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Related Experiment Videos

Measuring human beta-secretase (BACE1) activity using homogeneous time-resolved fluorescence.

Matthew E Kennedy1, Wenyan Wang, Lixin Song

  • 1CNS/Cardiovascular Research, Schering Plough Research Institute, Kenilworth, NJ 07033, USA. matthew.kennedy@spcorp.com

Analytical Biochemistry
|July 5, 2003
PubMed
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Researchers developed a new assay to measure BACE1 enzyme activity for Alzheimer's disease (AD) drug discovery. This high-throughput method accurately detects BACE1 inhibitors, aiding in the development of A beta-lowering therapies for AD.

Area of Science:

  • Biochemistry
  • Neuroscience
  • Pharmacology

Background:

  • Alzheimer's disease (AD) is characterized by A beta peptide accumulation, leading to neuronal death and amyloid plaques.
  • BACE1 enzyme activity is a critical target for reducing A beta production and developing AD therapeutics.
  • Existing methods for measuring BACE1 activity may not be suitable for high-throughput screening.

Purpose of the Study:

  • To design and validate a homogeneous time-resolved fluorescence (HTRF) assay for high-throughput measurement of BACE1 enzymatic activity.
  • To compare the activity of mature BACE1 (sautoBACE1) and proBACE1.
  • To evaluate the inhibition of BACE1 activity by known inhibitors using the developed HTRF assay.

Main Methods:

  • Development of a homogeneous time-resolved fluorescence (HTRF) assay using a FRET pair (Europium and Allophycocyanin).

Related Experiment Videos

  • Utilized an N-terminally labeled, C-terminally biotinylated APP Swedish-synthetic peptide incubated with streptavidin-coupled allophycocyanin (SA-XL665).
  • Measured BACE1 enzyme activity and inhibition by comparing sautoBACE1 and sproBACE1 activities and IC50 values of inhibitors.

    • Main Results:

    • The HTRF assay demonstrated robust FRET signals, reflecting time- and concentration-dependent BACE1 activity at low nanomolar concentrations.
    • Mature BACE1 (sautoBACE1) showed slightly increased activity compared to proBACE1, with differential inhibition by the BACE1 prodomain peptide.
    • IC50 values for BACE1 inhibitors OM99-2 and OM-003 obtained via HTRF assay correlated well with literature values.

    Conclusions:

    • The developed HTRF assay provides a sensitive, reliable, and high-throughput method for measuring BACE1 activity.
    • The assay accurately reflects native BACE1 activity and is suitable for screening potential AD therapeutics.
    • Intrinsic quench correction via ratiometric measurements enhances assay robustness and data reliability.