences > School of Life Sciences
Depositing User: Lisa Bailey
Date Deposited: 08 Jun 2018 11:46
Last Modified: 08 Jun 2018 11:46
URI: http://eprints.keele.ac.uk/id/eprint/4998

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