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.

autophagy; medicinal chemistry; neurodegenerative diseases; phenotypic high-throughput screen; small molecules; target identification.