PROJECT SUMMARY – RP5 Small molecules that enhance autophagy have the potential to serve as therapeutics against a phylogenetically diverse and broad range of pathogens. Nonetheless, most of the proteins that participate in, or regulate, autophagy lack chemical probes and some may even be viewed as undruggable. To accelerate the discovery of chemical probes, and ultimately drugs that promote anti-infective autophagy, new platforms are needed that can broadly and efficiently evaluate small molecule interactions for autophagy-related proteins from diverse structural and functional classes. Our laboratory has recently introduced the first chemical proteomic platforms to globally assess the druggability of proteins directly in native biological systems. These quantitative mass spectrometry-activity-based protein profiling (MS-ABPP) platforms can be applied to any cell type or state and have identified small-molecule hit ligands for many hundreds of proteins across a wide range of classes, including those previously considered undruggable (e.g., adaptors, transcription factors). Mining our current MS-ABPP data sets of the human proteome, which contain 30,000+ sites on 10,000+ proteins, has uncovered evidence of druggability for several proteins involved in degradative autophagy and/or ATG gene- dependent immunity. In RP5, we will use our MS-ABPP platforms in close collaboration with RP1-4 to discover and optimize small-molecule probes that enhance autophagy-mediated defense against infectious diseases. We will pursue the following Specific Aims: 1) the chemical proteomic identification of protein targets of autophagy-stimulating small molecules; 2) the chemical proteomic discovery of hit ligands for prioritized autophagy proteins; 3) the chemical proteomic discovery of novel, druggable proteins that regulate autophagy; and 4) the optimization of chemical probes for prioritized autophagy proteins. RP5 should furnish potent and selective chemical probes for several prioritized autophagy proteins, and these probes will be provided to RP1- 4 for biological studies. The chemical probes developed in RP5 should also serve as much needed tools for the greater autophagy and infectious disease communities, as well as valuable starting points for the development of broad-spectrum anti-infective therapies.