Discovery of a Small-Molecule Modulator of the Autophagy-Lysosome Pathway That Targets Lamin A/C and LAMP1, Induces Autophagic Flux, and Affects Lysosome Positioning in Neurons

Autophagy is a major
catabolic degradation and recycling process
that maintains homeostasis in cells and is especially important in
postmitotic neurons. We implemented a high-content phenotypic assay
to discover small molecules that promote autophagic flux and completed
target identification and validation studies to identify protein targets
that modulate the autophagy pathway and promote neuronal health and
survival. Efficient syntheses of the prioritized compounds were developed
to readily access analogues of the initial hits, enabling initial
structure–activity relationship studies to improve potency
and preparation of a biotin-tagged pulldown probe that retains activity.
This probe facilitated target identification and validation studies
through pulldown and competition experiments using both an unbiased
proteomics approach and western blotting to reveal Lamin A/C and LAMP1
as the protein targets of compound RH1115. Evaluation
of RH1115 in neurons revealed that this compound induces
changes to LAMP1 vesicle properties and alters lysosome positioning.
Dysfunction of the autophagy–lysosome pathway has been implicated
in a variety of neurodegenerative diseases, including Alzheimer’s
disease, highlighting the value of new strategies for therapeutic
modulation and the importance of small-molecule probes to facilitate
the study of autophagy regulation in cultured neurons and in vivo.