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Title: Small molecule effects on amyloidogenic, intrinsically disordered peptides
Author: Barber, Lucy Claire Elizabeth
ISNI:       0000 0004 8500 9142
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2019
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In recent years there has been an explosion of interest in the physiological functions of intrinsically disordered peptides (IDPs) and how they are involved in diseases, specifically amyloid diseases. A fascinating aspect of amyloid is that rigid, ordered fibrils can be formed from highly flexible IDPs such as, Amyloid-beta (AB) and human Islet Amyloid Polypeptide (hIAPP). These two peptides aggregate to form amyloid in two, currently incurable diseases: Alzheimer's disease and Type II Diabetes Mellitus (TIIDM) respectively. The early steps of how AB and hIAPP transition from disordered monomers to conformers compatible with amyloid formation is an enigma which remains a challenge to understand in molecular detail. The combination of IDP structural fluidity and the complexities of amyloid formation makes structural analysis and study of this area challenging to investigate but has the potential to reveal invaluable information. The strategy for such investigation presented here focuses on searching for small molecules able to stabilise monomeric conformers of these peptides and hence to potentially disfavour amyloid formation. This work presents a methodological strategy to assess small molecule effects on recombinantly expressed and purified AB40 amyloid aggregation. The strategy is then implemented on a carefully selected set of lead molecules; a library of 67 compounds were selected from in silico rapid overlay of chemical structures (ROCS) analysis based on structural similarity to either 1,2-naphthoquinone, adapalene, bexarotene, MM3003, or UV11352. These were screened for AB40 amyloid perturbation effects using a Thioflavin-T fluorescence assay and lead compounds were identified. Lead compounds which could modulate amyloid formation were then assessed by a carefully selected toolbox of methods including Electrospray ionisation ion mobility mass spectrometry, and electron microscopy. Finally a set of complementary NMR methods are presented which enable residue specific structural propensity (residual dipolar couplings, temperature coefficients and dCa measurements) and flexibility (transverse relaxation rates, heteronuclear nuclear Overhauser effects) of small molecule-induced conformers to be monitored and compared with the same proteins in the absence of bound ligand. The framework laid out in this work has great impact potential due to is applicability to the amyloid and IDP fields. The ability to study early species in the amyloid process will reveal insights on important structures and potential folding routes in the amyloid aggregation process.
Supervisor: Radford, Sheena ; Zhuravleva, Anastasia Sponsor: BBRSC
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available