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Peptidomimetic induced modulation of β-site APP cleaving enzyme (BACE1) - biochemical implications in alzheimer's disease

posted on 14.02.2017, 02:50 by Juliano, John Paul
The β-site APP Cleaving enzyme 1 (BACE1) is a membrane-associated aspartyl protease which mediates the production of Amyloid-β (Aβ), a major neurophysiological characteristic in Alzheimer’s disease brains. Performing the primary cleavage within Amyloid Precursor Protein (APP) of the amyloid-forming pathway, BACE1 serves as a favourable therapeutic target. Aside from APP, BACE cleaves numerous alternate substrates in vivo. BACE1 protease activity has been shown to be mediated by sub-cellular localisation and its own post-translational and catalytic structural elements. Additionally, BACE activity can be modulated allosterically by heparin and non-active site interacting peptides. The incorporation of transition state isostere (TSI) mimetics at the scissile site within active site substrates of BACE1 is a common strategy for development of compounds which inhibit BACE1. Many BACE1 TSIs are hydroxylated β-amino acid analogues shown to be effective against a number of aspartyl proteases. The use of iterative design and structural-activity relationship studies have resulted in the development of potent and specific small molecule BACE1 inhibitors with significantly reduced peptidic character which reduce cereberal Aβ levels in vivo. Despite these successes, the relationship between BACE1 substrates and the mechanisms that regulate BACE1 activity that result in the disease and non-disease states of Alzheimer’s disease are poorly understood. In this study, the relative activities of recombinant human BACE1 variants and the role of substrate derived non-TSI β-peptidomimetics as modulators of BACE1 activity was explored. Firstly, systems for the expression, refolding and purification of BACE1 from bacterial and yeast sources were developed. Functional and pure un-glycosylated bacterially-derived mature rhBACE homogenate at high yield was obtained and a heterogenous glycosylated rhBACE1 from yeast were successfully expressed. The amino acid sequences flanking the β-cleavage site within APP carrying the Swedish double mutation (APPSW), Neuregulin, the synthetic hydroxyethylene-based TSI peptide inhibitor OM99-2 and the high affinity peptide sequence SEISYEVER, served as the four substrate templates from which over 60 peptides were designed and synthesised by solid phase peptide synthesis. A Quenched Fluorescent Substrate BACE1 assay in conjunction with LC-MS analysis was established to investigate cleavage susceptibility and inhibition potency under competitive and non-competitive conditions. It was determined that β-amino acids substituted at the P1 scissile site position within known peptide substrates were resistant to proteolysis, and particular substitutions induced a concentration dependent stimulation of BACE1. Modulation of BACE1 has been observed only via allosteric interaction by non-peptide compounds and peptide substrates of poor affinity for the BACE active site. The results of this study indicate a possible modulatory role of native BACE1 substrates in vivo via active site and allosteric interaction. Further experimentation may deduce the complex mechanisms which regulate both BACE1 activity toward alternate in vivo substrates and the increased Aβ processing involved in Alzheimer’s disease.


Principal supervisor

Marie-Isabel Aguilar

Year of Award


Department, School or Centre

Biochemistry and Molecular Biology

Campus location



Doctor of Philosophy

Degree Type



Faculty of Medicine Nursing and Health Sciences