PROJECT SUMMARY Substance abuse disorders including opioid dependence are characterized by brain oxidative stress and neu- roinflammation. In addition to dopamine autooxidation, chronic drug consumption increases the expression of pro-oxidant enzymes such as NADPH-oxidase (NOX), leading to excessive production of reactive oxygen spe- cies (ROS) primarily in the form hydrogen peroxide (H2O2). Endogenous H2O2 can transiently and/or irreversibly oxidize cysteinyl residues depending upon its concentration and duration of exposure. In proteins, H2O2 reacts with redox-sensitive cysteine thiols to form sulfenic acid (Cys-SOH). Cysteine S-sulfenation has emerged as a major post-translational modification that exerts significant effects on protein function, analogous to phosphory- lation. A small but compelling literature suggests that changes in cysteine redox state affect mu opioid receptor (MOR) function, as thiol alkylation, site-directed mutagenesis, and redox-modulating agents can alter ligand binding and downstream signaling events. In addition to structural and regulatory roles, oxidation of the cysteine thiol blocks the reaction of this residue with a,b-unsaturated carbonyls and alkyl halides, significantly limiting the utility of conventional electrophile-fragment screening as a tool for covalent ligand discovery. To address this issue, we have recently developed a strategy that employs nucleophilic covalent fragments to target S-sulfenated (oxidized) cysteines. These sulfenic acid-reactive activity-based protein profiling (ABPP) probes have been cou- pled with state-of-the-art quantitative proteomics to identify S-sulfenated cysteines in human cells, which presage the development of covalent fragments therapeutically targeting redox-active cysteines. Here, the following Spe- cific Aims are proposed: 1) Increase the size and structural diversity of our nucleophile-fragment libraries; 2) Map cysteine redox reactivity changes and S-sulfenated (oxidized) cysteine ligandability in differentiated mature SH- SY5Y neurons that are unstimulated (control) or treated with morphine agonist (stimulated). These studies are rationalized based on proof-of-concept experiments which demonstrate that unique ligandable sites are identified when fragments are functionalized with nucleophilic reactive groups that react with S-sulfenated (oxidized) cys- teine residues. Deliverables from these studies are a novel chemoproteomic method, chemical matter that can be mined as a source of small-molecule probes and as starting points for drug discovery.