SUBSTRATE BINDING INHIBITS γ-SECRETASE CLEAVAGE OF AMYLOID PRECURSOR PROTEIN

Abstract

Alzheimer's disease (AD) has become a severe public health challenge with no disease-modifying treatment available. The inhibition of amyloid precursor protein (APP) processing by γ-secretase is a prominent strategy, while clinical trials of γ-secretase inhibitors largely failed due to serious side effects. A Compound C1 was discovered by NMR binding based on a previously solved NMR structure of the transmembrane domain of APP (APPTM) in micelle. MALDI-TOF-MS and LC-ESI-MS were employed to access the C1 modification pattern and mechanism on APPTM. A gel-based cleavage assay and AlphaLISA assay were used to test if C1-modified substrate can inhibit γ-secretase cleavage of APP. The effect of C1 on the Aβ production was also detected in HEK 293 cells. C1 covalently modifies the C-terminal juxtamembrane lysines (K53 K54 K55) side chain of APPTM. C1-modified substrate inhibits γ-secretase cleavage with an IC50 value of 1.9 μM for the inhibition on Aβ42 production and 3.9 μM for Aβ40. In agreement with the smaller IC50 for Aβ42 in AlphaLISA assay, C1 reduces the level of Aβ42 more than that of Aβ40 in HEK 293 cells, resulting in a reduced Aβ42/Aβ40 ratio. By mutagenesis, K55 was found to be the most reactive lysine in APPTM and plays crucial role in intramembrane proteolysis. Our work demonstrates that targeting the substrate of γ-secretase to reduce the production of Aβ is viable and novel strategy. This is to our knowledge the first example of small molecule that inhibits γ-secretase cleavage and Aβ production by interacting with APPTM. Our results point to a new direction in AD drug discovery for reducing amyloid load as disease-modifying therapy.