A Novel High Throughput 1536-Well Notch1 γ -Secretase AlphaLISA Assay

Author(s): De-Ming Chau, David Shum, Constantin Radu, Bhavneet Bhinder, David Gin, M. Lane Gilchrist, Hakim Djaballah, Yue-Ming Li.

Journal Name: Combinatorial Chemistry & High Throughput Screening

Volume 16 , Issue 6 , 2013

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Abstract:

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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Article Details

VOLUME: 16
ISSUE: 6
Year: 2013
Page: [415 - 424]
Pages: 10
DOI: 10.2174/1386207311316060001
Price: $58

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