Catalytic Site-Directed γ-Secretase Complex Inhibitors Do Not Discriminate Pharmacologically between Notch S3 and β-APP Cleavages

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Abstract
The generation of γ-secretase inhibitors which block the release of β-amyloid peptide (Aβ) has long been an attractive therapeutic avenue for treatment or prevention of Alzheimer's disease (AD). Such inhibitors would reduce levels of Aβ available for aggregation into toxic assemblies that lead to the plaque pathology found in affected brain tissue. Cumulative evidence suggests that the S3 cleavage of Notch is also dependent on presenilins (PS) and is carried out by the multimeric PS-containing γ-secretase complex. It is therefore possible that Notch function could be affected by γ-secretase inhibitors. To assess the relationship between the cleavage of these substrates in the same system, Western blot cleavage assays have been established using a human cell line stably expressing both the β-amyloid precursor protein (β-APP) and the truncated Notch1 receptor fragment NotchΔE. Thus, a direct correlation may be made, following inhibitor treatment, of the decrease in the levels of the cleavage products, Aβ peptide and the Notch intracellular domain (NICD), as well as the increase in stabilized levels of both substrates. This analysis has been performed with a range of selected γ-secretase inhibitors from six distinct structural classes. Changes in all four species usually occur in concert and with remarkably good agreement. A significant cleavage window is not clearly apparent in any case. Thus, these Notch and β-APP cleavages cannot be dissected apart easily since they show the same pharmacological profile of inhibition. Whether this translates into proportionally reduced Notch signaling in vivo, however, remains to be seen.