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Title: Peptides as therapeutics
Author: Lopez Aguilar, Aime
ISNI:       0000 0004 2728 1463
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2011
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Peptides have attracted increasing attention as therapeutics in recent years, at least partially as a consequence of the widespread acceptance of protein therapeutics; but also as possible solutions to problems such as short half-life and delivery of molecules, and as therapeutics in their own right. The current work presents three projects that involve applications of peptides in a therapeutic environment. The first project studies the use of ER retaining peptides and CPPs (Cell penetrating peptides) in enhancing the effective concentration of DNJ (1-deoxynojirimycin), an α-glucosidase inhibitor, in cells. DNJ constructs with ER retaining peptides (6-[N-(1-deoxynojirimycino)]-hexanoyl-KDEL and 6-[N-(1-deoxynojirimycino)]-hexanoyl-KKAA) and CPPs (6-[N-(1-deoxynojirimycino)]-hexanoyl-TAT and 6-[N-(1-deoxynojirimycino)]-hexanoyl-MAP) were synthesised and analysed for their inhibitory activity against α-glucosidase I and II in vitro. The constructs were then analysed in a cell-based assay to determine their inhibitory activity on α¬-glucosidase-mediated hydrolysis of N-linked oligosaccharides. FITC-labelled ER retaining peptides were also synthesised to determine the internalisation and trafficking of the constructs by FACS and IF-microscopy. While none of the DNJ-constructs showed higher cellular inhibition than NB-DNJ (N-butyl DNJ; Miglustat), the CPP construct 6-[N-(1-deoxynojirimycino)]-hexanoyl-TAT showed comparable activity and the ER retaining construct 6-[N-(1-deoxynojirimycino)]-hexanoyl-KDEL showed a small but significant increase in activity following long-term administration. The second project focuses on beauveriolides, a cyclic depsipeptide family shown to have activity as ACAT inhibitors and thus a possible treatment for Alzheimer’s disease by the decrease in the production of Amyloid β (Aβ). A published total synthetic method was improved by the use of a cross-metathesis to reduce the total synthesis by 5 steps and increase its flexibility to allow the production of analogues. The synthesised beauveriolide III was used in attempts to develop an IF-FACS-based assay to measure the intracellular concentrations of Aβ. However, the location of γ-secretase in the used cell-line meant that levels of intracellular Aβ were not sufficient to track any decrease caused by ACAT inhibition. The third project involves the design of a cyclic peptide that could block the binding site for the influenza virus in the host cell. The cyclic peptide (cGSGRGYGRGWGVGA) was developed from a comparative study of four different sialic acid-binding proteins and synthesised by solution cyclisation of the linear peptide synthesised by traditional solid phase peptide synthesis (SPPS). An in silico study showed that the cyclic peptide allowed overlap with the binding site of Hemagglutinin. A 1H NMR titration determined the dissociation constant of the cyclic peptide to sialic acid. The KD corresponded to a low binding affinity, however the observed binding seemed to be specific and caused by a single bound conformation.
Supervisor: Butters, Terry ; Wentworth, Paul Sponsor: CONACYT Mexico
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Biochemistry ; Glycobiology ; Medical Sciences ; Infectious diseases ; Chemical biology ; Polymers Amino acid and peptide chemistry ; Organic synthesis ; Natural products ; Life Sciences ; Disease prevention ; Viruses