Development of a novel β-secretase binding assay using the AlphaScreen platform

J Biomol Screen. 2013 Jul;18(6):695-704. doi: 10.1177/1087057113482138. Epub 2013 Mar 29.


Alzheimer's disease (AD) is a devastating neurodegenerative disease affecting millions of people. β-secretase-1 (BACE1), an enzyme involved in the processing of the amyloid precursor protein (APP) to form Aβ is a validated target for AD. Herein, the authors develop and validate a novel binding assay for BACE1 using the AlphaScreen platform that is amenable for high-throughput screening (HTS). Small-molecule BACE1 inhibitors of the hydroxyethylamine, hydantoin, and sulfamide classes were functionalized by biotin PEG linkers of varying lengths forming probes that were bound to streptavidin donor beads. BACE1 was coupled to nickel-chelate acceptor beads. Upon mixing, probes designed from all three classes registered high signal-to-background values in the AlphaScreen binding assay, where the interaction between probe and BACE1 was completely blocked by free parent compound. A probe from the hydantoin class was chosen for further optimization, where the final assay conditions of 50 nM BACE and 250 nM probe were used and Z(') values >0.75 were commonly observed. IC50 values determined by the AlphaScreen assay format exhibited ~10-fold greater sensitivity when compared with a fluorescence polarization-based activity assay. The assay was miniaturized to a 1536-well format for HTS, in which 525 000 compounds were screened.

Keywords: AlphaScreen; Alzheimer’s disease; binding assay; high-throughput screening; β-secretase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alzheimer Disease / enzymology
  • Alzheimer Disease / metabolism
  • Amyloid Precursor Protein Secretases / antagonists & inhibitors
  • Amyloid Precursor Protein Secretases / metabolism*
  • Aspartic Acid Endopeptidases / antagonists & inhibitors
  • High-Throughput Screening Assays / methods*
  • Humans
  • Hydantoins
  • Inhibitory Concentration 50
  • Sensitivity and Specificity
  • Small Molecule Libraries / pharmacology*


  • Hydantoins
  • Small Molecule Libraries
  • Amyloid Precursor Protein Secretases
  • Aspartic Acid Endopeptidases
  • BACE1 protein, human