Probing Biological Performance Diversity and the Discovery of Autophagy-Specific Inhibitors

The current generation of high-throughput screening (HTS) involves the parallel processing of hundreds of thousands of molecules. Despite such large-scale efforts, low hit rates for challenging targets slow the overall rate of novel therapeutic discovery. The success of future screening campaigns for unmet needs can be supported by advances in the quantification of the biological performance diversity and the prediction of chemical features that enhance this diversity. Compound libraries that are biologically performance-diverse contain molecules that have broad, nonredundant coverage of a large set of biological targets. These libraries would accelerate drug discovery by increasing hit rates while reducing the overall footprint of HTS. In this work we present the profiling of two flavonoid-based libraries using the Cell Painting assay to establish which types of chemical and physical features are most relevant for the composition of biologically performance-diverse libraries. We conclude that the impact of varying features is highly dependent on the scaffold around which a library is based further emphasizing the need to screen libraries as they are synthesized to inform synthetic efforts. Autophagy is a catabolic process implicated in a variety of human diseases due to its role in the maintenance of cellular homeostasis. During autophagy, cellular cargo is sequestered within double-membrane autophagosomes which traffic these contents to the lysosome for recycling. Many autophagy modulators exist, but these modulators lack overall specificity, either impacting autophagy at several points within the pathway or affecting other pathways within the cell. To better understand the role of autophagy in various diseases, we need to develop autophagy-specific probes that can be used to evaluate the underlying biology and serve as the starting points for the creation of effective therapies. We approached this problem by targeting two protein-protein interactions (PPIs) found within the autophagy pathway, the ATG14L-Beclin 1 PPI and the ATG5-ATG16L1 PPI. To target these interfaces, we developed two HTS-compatible assays utilizing NanoBRET and fluorescence polarization, respectively. We successfully implemented the NanoBRET HTS to discover a novel molecule that inhibits the formation of VPS34 Complex I and disrupts autophagy more selectively than known autophagy inhibitors that target the VPS34 kinase directly.