Novel and Specific Agonists to Protease‐activated Receptor‐2 (PAR‐2) Stimulate Multiple Signaling Pathways to Induce a Physiological Response in vitro and in vivo

PAR‐2 is a protease‐activated GPCR and contributes to the cellular response to endogenous and exogenous proteases. Proteolytic cleavage of a tethered ligand activates PAR‐2 and two signaling pathways: MAP kinase and intracellular Ca 2+ . Peptides or peptidomimetics can mimic tethered ligand binding to activate signaling without non‐specific effects. The most common peptide activators lack potency at the receptor. However, the potency of 2‐furoyl‐LIGRLO (2‐f‐LIGRLO) underscores the use of peptidomimetics as a mechanism to enhance PAR‐2 activation. We sought to develop potent, specific PAR‐2 agonists and a high‐throughput method to evaluate them. We identified two novel agonists, 2‐aminothiazol‐4‐yl‐LIGRL (2‐at‐LIGRL) and 6‐aminonicotinyl‐LIGRL (6‐an‐LIGRL), specific for PAR‐2. Both agonists stimulated Ca 2+ and MAPK signaling, and a physiological response in in vitro , with similar potency as 2‐f‐LIGRLO. Because PAR‐2 plays a major role in pathological pain and to test potency of the novel agonists in vivo , we evaluated each agonist in nociceptive models. 2‐at‐LIGRL and 6‐an‐LIGRL activated Ca 2+ signaling in nociceptors and 2‐at‐LIGRL and 2‐f‐LIGRLO stimulated thermal hyperalgesia in vivo . We have identified two novel PAR‐2 agonists using a three‐tiered screening methodology. We propose to use these novel agonists to explore PAR‐2 physiology in various systems and pathologies.