Project Summary Ferroptosis is a recently discovered type of regulated cell death that is biochemically distinct from apoptosis. Ferroptosis does not depend on the mechanisms by which cancer cells frequently evade apoptosis, such as overexpression of the apoptosis-inhibitor protein Bcl-2. Therefore, inducing ferroptosis represents a potentially orthogonal approach to kill cancer cells that have developed resistance to apoptosis. Additionally, ferroptosis has been proposed to be the mechanism of neuronal death in many neurodegenerative diseases, including Parkinson’s disease. A defining feature of ferroptosis is the iron-dependent accumulation of peroxidized membrane phospholipids containing polyunsaturated fatty acids. However, our understanding of the pathways downstream of peroxidized phospholipids that execute ferroptosis remains very limited. For instance, it is unclear whether peroxidized phospholipids are themselves cytotoxic or instead are metabolized into products, such as lipid-derived electrophiles (LDEs), which function as the primary drivers of ferroptosis. The goal of this proposal is to investigate these important questions using an innovative suite of chemical probes and activity-based proteomic methods. In Specific Aim 1, we will take advantage of an advanced library of selective and cell-active serine phospholipase inhibitors developed in the Cravatt lab to deduce the role of individual enzymes in regulating oxidized phospholipid metabolism and function. We will screen our inhibitor library to identify compounds that enhance or inhibit ferroptosis in human cancer cells. We will then use activity-based probes to confirm the targets of these active inhibitors. We will also perform lipidomic studies to determine how ferroptosis-modulating phospholipase inhibitors alter the oxidized phospholipid network of cancer cells. In Specific Aim 2, we will use advanced chemical proteomic methods to globally map cysteine reactivity during ferroptosis. Oxidized phospholipids are known to generate LDEs that covalently react with cysteine residues in proteins. We will globally quantify cysteine reactivity during ferroptosis, and reductions in cysteine reactivity will designate likely sites of LDE action. We will follow up on target proteins harboring ferroptosis-sensitive cysteines to determine how modification of these cysteines affects protein function. Finally, we will evaluate changes in cysteine reactivity that occur in the presence of ferroptosis-modulating phospholipase inhibitors. From a translational perspective, determining the biochemical mechanism of ferroptosis may identify therapeutic targets to promote or restrain this form of cell death for the treatment of human disease.