Identification of potential autophagy inducers through high-throughput and high-content imaging analysis

Autophagy is a cellular degradation process that plays a critical role in maintaining homeostasis and preventing stress-induced damage, making it a promising therapeutic target for cancer and neurodegenerative diseases. In this study, we utilized mouse embryonic fibroblasts expressing GFP-labeled microtubule-associated protein 1 light chain 3, a widely used biomarker of autophagy activation, to screen 3733 clinically approved or investigational drugs using a high-throughput and high-content screening platform. From the primary screening, 117 compounds were identified as potential autophagy inducers. Subsequent confirmation studies narrowed this group to 5 previously uncharacterized autophagy-inducing candidates for further investigation. Follow-up studies assessed the mechanisms underlying autophagy modulation by these compounds, with a focus on key pathways such as mechanistic target of rapamycin inhibition, endoplasmic reticulum stress activation, and p53 activation. To further explore their therapeutic potential in cancer, we conducted an angiogenesis inhibition assay. This study successfully identified several autophagy inducers that may be repurposed for the treatment of cancer, highlighting their potential for future therapeutic development. SIGNIFICANT STATEMENT: This study identifies novel autophagy inducers from high-throughput screening of approved and investigational drugs. The findings highlight key pathways such as mechanistic target of rapamycin inhibition and endoplasmic reticulum stress activation, and demonstrate the ability of these compounds to inhibit angiogenesis, suggesting their potential for repurposing in cancer therapy.