Multicolor, Cell-Impermeable, and High Affinity BACE1 Inhibitor Probes Enable Superior Endogenous Staining and...

Area of Science:

• Neuroscience
• Biochemistry
• Molecular Biology

Background:

• The amyloid cascade hypothesis implicates beta-site APP cleaving enzyme 1 (BACE1) in Alzheimer's Disease (AD) pathogenesis.
• Understanding BACE1's functional properties is crucial for developing AD therapies.

Purpose of the Study:

• To investigate the functional properties of BACE1 using novel labeling tools.
• To characterize BACE1 interactions and behavior at the single-molecule level.

Main Methods:

• Development of novel fluorescent small molecules (Alexa-C3) by conjugating BACE1-inhibitor IV (C3) to Alexa Fluor dyes.
• Utilizing tag- and antibody-free labeling for specific BACE1 binding and 1:1 stoichiometry.
• Employing Förster resonance energy transfer (FRET) and single-particle imaging in cell systems and native neuronal tissue.

Main Results:

• Alexa-C3 compounds bind specifically to BACE1 at its orthosteric site with a 1:1 stoichiometry.
• Demonstrated utility of Alexa-C3 for single-molecule and super-resolution microscopy.
• Evaluated BACE1 multimerization and diffusion behavior using FRET and single-particle imaging.

Conclusions:

• Novel fluorescent inhibitors (Alexa-C3) provide unprecedented insights into BACE1 protein-protein interactions.
• These tools facilitate detailed characterization of BACE1 dynamics at the single-molecule level.
• Potential applications in understanding Alzheimer's Disease mechanisms and drug development.