%0 Journal Article %A Yuhong Jiang %A Mei-Kwan Yau %A Junxian Lim %A Kai-Chen Wu %A Weijun Xu %A Jacky Y Suen %A David P Fairlie %T A potent antagonist of protease-activated receptor 2 that inhibits multiple signaling functions in human cancer cells %D 2017 %R 10.1124/jpet.117.245027 %J Journal of Pharmacology and Experimental Therapeutics %P jpet.117.245027 %X Protease-activated receptor 2 (PAR2) is a cell surface protein linked to G-protein dependent and independent intracellular signaling pathways that produce a wide range of physiological responses, including those related to metabolism, inflammation, pain and cancer. Certain proteases, peptides and nonpeptides are known to potently activate PAR2. However, no effective potent PAR2 antagonists have been reported yet despite their anticipated therapeutic potential. This study investigates antagonism of key PAR2-dependent signaling properties and functions by an imidazopyridazine compound, I-191, in cancer cells. At nanomolar concentrations, I-191 inhibited PAR2 binding of, and activation by, structurally distinct PAR2 agonists (trypsin, peptide, nonpeptide) in a concentration-dependent manner in HT-29 cells. I-191 potently attenuated multiple PAR2-mediated intracellular signaling pathways leading to Ca2+ release, ERK1/2 phosphorylation, RhoA activation and inhibition of forskolin-induced cAMP accumulation. The mechanism of action of I-191 was investigated using binding and calcium mobilization studies in HT29 cells where I-191 was shown to be non-competitive and a negative allosteric modulator of the agonist 2f-LIGRL-NH2. The compound alone did not activate these PAR2-mediated pathways, even at high micromolar concentrations, indicating no bias in these signaling properties. I-191 also potently inhibited PAR2-mediated downstream functional responses, including expression and secretion of inflammatory cytokines, cell apoptosis and migration, in human colon (HT-29) and breast (MDA-MB-231) cancer cells. These findings indicate that I-191 is a potent PAR2 antagonist that inhibits multiple PAR2-induced signaling pathways and functional responses. I-191 may be a valuable tool for characterising PAR2 functions in cancer and in other cellular, physiological and disease settings. %U https://jpet.aspetjournals.org/content/jpet/early/2017/12/20/jpet.117.245027.full.pdf