PROJECT SUMMARY/ABSTRACT Migraine is the most common neurological disorder in the world characterized by episodes of severe headache accompanied by a variety of symptoms such as nausea, vomiting, and depression of activity. Despite its prevalence, treatments for migraine are poor. Nonspecific analgesics such as opioids and nonsteroidal anti- inflammatory drugs (NSAIDs) produce pain relief, but repeated may produce addiction and gastric ulcers, respectively. Traditional treatments for migraine such as triptans (serotonin receptor agonists) are plagued by side effects, while repeated use of certain anti-migraine treatments worsen headache in a condition known as medication-overuse headache (MOH). Thus, there is a desperate need for new non-opioid anti-migraine agents with novel mechanisms of action. To this end, we aim to develop dual inhibitors, single small molecules that will simultaneously inhibit two pain-related enzymes: soluble epoxide hydrolase (sEH) and fatty acid amide hydrolase (FAAH). The most original and mechanistically distinct aspect of these compounds is their ability to inhibit two different enzymes that play significant roles in pain and inflammation within the trigeminovascular system simultaneously. Dual sEH/FAAH inhibitors described here have the potential to be used as a promising novel non-opioid anti-migraine strategy. Our undergraduate students have identified several very potent dual inhibitors and tested our lead inhibitor in vivo to demonstrate antinociception in a rat model of acute inflammatory pain. We also observed that this dual inhibitor produces antinociception at lower doses than traditional NSAID ketoprofen and does not produce behaviorally disruptive side effects. In the proposed studies we will rigorously evaluate our two lead dual inhibitors in a rat model of migraine pain, synthesize and evaluate novel libraries of ligands that are designed to interact simultaneously with both target enzymes, assess the anti-migraine effects of these new ligands, and evaluate whether administration of our dual inhibitors produce opioid-like rewarding effects or medication overuse headache. The compounds we propose to study represent a completely novel non-opioid, groundbreaking effort in anti-migraine treatments. Because this class has different biological targets from existing treatments, it represents an opportunity to solve long-standing problems that have been linked to the existing anti-migraine therapies (e.g., opioids, triptans). To our knowledge, this proposal is the first attempt to use the strategy of dual sEH/FAAH inhibitors as a rational anti-migraine therapeutic strategy. In addition, the design principles, synthetic and in vivo strategies behind these dual inhibitors are truly multi-disciplinary, rigorous, comprehensive, and innovative. Our studies will provide a firm foundation for the future investigation of using this dual ligand strategy as a treatment for migraine pain. This proposal represents collaborat...