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Yeast growth selection system for the identification of cell-active inhibitors of [beta]-secretase

Middendorp, Oliver M.. Yeast growth selection system for the identification of cell-active inhibitors of [beta]-secretase. 2004, Doctoral Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_6943

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Abstract

The production and deposition of the cytotoxic Aβ peptide is a central event in the
pathogenesis of Alzheimer’s disease (AD). Aβ is excised from the amyloid precursor
protein (APP) through sequential actions of the β-secretase, which cleaves at the socalled
β-site, and the γ-secretase, which cleaves at the so-called γ-site of APP. Inhibition
the β-secretase BACE1 is a promising approach for AD therapy, but the search for small
molecule inhibitors has proven to be challenging. In this thesis I describe a novel
screening assay to identify cell-active BACE1 inhibitors by a positive yeast growth
selection system, which combines the practicability of in vitro assays with the advantages
of a cell-based assay. Analogue to the situation in mammalian cells, the β-site cleavage
reaction was reconstituted in the secretory pathway of yeast cells using membrane-bound
BACE1 and a membrane-bound APP-derived substrate. In contrast to conventional
mammalian cell-based assays, false positive compounds can be rapidly excluded by the
use of simple specificity controls that mimic the readout in the absence of BACE1.
Furthermore, there is counter-selection for toxic compounds due to the positive growth
readout upon inhibition of BACE1. The system was initially validated with two bona fide
BACE1 inhibitors that stimulated the growth of BACE1-expressing cultures in a
concentration-dependent manner, whereas the growth of control cultures remained
unaffected in the presence of these inhibitors. In order to identify novel BACE1 inhibitors
and to further validate the system, we screened a library of 15’000 small molecules. This
screening revealed six compounds, which significantly reduced the secretion of Aβ from
a human cell line overexpressing APP.
Advisors:Riezman, Howard
Committee Members:Rüegg, Markus A.
UniBasel Contributors:Rüegg, Markus A.
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:6943
Thesis status:Complete
Bibsysno:Link to catalogue
Number of Pages:85
Language:English
Identification Number:
Last Modified:22 Apr 2018 04:30
Deposited On:13 Feb 2009 14:58

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