Applying an high-throughput drug screen for novel autophagy-specific PPI inhibitors for personalized cancer treatment

Autophagy is a cellular catabolic pathway, conserved in all eukaryotic cells. Its specific role in regaining and maintaining homeostasis under stressful conditions makes autophagy highly important for cells survival and viability. Current research has established that in many tumors, especially during chemotherapy or targeted cancer therapy, autophagy is upregulated and assists the cancer cells to cope with various stress conditions that develop in the tumor microenvironment. Hence, inhibition of autophagy is beneficial for cancer therapy, as it can be used in combination with other conventional treatments. Currently a few indirect autophagy inhibitors are being used in clinical trials, such as Chloroquine and its derivatives, which have many off target effects, underscoring that the field lacks specific autophagy inhibitors. Here we recruited the protein-fragment complementation assay (PCA) to establish a robust high-throughput system to screen for novel autophagy compound inhibitors, specifically targeting protein-protein interactions (PPI). We focused on PPI that form part of the core machinery of autophagy. A careful choice of the ultimate pair of proteins to be targeted in this screen was based on the solved crystal structure of the interaction interface between them, which revealed a promising ‘hotspot’ that is amenable to inhibition by small molecules, and which also guaranteed less off- target effects. The platform was adapted to large robotic compound screens. Potential hits will be tested in cancer organoid assays that we intend to develop from patients’ tumors and in xenograft mouse models based on these same tumors.