Chemical genetics aims to elucidate the molecular mechanisms behind complex and/or poorly characterized biological processes by perturbing them with small-molecules. Such chemical probes can be powerful tools to modulate complex physiologies involved in disease and basic biological processes. However, the tendency of small molecules to interact with multiple proteins and the lack of robust technologies to systematically characterize these interactions hinders the molecular understanding of their pharmacology. To address this deficiency in chemical genetics, we propose to synthesize a small-molecule library based on an alkynyl-7-benzoyl-benzo-1,4-diazepin-2,5-dione scaffold such that every library member can be photocrosslinked to its protein binding target(s) directly in living cells without requiring any further probe modification. We will apply our library to three different phenotypic-based screens to identify small-molecules that affect biochemical pathways relevant to aging, metabolism, and adipogenesis. We will then identify the targets of bioactive probes using established Cravatt lab proteomics techniques involving in situ UV-photocrosslinking, bioorthogonal conjugation chemistry, and LC-MS. Overall, this library design and chemical proteomics platform constitute a potentially universal strategy to generate novel chemical probes, identify hit-target interactions, and elucidate key protein components in poorly characterized and/or complex pathways. PUBLIC HEALTH RELEVANCE: The aim of this project is to develop an advanced screening platform that enables rapid, simultaneous discovery of disease-relevant protein targets and selective small-molecules that perturb these targets. This technology has the potential to yield new chemical probes and drugs that can be used to elucidate the underlying components of disease in a systematic way and affect their treatment to restore health.