Autophagy is an evolutionarily conserved, catabolic process through which cytosolic cargo is engulfed in an autophagosome that fuses with a lysosome to recycle the contents and maintain cellular homeostasis. Autophagy has a role in a wide range of human diseases, and the development of potent and specific modulators could help illuminate the therapeutic potential of this pathway. To evaluate autophagy in different diseases, including cancer and neurodegenerative diseases, modulators were identified in high-throughput using both targeted and phenotypic approaches. A compound-multiplexed, fluorescence polarization assay was developed to identify small molecules that disrupt the ATG5-ATG16L1 protein-protein interaction (PPI) involved in autophagosome formation. One molecule was able to disrupt this PPI with 70% inhibition and will serve as a starting point for chemical optimization. A high-content phenotypic screen was also conducted which identified small-molecule autophagy modulators. These modulators will ultimately be evaluated in models of Niemann Pick Type C (NPC) disease, a rare, autosomal recessive, neurodegenerative disease with no FDA approved therapy. Mutations in the NPC1 or NPC2 genes result in trafficking defects for cholesterol and other lipids, so a secondary screen was performed in NPC patient-derived fibroblasts to measure unesterified cholesterol clearance using filipin staining. Two autophagy modulators with different scaffolds were identified that lower cholesterol levels, and these molecules were determined to be mTOR-independent and not cytotoxic. Current work is focused on elucidation of the mechanisms of action of these two molecules and their optimization for <i>in vivo</i> studies.

High-throughput discovery of autophagy modulators through targeted and phenotypic approaches

ACS Spring Meeting 2021

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