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The Mechanism Of Action Of Novel Gamma-Secretase Modulators

Author
Paresi, Chelsea
Abstract
Gamma-secretase (GS) is a multi-protein, aspartyl protease complex consisting of presenilin (PS1), nicastrin (NCT), anterior pharynx-defective-1 (APH-1) and presenilin enhancer 2 (PEN-2)1,2. GS cleaves a variety of substrates, all of which are type I transmembrane proteins. The activity of GS has been most extensively studied in relation to the processing of amyloid precursor protein (APP) and Notch due to their implication in Alzheimer’s disease and cancer respectively. The role of GS in these pathways makes the enzyme an attractive drug target; however, a major obstacle of using GS as a pharmacological target to treat these diseases is the development of specific inhibitors that selectively target the cleavage of only one substrate. In the case of APP processing, an ideal pharmaceutical would not only conserve cleavage of other gamma-secretase substrates, but would also maintain production of nonamyloidogenic amyloid beta (A?) peptides. In an effort to discover novel GS modulators we have developed and utilized an efficient in vitro gamma-secretase activity assay that can be used to measure GS activity on both Notch and APP concurrently. We have shown that recombinant substrates of gamma-secretase compete for cleavage in our assay and that measured IC50 values are significantly different when determined in the presence of an additional substrate. This work demonstrates the importance of using accurate biochemical assays when calculating selectivity margins. We have utilized this assay to study novel gamma-secretase inhibitors and modulators, specifically, benzimidazoles which inhibit Notch processing while simultaneously altering APP cleavage specificity. The data presented herein suggest that similar to their mechanism of proton pump inhibition, benzimidazoles modulate gamma-secretase activity by binding to cysteine residues on Notch, PS1-NTF, PS1- CTF, and Pen-2. A large portion of the evidence for benzimidazole binding to GS and Notch consists of protein labeling experiments performed with benzimidazole based probes. In addition to using these probes for target identification, we have also undergone a systematic comparison of available copper-free click chemistries. We have validated the use of molecular probes for target ID by showing direct labeling of the known benzimidazole target, the gastric proton pump, and have identified several novel binding partners of benzimidazole com
Date Issued
2016Subject
APP; Benzimidazole; gamma-secretase; gamma-secretase modulator; Notch; Proton pump inhibitors
Degree Discipline
Pharmacology
Degree Level
Doctor of Philosophy
Rights
Attribution-NonCommercial-NoDerivatives 4.0 International
Type
dissertation or thesis
Except where otherwise noted, this item's license is described as Attribution-NonCommercial-NoDerivatives 4.0 International