PROJECT SUMMARY Migraine affects over 36 million people in the US, particularly those of working age (18-55 years), negatively impacting workplace productivity and presenting an enormous healthcare burden. For almost half of those who suffer from migraine, current treatments fail to provide relief and have limited ability to reverse pain once it has started. The most effective medications either require injection (CGRP antibodies) or are associated with increased cardiovascular risk (triptans). In addition, none of these provide complete relief to most patients. Migraine pain results from aberrant activation of specialized neurons that innervate the meninges, resulting in the release of neurotransmitters that cause local inflammation, and trigger a pain signal to central terminals in the brainstem. This signal is then carried to higher brain centers resulting in the perception of pain, and the continued presence of inflammatory mediators in the periphery perpetuate this painful signal. Protease-activated-receptor-2 (PAR2) is a membrane receptor that is activated by enzymes that are released from immune cells in the meninges, over the course of a migraine attack. Its activation contributes to migraine pain at multiple points: increasing the release of excitatory neurotransmitters; lowering the threshold for activation of pain transmitting neurons; and promoting the synthesis of additional inflammatory mediators. The main site of action of PAR2 is in the peripheral nerve terminals. This is in contrast to the targets of current migraine therapeutics that also play major roles in the central nervous system. We have identified two small molecule inhibitors of PAR2 that reduce migraine-like pain in animals. Significantly, these molecules act without crossing the blood brain barrier (BBB), even when administered after the onset of an attack. In this proposal, we aim to modify the chemical scaffold of our lead compound to improve its potential for oral delivery. We will use a high throughput screen to examine inhibition of receptor activation and downstream signaling pathways. Selected compounds will then be tested for favorable pharmacokinetics (e.g., stability, oral bioavailability, and low BBB penetrance) and pain-reduction in animal models of migraine. The overall goal is to identify a lead compound that can be delivered orally to move into IND-enabling studies for the treatment of migraine pain.