A novel high throughput 1536-well Notch1 γ -secretase AlphaLISA assay

Comb Chem High Throughput Screen. 2013 Jul;16(6):415-24. doi: 10.2174/1386207311316060001.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amyloid Precursor Protein Secretases / antagonists & inhibitors*
  • Amyloid Precursor Protein Secretases / metabolism
  • Enzyme Inhibitors / chemistry*
  • Enzyme Inhibitors / pharmacology
  • Enzyme-Linked Immunosorbent Assay*
  • High-Throughput Screening Assays / instrumentation
  • High-Throughput Screening Assays / methods*
  • Molecular Structure
  • Receptor, Notch1 / metabolism*
  • Structure-Activity Relationship

Substances

  • Enzyme Inhibitors
  • Receptor, Notch1
  • Amyloid Precursor Protein Secretases