Modulating human proteinase activated receptor 2 with a novel antagonist (GB88) and agonist (GB110)

BACKGROUND AND PURPOSE Many cells express proteinase activated receptor 2 (PAR2) on their plasma membrane. PAR2 is activated by proteolytic enzymes, such as trypsin and tryptase that cleave the receptor N‐terminus, inititating signalling to intracellular G proteins. Studies on PAR2 have relied heavily upon activating effects of proteases and peptide agonists that lack stability and bioavailability in vivo. EXPERIMENTAL APPROACH A novel small molecule agonist GB110 and an antagonist GB88 were characterized in vitro against trypsin, peptide agonists, PAR2 antibody, PAR1 agonists and flow cytometry,in seven cell lines using intracellular Ca2+ mobilization and examined in vivo against PAR2‐ and PAR1‐induced rat paw oedema. KEY RESULTS GB110 is a potent non‐peptidic agonist activating PAR2‐mediated Ca2+ release in HT29 cells (EC50∼200 nM) and six other human cell lines, inducing PAR2 internalization. GB88 is a unique PAR2 antagonist, inhibiting PAR2 activated Ca2+ release (IC50∼2 µM) induced by native (trypsin) or synthetic peptide and non‐peptide agonists. GB88 was a competitive and surmountable antagonist of agonist 2f‐LIGRLO‐NH2, a competitive but insurmountable antagonist of agonist GB110, and a non‐competitive insurmountable antagonist of trypsin. GB88 was orally active and anti‐inflammatory in vivo, inhibiting acute rat paw oedema elicited by agonist GB110 and proteolytic or peptide agonists of PAR2 but not by corresponding agonists of PAR1 or PAR4. CONCLUSIONS AND IMPLICATIONS The novel PAR2 agonist and antagonist modulate intracellular Ca2+ and rat paw oedema, providing novel molecular tools for examining PAR2‐mediated diseases.