Project Summary Protein lipidation is a dynamic post-translational modification (PTM) that affects subcellular trafficking, co-factor binding affinity, and membrane localization of proteins, which in turn influence downstream signaling cascades. In particular, cyclic S-acylation and -deacylation of proteins at specific cysteine residues is emerging as a key link between circulating lipid levels and the regulation of essential biological processes, including those involved in cellular growth, metabolism, and neurological health. In-depth study of this PTM, however, has proven technically difficult, in large part due to the paucity of selective, effective chemical inhibitors for the enzymes that catalyze its installation and removal. The proposed research program is designed to generate novel chemical technologies, namely small molecule probes and inhibitors, in the service of illuminating the involvement of regulated protein S-acylation in both normal and pathophysiological contexts. These goals will be realized through two complementary chemical and cellular biology research areas. One area will involve measuring, manipulating, and determining the targets of the “writers” of S-acylation, DHHCs. To do so, pan-active DHHC inhibitors will be identified using newly developed and optimized screening and selectivity profiling assays in combination with rationally and computationally designed molecules, as well as a library of putative inhibitors. Validated inhibitors and proteomics-based methods will then be used to identify the specific protein targets of DHHCs in live cells to more precisely describe their involvement in various disease states. The second research area will utilize our recently validated chemical tools to study the biological function of the “erasers” of S-acylation, APTs, with particular emphasis on the involvement of these enzymes in cellular redox homeostasis and metabolic disease. The expected outcome of this multidisciplinary research program is two-fold: generating a collection of chemical tools and assays for the study of this important PTM, and describing its biological function and influence in normal and disease states.