PROJECT SUMMARY: Surgical pain is caused by tissue injury and inflammation. To treat surgical pain, people are given opioids which is leading to secondary health problems after surgery including opioid abuse, dependence, and overdose that is driving the United States opioid epidemic. This is particularly important for older adults as treating pain with opioids also present challenges with age-related changes in drug metabolism in addition to drug-drug interactions due to polypharmacy. Therefore, discovering new non-opioid targets and developing non-opioid treatments that are as effective in the young and elderly populations to alleviate surgical pain are urgently needed. For this HEAL proposal, the goal of the research is to develop a peptidomimetic to block the interaction of NF-κB essential modulator (IKGKB, UniProt #Q9Y6K9) with IKKβ and further determine whether a peptide targeting NF-κB essential modulator (NEMO) reduces surgical pain in young and old rodents. NEMO is a promising non-opioid target to develop drugs to treat pain since NEMO is a critical protein regulating the canonical pathway of NF-κB-mediated inflammation. Here we will leverage new key findings regarding the NEMO interaction site with IKKβ based upon the crystal structure to develop a peptidomimetic to block the NEMO interaction with IKKβ. In order to carry out this work, we developed an assay to screen NEMO peptide modifications by using a mouse skin fibroblast NIH-3T3 cell line that contains a stable NF-κB luciferase reporter. Further, we will determine whether a NEMO binding peptide will limit pain and inflammation after injury using a surgical incision model. To carry out these studies, we developed a rodent paw surgical incision model that increases phosphorylated NF-κB 3-fold in addition to a 10-fold increase in NF-κB-regulated pro- inflammatory cytokines including IL-6 and IL-1β. Taken together, this proposal can advance the field by developing a potential non-opioid therapeutic to treat pain and test whether a peptide binding NEMO is effective in reducing surgical pain in young and old rodents. Additionally, since the studies performed for this proposal will determine whether binding NEMO limits NF-κB activation and production of NF-κB-mediated proinflammatory genes, these studies also have a broad importance to aging. This is because inflammation is a hallmark of aging and the cellular senescence- associated secretory phenotype described in aging cells is driven by increases in IL-6 and IL-1β; where IL-6 is the major driver of this phenotype. Therefore, developing a peptidomimetic that binds NEMO may potentially have broader implications besides treating surgical pain and useful in limiting inflammatory pain for the elderly.