Summary: There is emerging need in new medical products that can mitigate and/or treat the short- and long-term consequences of radiation exposure after a radiological or nuclear terrorist event that may cause a public health emergency. Ionizing radiation causes radiolysis of water with production of free radicals. While direct effects of ionizing radiation and water radiolysis products are realized primarily via apoptotic cell death pathways in rapidly proliferating cells within the initial 1-2 days after the exposure, the subsequent mechanisms of damage seem to be triggered by secondary responses whereby inflammatory cytokines, chemotactic factors and lipid mediators are the major players. At least two novel regulated necrotic cell death pathways, necroptosis and ferroptosis, are executed in response to inflammatory cytokines and lipid mediators. Necroptosis is dependent on receptor interacting protein kinase (RIPK) 1 and can be inhibited by Necrostatin- 1. Ferroptosis is an iron dependent cell death regulated by glutathione peroxidase 4 (Gpx4). The goal of our project is to decipher RIPK and Gpx4 driven signaling pathways associated with necroptosis and ferroptosis in mechanism-based discovery of radiomitigators based on our exciting Preliminary data: 1) discovery of radiomitigative potency of necrostatins (inhibitors of RIPK1) and a mitochondria-targeted Gpx4 mimic, Mito- Ebselen; and 2) detection of phospholipid oxidation/hydrolysis products, including cardiolipin-derived products ? during execution of ferroptosis in Gpx4 conditional knock out mice. During the last funding cycle we identified a new Ca2+-independent pathway for biosynthesis of lipid mediators after total body irradiation (TBI) with potential signaling roles in necroptosis and ferroptosis. Our central hypothesis is that necroptosis and ferroptosis are important pathogenic mechanisms of acute radiation injury syndrome triggered by pro- inflammatory responses (cytokines, lipid mediators) thus representing new targets for radiomitigation. Specific Aim 1 will perform detailed characterization of irradiation-triggered and pro-inflammatory cytokine- induced necroptosis and ferroptosis, including their association with phospholipid oxidation products and lipid mediators in the context of the design and development of new radiomitigators. Specific Aim 2 will explore the radiomitigative effectiveness of small molecule inhibitors of necroptosis. Specific Aim 3 will establish the contribution of ferroptosis to the pathogenesis of acute radiation injury and determine the effectiveness of several classes of ferroptosis inhibitors as radiomitgators. Experiments described in the above three Specific Aims include in vitro as well as in vivo studies with quantitation of both tissue and plasma signatures of regulated necrotic cell death pathways, cytokine and lipid mediators, and organ specific drug distribution. These studies will establish and optimize mechanism based novel therapies for radiation damage mitigation applicable for use in radiation counter terrorism.