The Notch pathway plays a crucial role in cell fate decisions through controlling various cellular processes.
Overactive Notch signal contributes to cancer development from leukemias to solid tumors. γ-Secretase is an
intramembrane protease responsible for the final proteolytic step of Notch that releases the membrane-tethered Notch
fragment for signaling. Therefore, γ-secretase is an attractive drug target in treating Notch-mediated cancers. However, the
absence of high throughput γ-secretase assay using Notch substrate has limited the identification and development of γ-
secretase inhibitors that specifically target the Notch signaling pathway. Here, we report on the development of a 1536-
well γ-secretase assay using a biotinylated recombinant Notch1 substrate. We effectively assimilated and miniaturized this
newly developed Notch1 substrate with the AlphaLISA detection technology and demonstrated its robustness with a
calculated Z’ score of 0.66. We further validated this optimized assay by performing a pilot screening against a chemical
library consisting of ~5,600 chemicals and identified known γ-secretase inhibitors e.g. DAPT, and Calpeptin; as well as a
novel γ-secretase inhibitor referred to as KD-I-085. This assay is the first reported 1536-well AlphaLISA format and
represents a novel high throughput Notch1-γ-secretase assay, which provides an unprecedented opportunity to discover
Notch-selective γ -secretase inhibitors that can be potentially used for the treatment of cancer and other human disorders.
Keywords: Alzheimer disease, AlphaLISA, cancer, γ-secretase, γ-secretase modulators, Notch signaling.
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