A Novel High-Throughput 1536-well Notch1 γ-Secretase AlphaLISA Assay.
A Novel High-Throughput 1536-well Notch1 γ-Secretase AlphaLISA Assay.
Comb Chem High Throughput Screen. 2013 Feb 27;
Authors: Chau DM, Shum D, Radu C, Bhinder B, Gin D, Gilchrist ML, Djaballah H, Li YM
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 tumor. 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 f...
Source: Combinatorial Chemistry and High Throughput Screening - Category: Chemistry Authors: Chau DM, Shum D, Radu C, Bhinder B, Gin D, Gilchrist ML, Djaballah H, Li YM Tags: Comb Chem High Throughput Screen Source Type: research