Hepta and octapeptide agonists of protease‐activated receptor 2

Protease-activated receptor 2 (PAR(2)) is a G protein-coupled cell surface receptor for trypsin-like enzymes. Proteolytic cleavage at a specific site in the extracellular N-terminus exposes a receptor-activating sequence, the 'tethered ligand', which binds intramolecularly to initiate receptor signalling. Peptide or small molecule agonists for PAR(2), devoid of the non-specific and proteolytic effects of enzyme activators, may be promising therapeutic agents for proliferative and inflammatory diseases reportedly mediated by PAR(2). Synthetic hexapeptides that correspond to the native tethered ligand of human or rodent PAR(2) (SLIGKV and SLIGRL, respectively) can activate the receptor independently of proteolytic cleavage; however, known peptide agonists have much lower potency compared to protease-mediated activation. Here, we investigated the agonist activity of 94 hepta and octapeptide derivatives of the human and rodent PAR(2)-tethered ligand sequences in human airway epithelial (A549) cells which endogenously express PAR(2). Thirty synthetic peptides were found to be as potent as or more potent than SLIGRL on the basis of intracellular Ca(2+) responses. The more active peptide agonists were also examined for agonist cross-reactivity at PAR(1) in Chinese Hamster Ovary (CHO) cells that endogenously express functional PAR(1) but not PAR(2). Two potent and PAR(2)-selective agonists were further examined for their capacity to relax phenylephrine-contracted rat aortic rings. Our findings reveal an important role for carboxyl extensions to native PAR(2) activating peptides in potentiating agonist activity.