title: A novel high throughput 1536-well Notch1 γ -secretase AlphaLISA assay.
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.
authors: Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York, NY 10065, USA.
authors: De-Ming
authors: DM
authors: Chau
authors: David
authors: D
authors: Shum
authors: Constantin
authors: C
authors: Radu
authors: Bhavneet
authors: B
authors: Bhinder
authors: David
authors: D
authors: Gin
authors: M Lane
authors: ML
authors: Gilchrist
authors: Hakim
authors: H
authors: Djaballah
authors: Yue-Ming
authors: YM
authors: Li
mesh_terms: D053829:Amyloid Precursor Protein Secretases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D057166:High-Throughput Screening Assays / Q000295:instrumentation / Q000379:methods*; D015394:Molecular Structure; D051881:Receptor, Notch1 / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D004791:Enzyme Inhibitors; D051881:Receptor, Notch1; D053829:Amyloid Precursor Protein Secretases