Multicolor, Cell-Impermeable, and High Affinity BACE1 Inhibitor Probes Enable Superior Endogenous Staining and Imaging of Single Molecules

The prevailing but not undisputed amyloid cascade hypothesis
places
the β-site of APP cleaving enzyme 1 (BACE1) center stage in
Alzheimer′s Disease pathogenesis. Here, we investigated functional
properties of BACE1 with novel tag- and antibody-free labeling tools,
which are conjugates of the BACE1-inhibitor IV (also referred to as
C3) linked to different impermeable Alexa Fluor dyes. We show that
these fluorescent small molecules bind specifically to BACE1, with
a 1:1 labeling stoichiometry at their orthosteric site. This is a
crucial property especially for single-molecule and super-resolution
microscopy approaches, allowing characterization of the dyes′
labeling capabilities in overexpressing cell systems and in native
neuronal tissue. With multiple colors at hand, we evaluated BACE1-multimerization
by Förster resonance energy transfer (FRET) acceptor-photobleaching
and single-particle imaging of native BACE1. In summary, our novel
fluorescent inhibitors, termed Alexa-C3, offer unprecedented
insights into protein–protein interactions and diffusion behavior
of BACE1 down to the single molecule level.