A novel high throughput 1536-well notch1 γsecretase AlphaLISA assay Journal Article
| Authors: | Chau, D. M.; Shum, D.; Radu, C.; Bhinder, B.; Gin, D.; Gilchrist, M. L.; Djaballah, H.; Li, Y. M. |
| Article Title: | A novel high throughput 1536-well notch1 γsecretase 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. γSecretase is an intramembrane protease responsible for the final proteolytic step of Notch that releases the membrane-tethered Notch fragment for signaling. Therefore, γsecretase is an attractive drug target in treating Notch-mediated cancers. However, the absence of high throughput γsecretase 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 γsecretase 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 γsecretase inhibitors e.g. DAPT, and Calpeptin; as well as a novel γsecretase inhibitor referred to as KD-I-085. This assay is the first reported 1536-well AlphaLISA format and represents a novel high throughput Notch1-γsecretase assay, which provides an unprecedented opportunity to discover Notch-selective γsecretase inhibitors that can be potentially used for the treatment of cancer and other human disorders. © 2013 Bentham Science Publishers. |
| Keywords: | signal transduction; controlled study; unclassified drug; human cell; reproducibility; protein degradation; signal noise ratio; drug potency; high throughput screening; in vitro study; information processing; reliability; immunoassay; ic 50; alzheimer disease; concentration response; gamma secretase inhibitor; notch1 receptor; gamma secretase; polyclonal antibody; notch signaling; chemoluminescence; calpeptin; γsecretase; γsecretase modulators; alphalisa; kd i 085; alphalisa assay; receptor solubilization |
| Journal Title: | Combinatorial Chemistry and High Throughput Screening |
| Volume: | 16 |
| Issue: | 6 |
| ISSN: | 1386-2073 |
| Publisher: | Bentham Science Publishers |
| Date Published: | 2013-07-01 |
| Start Page: | 415 |
| End Page: | 424 |
| Language: | English |
| DOI: | 10.2174/1386207311316060001 |
| PROVIDER: | scopus |
| PMCID: | PMC3664143 |
| PUBMED: | 23448293 |
| DOI/URL: | |
| Notes: | --- - "Export Date: 1 July 2013" - "CODEN: CCHSF" - "Source: Scopus" |
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