A Novel High Throughput 1536-Well Notch1 γ -Secretase AlphaLISA Assay
| Autor: | Hakim Djaballah, Yue-Ming Li, David Shum, Deming Chau, Bhavneet Bhinder, Constantin Radu, David Y. Gin, M. Lane Gilchrist |
|---|---|
| Rok vydání: | 2013 |
| Předmět: |
biology
Intramembrane protease Organic Chemistry Notch signaling pathway General Medicine Bioinformatics Computer Science Applications Chemical library Cell biology law.invention chemistry.chemical_compound chemistry law Biotinylation Drug Discovery High-Throughput Screening Assays biology.protein Recombinant DNA Structure–activity relationship Amyloid precursor protein secretase |
| Zdroj: | Combinatorial Chemistry & High Throughput Screening. 16:415-424 |
| ISSN: | 1386-2073 |
| DOI: | 10.2174/1386207311316060001 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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