PROJECT SUMMARY/ABSTRACT Substance abuse behaviors result from molecular changes to gene expression programs in neurons over time. Morphine and heroin are highly addictive opioids that primarily stimulate the mu opioid receptor resulting in short- term euphoria. The addictive nature of morphine and heroin, unlike natural opioid peptides, may result from their cell permeability and thus direct influence on gene expression programs leading to addiction. However, understanding these precise molecular mechanisms is typically challenging due to the dearth of technologies to precisely map global molecular targets and pathways of a small molecule. We have developed an interdisciplinary precision pharmacology strategy to map the direct and indirect effects of a small molecule in the cellular proteome that we propose to apply to these addictive opioids. Our approach draws on the fields of chemical biology, mass spectrometry and data science to enable insight to the full range of molecular interactions, the structural biology underlying the interaction site, and changes to downstream pathways in a single experiment. The strategy involves: (1) treatment of whole cells with the small molecule of interest, (2) isolation of the resulting global molecular binding sites and (3) confident mass spectrometry-based assignment. We recently applied this platform to study three non-steroidal anti-inflammatory drugs (NSAIDs). Our results revealed several protein complexes involved in gene expression that the NSAIDs interact with, including a directly with the nucleosome. These results point to the vast web of molecular mechanisms that is now observable by precision pharmacology. In this proposal, we will apply our technology to morphine and heroin to determine their direct influence on gene expression leading to substance abuse. We will first develop a set of “click opioids” and “photo-click opioids” as generally useful probes for tracking opioid mechanisms in biology. We will specifically apply these probes to characterize opioid-driven genetic and epigenetic regulation in neuronal cells first in tissue culture and subsequently to mouse models of addiction. With a global map of the opioid interactome, these data will reveal direct opioid interactions with nucleosomes by binding, covalent modification, or mediating acetylation marks, and the indirect influence on upstream transcriptional programs that drive gene expression changes. By characterizing the broader interactions of the opioids, we are poised to expose molecular mechanisms leading to addiction, identify novel targets for new therapeutics and diagnostics, and open new paradigms in biological regulation by small molecules. The outlined research is a conceptually novel approach to perform mechanism of action studies that is suitable for the Avenir Award due to the broad potential for impact and early stage of this research.