Uncovering active modulators of native macroautophagy through novel high-content screens
Autophagy is an evolutionarily conserved pathway mediating the breakdown of cellular proteins and organelles. Emphasizing its pivotal nature, autophagy dysfunction contributes to many diseases; nevertheless, development of effective autophagy modulating drugs is hampered by fundamental deficiencies in available methods for measuring autophagic activity, or flux. To overcome these limitations, we introduced the photoconvertible protein Dendra2 into the MAP1LC3B locus of human cells via CRISPR/Cas9 genome editing, enabling accurate and sensitive assessments of autophagy in living cells by optical pulse labeling. High-content screening of 1,500 tool compounds provided construct validity for the assay and uncovered many new autophagy modulators. In an expanded screen of 24,000 diverse compounds, we identified additional hits with profound effects on autophagy. Further, the autophagy activator NVP-BEZ235 exhibited significant neuroprotective properties in a neurodegenerative disease model. These studies confirm the utility of the Dendra2-LC3 assay, while simultaneously highlighting new autophagy-modulating compounds that display promising therapeutic effects.